Functional Tissue Engineering of Ligament and Tendon Injuries

C H A P T E R

67 Functional Tissue Engineering of Ligament and Tendon Injuries Savio L-Y. Woo1, Jonquil R. Mau1, Huijun Kang1, Rui Liang1, Alejandro J. Almarza1, Matthew B. Fisher2 1

University of Pittsburgh, Pittsburgh, PA, United States; 2North Carolina State University, Raleigh, NC, United States

INTRODUCTION Tendons and ligaments are soft connective tissues composed of closely packed, parallel collagen fiber bundles that connect bone to muscle and bone to bone, respectively. These unique tissues serve essential roles in the musculoskeletal system by transferring tensile loads to guide motion and stabilize diarthrodial joints. Injuries to tendons, such as the patellar tendon (PT) of the knee, or ligaments, such as the collateral and cruciate ligaments of the knee, upset the balance between mobility and stability of this joint. These injuries are often manifested in abnormal knee kinematics and damage to other tissues in and around the joint, such as meniscus and articular cartilage, which may lead to morbidity, pain, and osteoarthritis. With the high incidence of ligament and tendon injuries in sports and work-related activities, improvements in healing and repair of these tissues are of great interest [1]. There is dramatic variability in the propensity for healing of the medial collateral ligament (MCL) and anterior cruciate ligament (ACL) in the same knee joint. Clinical and laboratory studies have shown that injuries to the MCL could generally heal well such that nonsurgical management has become the treatment of choice and the structural properties of the femureMCLetibia complex (FMTC) in the functional range are restored within weeks [2e8]. However, the mechanical properties of the healed MCL (i.e., the stressestrain relationship) could not return to those of the normal MCL and the histomorphological appearance (e.g., uniform distribution of small collagen fibrils) and biochemical composition are altered (e.g., elevated type III and V collagens) [9e19]. As such, the healing MCL needed to be significantly larger (higher in quantity) to make up for the lack of quality. For the ACL, it is well-known that a midsubstance tear would not heal and the success of nonsurgical management has been limited. Thus, surgical reconstruction of the ACL using autografts harvested from the PT or hamstring tendons as a replacement is performed to restore knee stability and function. However, there are issues affecting patient outcome from the use of boneePTebone (BPTB) autografts, including a persistent palpable defect in the tendon, anterior knee pain, arthrofibrosis, changes to the remaining PT, and PT adhesion to adjacent tissues (i.e., the fat pad) [20,21]. For hamstring tendon autografts, there are issues including a slower healing rate at the soft tissue to bone interphase, less long-term stability of the knee [22], significant hamstring muscle weakness [23,24], as well as an increased prevalence of bone tunnel enlargement after reconstruction [25e28]. Long-term clinical follow-up studies for patients who underwent ACL reconstruction for 10 or more years demonstrate up to 25% of unsatisfactory results that include a prevalence of osteoarthritis [29e31]. Hence, functional tissue engineering (FTE) and regenerative medicine approaches are attractive to improve the suboptimal quality of the healing MCL and issues related to ACL graft harvest and healing after reconstruction. In addition, healing of the injured ACL using FTE as an alternative to reconstruction is a subject of intense interest. These same principles could also be applied to aid the repair of other ligaments and tendons [17,32e37]. In this chapter, we will review the properties of normal and healing ligaments and tendons and discuss the current FTE methods, including the use of growth factors, gene delivery, stem cell therapy, and biological augmentation as well as mechanical augmentation, aimed at enhancing tendon and ligament healing. The goal is not only to restore Principles of Regenerative Medicine, Third Edition https://doi.org/10.1016/B978-0-12-809880-6.00067-9

1179

Copyright © 2019 Elsevier Inc. All rights reserved.

1180

67. FUNCTIONAL TISSUE ENGINEERING OF LIGAMENT AND TENDON INJURIES

the normal histomorphological appearance, biochemistry, and mechanical properties of the healing ligament, but most important, to regain normal joint function. New technologies and research avenues are suggested that have the potential to enhance treatment strategies for ligament and tendon injuries.

NORMAL LIGAMENTS AND TENDONS Biology Ligaments and tendons are hypovascular [38e40] and hypocellular with less than 5% of the total volume occupied by cells [39,41,42]. The cells in these tissues, fibroblasts, or tenocytes, exert important functions in maintaining and remodeling the extracellular matrix (ECM) in which they reside, which consists of both fibrillar (e.g., collagen and elastin) and nonfibrillar (e.g., proteoglycans, elastin, glycolipids, and water) components. The collagen fibers are organized and well-oriented along the longitudinal direction. Normally, the cells are arranged in rows along these fibers [43,44]. Collagen fibers in normal ligaments or tendons are mainly formed by type I collagen (roughly 70e80% dry weight). When viewed histologically under unloaded conditions, these collagen fibers display a crimp pattern with a regular wavy appearance [41,42,45,46]. Under transmission electron microscopy, a bimodal distribution of collagen fibril size can be observed with one group of fibrils measuring 40e75 nm in diameter, and the other, larger fibrils 100e150 nm in diameter [47e50]. Such a bimodal distribution is functionally significant because the incorporation of a high fraction of small-diameter fibrils ensures better interfibrillar binding by virtue of their higher surfaceevolume ratio whereas the large-diameter fibrils meet the strength requirements. In addition, a bimodal distribution can improve fibril packingdthe smaller fibrils wedge themselves in the spaces left among the larger ones [51]. Besides type I collagen, many other collagen subtypes, including III, V, X, XI, and XII, are present in much smaller amounts but still play important roles in maintaining the structure and functions of ligaments and tendons. For example, type III collagen is involved in tissue healing and remodeling [52]; type V collagen has been found to exist in association with type I collagen and serve as a regulator of collagen fibril diameter [53,54]; and type XII collagen provides lubrication between collagen fibers [55]. Interestingly, collagen types IX, X, and XI have been identified to coexist with type II collagen at the fibrocartilaginous zone of the ligamentebone and tendonebone interfaces [56e58]. The significance of the coexistence has been related to the minimization of stress concentrations when loads are transmitted from soft tissue to bone [59,60]. The ground substance makes up only a small percentage of the total dry tissue weight of ligaments or tendons but it is nevertheless pivotal in its function because of its ability to intake water. Water and proteoglycans are crucial to the gliding function of fibers in the tissue matrix by providing lubrication and spacing of collagen fibers. Elastin, another fibrillar macromolecule in matrix, is also present in ligaments and tendons at a few percent by weight. Although its detailed significance has yet to be elucidated, elastin has been implicated in its ability to allow the tissue to return to its prestretched length and shape upon unloading. Collectively, these constituents are indispensable in the normal functioning of collagen fibers by distributing load optimally in response to mechanical stress. Although ligaments and tendons are morphologically similar, they are different in their compositions. For example, ligaments are composed primarily of water (65e70% of wet weight) and collagen (70e80% of dry weight); type I collagen is the most abundant collagen subtype. Type III collagen (8% dry weight), type V collagen (12% dry weight), and other minor subtypes such as II, IX, X, XI, and XII, have also been found to be present [56e58,61]. Tendons, on the other hand, generally contain less water (55% wet weight) and slightly more type I collagen (85% dry weight), along with much smaller amounts of other collagen subtypes, such as III, V, XII, and XIV [49]. In addition, most ligaments are more metabolically active. They have more cellular nuclei, a higher DNA content, and greater amounts of reducible cross-links between collagen fibers [45].

Biomechanics The major function of ligaments and tendons includes maintaining the proper anatomic alignment of the skeleton and guiding joint movements. They also transmit forces along their longitudinal axis; hence, their biomechanical properties are measured in uniaxial tension and they exhibit nonlinear behavior governed by the recruitment of collagen. These unique properties allow ligaments to maintain smooth normal joint motion that requires low loads. In response to high loads, stiffness increases dramatically to limit excessive joint displacements. Thus, when large

1181

NORMAL LIGAMENTS AND TENDONS

muscle forces are applied, the bones are well-aligned to allow them to glide smoothly on each other. Ligaments and tendons also exhibit time- and history-dependent viscoelastic behavior that could be attributed to the complex interactions of tissue constituents such as collagen, proteoglycans, water, and ground substance [62e65]. Viscoelastic properties of ligaments and tendons are important and clinically relevant. For instance, during regular activities such as walking and jogging, tissues have the ability to soften over time. This phenomenon reduces susceptibility to damage related to fatigue [63e65]. However, after injury, ligaments and tendons generally fail to recover their normal mechanical and viscoelastic properties. Thus, abnormal joint kinematics may result that can lead directly to excessive forces on surrounding tissues (e.g., articular cartilage and meniscus in the knee). This excess loading could result in further injury causing degeneration such as osteoarthritis. Because the ultimate goal is to restore the properties of ligaments and tendons, and thus the function of the injured joint, it is necessary to understand their normal mechanical behavior and how they contribute to joint function. To do this, two common mechanical tests have been designed: (1) uniaxial tensile testing, which is a test to measure the structural properties of the boneeligamentebone complex and mechanical properties of the tissue substance; and (2) functional testing, which determines the contribution of the ligament or tendon to joint kinematics and the in situ forces in response to externally applied loading conditions. Uniaxial Tensile Testing Tendons are generally long and can be tested in their isolated state using sinusoidal-shape or frozen grips to limit slippage. Isolated ligaments, on the other hand, are shorter in length, which makes it difficult to clamp them independently. Hence, a uniaxial tensile test is generally conducted on the entire boneeligamentebone complex (e.g., FMTC) with tissue insertion sites left anatomically intact. With cross-sectional area (CSA) measurements and the use of tissue markers to measure tissue strain, the structural properties of the boneeligamentebone complex as well as mechanical properties of the ligament substance can be measured from a load to failure test [66,67]. Structural properties (Fig. 67.1) of the boneeligamentebone complex (i.e., a loadeelongation curve) are generally described by four parameters including stiffness (slope of the linear portion of the loadeelongation curve), ultimate load (maximum load at which the complex fails), ultimate elongation (elongation corresponding to the maximum load), and energy absorbed at failure (area under the curve to the maximum load). These data reflect the behavior of the entire boneeligamentebone complex, which includes tissue size, orientation of collagen fibers to applied loads, and as the contribution of the bony insertions [67]. Measuring the mechanical properties (Fig. 67.2) of the ligament substance (i.e., a stressestrain curve), on the other hand, requires knowledge of the CSA of the ligament, commonly measured using a laser micrometer system [66], and tissue strain, commonly measured using video techniques to track two or more reflective markers placed on the tissue midsubstance [8]. Stress in the tissue is obtained by dividing load by the CSA, and strain is obtained by computing change in the marker distance during the test relative to their original distance. Parameters describing the mechanical properties of the ligaments and tendons (Fig. 67.2) include the tangent modulus (slope of the linear portion of the stressestrain curve), tensile strength (stress at failure), ultimate strain (strain corresponding to the tensile strength), and strain energy density (area under the stressestrain curve until failure). These data represent the quality of the tissue, irrespective of tissue size. The viscoelastic properties of ligaments and tendons include stress relaxation (decrease in stress over time in response to a constant elongation) and creep (increase in elongation over time in response to a constant load). In addition, they display a phenomenon called “hysteresis” in response to cyclic loading (Fig. 67.3). This results from a loss of Failure

Ultimate load 750

Load (N)

600 Linear stiffness

450 300

Ultimate elongation

Energy absorbed

150 0 0

1

2

3

4

5

6

Elongation (mm)

FIGURE 67.1 Typical loadeelongation curve representing the structural properties of the femureanterior medial bundleetibia complex of the human anterior cruciate ligament.

1182

67. FUNCTIONAL TISSUE ENGINEERING OF LIGAMENT AND TENDON INJURIES

Failure

Tensile strength

Stress (MPa)

25 20 Tangent modulus

15

Strain energy density

10 5

Ultimate strain

0 0

5

15

10 Strain (%)

FIGURE 67.2

Atypical stressestrain curve representing the mechanical properties of the anterior medial bundle of the human anterior

cruciate ligament.

16 1st Cycle 14

10th Cycle

Load (N)

12 10 8 6

a Lo

4

din U

2

g

a nlo

din

g

0 0

0.2

0.4

0.6

0.8

1

Elongation (mm)

FIGURE 67.3 Hysteresis loops (first and 10th cycles) obtained from cyclic loading of the femureanterior medial bundleetibia complex of the human anterior cruciate ligament in uniaxial tension. The area of hysteresis (area between the loading and unloading curves) decreases with repetitive cycling, demonstrating the phenomenon of “preconditioning.”

internal energy causing the loading and unloading paths to be different. The area of hysteresis reduces as the tissue undergoes several cycles of loading and unloading and the tissue is said to be “preconditioned,” a state desired for a tissue before mechanical testing. Nonlinear viscoelastic models such as the quasilinear viscoelastic theory: Z t vse ðεÞ vε vs; (67.1) Gðt  sÞ sðtÞ ¼ vε vs N and single integral finite strain theory: 

2



T ¼ pI þ C0 ½1 þ mI ðtÞBðtÞ  mB ðtÞ  C0 ð1  gÞ 

Z

t

  _  sÞ ½1 þ mIðsÞBðtÞ  mFðtÞCðsÞFT ðsÞ ds; Gðt

(67.2)

0

have been used to model these behaviors in ligaments and tendons [62e65,68]. These basic testing methodologies described have been employed for a number of decades. In our research center, much work has been devoted to finding the most appropriate testing procedures, which include specimen orientation [69], handling, storage, and hydration [70,71]. These carefully developed methodologies have led to important findings regarding physiological changes associated with growth and development [69e71], the adaptation of ligaments and tendons to mobility [5,6,63e65,72], and the effects of injury and treatment [5,6,19]. Contribution to Joint Function Joint motion is governed by the direction and magnitude of externally applied loads, ligament forces, contact between joint surfaces, and muscle activity. For the knee, motions include a combination of translations (proximodistal,

1183

HEALING OF LIGAMENTS AND TENDONS

6-DOF robotic manipulator UFS AP VV ML

IE FE

PD

Tibla and Tibial clamp

Femur and Femoral clamp

FIGURE 67.4 Schematic drawing illustrating the six degrees of freedom of motion of the human knee joint, indicating anteroposterior (AP), mediolateral (ML), and proximodistal (PD) translation as well as flexioneextension (FE), internaleexternal (IE), and varusevalgus (VV) rotation. DOF, degrees of freedom.

mediolateral, and anteroposterior) and rotations (internaleexternal, flexioneextension, and varusevalgus). In total, these translations and rotations describe motion in 6 degrees of freedom (DOF). While evaluating joint function, constraining DOF of the knee can have significant impact on the results obtained [73,74]. When knee motion was allowed in all directions, sectioning the MCL resulted in only small increases in valgus laxity (21%), which suggests that the ACL has a significant role as a joint restraint to this knee motion. However, when anteroposterior translation and internaleexternal rotation were constrained, valgus laxity increased significantly (171%) after sectioning of the MCL. For this reason, it is important to have a testing device that allows for unconstrained knee motion. For more than 2 decades, a roboticeuniversal force-moment sensor (UFS) testing system developed by our research center has been used to study knee kinematics as well as directly measure the in situ forces in the knee ligaments in response to external loading conditions (Fig. 67.4) [75,76]. This methodology has been employed to study the function of the ACL and a number of variables for ACL reconstruction, including surgical technique [77e79] and graft choice [80]. In addition, knee function after ligament injury has been studied for both the MCL and ACL [8,81e84].

HEALING OF LIGAMENTS AND TENDONS The events of healing of ligaments and tendons can be roughly divided into four overlapping phases: hemorrhage, inflammation, repair (proliferation), and remodeling. Minutes after the ligament injury, blood collects and forms a platelet-rich fibrin clot at the injury site and the hemorrhagic and inflammatory phases occur over several days. In the hemorrhage phase, a cascade of cellular events occurs that includes release of cytokines within the clot followed by the appearance of polymononuclear leukocytes and lymphocytes. These cells respond to autocrine and paracrine signals to expand the inflammatory response and recruit other types of cells to the wound [85]. The reparative phase follows over the next couple of weeks to months. During this phase, fibroblasts recruited to the injury site start forming healing tissue. Growth factors, including transforming growth factor-b (TGF-b) and platelet-derived growth factor (PDGF) isoforms, are involved in modulating healing [86]. Meanwhile, increased neovascularization brings in circulating cells and nutrients to enhance the healing process further. The blood clot quickly turns into newly formed healing tissue that is composed of an aggregation of cells surrounded by a matrix.

1184

67. FUNCTIONAL TISSUE ENGINEERING OF LIGAMENT AND TENDON INJURIES

However, its histomorphological appearance and biochemical composition are different from those of an uninjured ligament. Notably, there is a homogeneous distribution of smaller-diameter collagen fibrils, which is in stark contrast to the bimodal distribution of the normal ligament [12,15,50]. Biochemically, it contains elevated amounts of proteoglycans, a higher ratio of type V to type I collagen, and a decrease in the number of mature collagen cross-links. Then the remodeling phase follows for months to years after the injury. In this phase, cellularity and levels of collagen type III are decreased, and the matrix is realigned in response to loading applied to the tissues. On the other hand, the diameter of collagen fibrils remains small, and the level of collagen type V remains elevated for years after injury [9,11,12,15,16]. Type V collagen has been shown to have a central role in regulating the lateral aggregation of smaller collagen fibrils. Thus, elevated type V collagen could be associated with the inferior mechanical properties of healing tissue [87,88].

Medial Collateral Ligament of the Knee The healing process of the MCL follows this general healing pathway, as described earlier. Thus, it serves as a good model for studying histological, biochemical, and biomechanical changes over time. It has been shown that the process of MCL healing is greatly affected by treatment [5,6,89e92]. Laboratory and clinical studies have shown that controlled mobilization is superior to immobilization [5,6,19,93]. As a result, nonoperative repairs have a better outcome than surgical repairs. In our research center, a severe “mop-end” injury model of the rabbit MCL that tears the midsubstance while damaging the insertion sites was developed and used to compare nonoperative treatment without mobilization with surgical repair with a brief period of mobilization [19]. After 12 weeks of healing, there were no significant differences in varusevalgus knee rotation, in situ force of the MCL, or tensile properties between repaired and nonrepaired MCL [19]. Based on these studies, clinical management shifted from surgical repair with immobilization to nonoperative management (i.e., bracing) with early controlled range of motion exercises as soon as pain subsides [94,95]. The MCL heals with nonoperative treatment, and the stiffness of the healing FMTC begins to approach normal levels, but the CSA of the healed tissue continues to increase with time, measuring as much as 2.5 times its normal size by 52 weeks after injury [93]. Meanwhile, the mechanical properties of the healing MCL remain consistently low compared with those for the normal ligament and do not improve with time. In other words, the healing process involves making a larger quantity of lesser-quality ligamentous tissue. Moreover, studies show that the rate of healing of the ligament is asynchronous with the insertion sites because of its anatomical and morphological complexity. There is also evidence that the activity level could influence the rate of healing [81e83]. A goat model was used because it has more robust activity, and it is larger than the rabbit. With this model, the stiffness and ultimate load of the healing goat FMTC are closer to control values at earlier periods than those from the rabbit model.

Anterior Cruciate Ligament of the Knee A midsubstance tear of the ACL has limited potential to heal on its own and can lead to chronic knee instability and damage to secondary stabilizers such as the meniscus. As a result, surgical reconstruction has become the treatment of choice. With the ultimate goal of ACL reconstruction being to restore knee function, the success of these procedures depends on a number of surgical, biomechanical, and biological factors. The most popular choice is autografts from the PT, i.e., bone central (or medial) third of the BPTB, or semitendinosus plus gracilis tendons. Allografts, including the Achilles tendon, BPTB, and hamstring tendons, have seen limited use except in revision surgery or for multiple ligamentous injuries. BPTB grafts are generally considered the reference standard for ACL reconstruction because they facilitate better initial fixation for bone-to-bone healing inside bone tunnels [96e101]. However, the major drawback is that the defect after the large incision does not completely heal for months [20,102e104]. This contributes to a higher incidence of complications including donor site morbidity, patella baja, arthrofibrosis, adhesion to the fat pad, and patellofemoral pain [105e110]. Studies designed to examine the healing PT after harvest have found a deterioration of structural properties of the remaining BPTB complex with a concomitant increase in the CSA of the PT tissue [111e115]. In the rabbit model, the ultimate load of the entire BPTB complex decreased by 38% [112] whereas there was an increase in CSA of 83e108% at 12 weeks after harvest [111,115]. For the central healing tissue, its tangent modulus and ultimate tensile strength were only 15% and 18% of controls, respectively, after 26 weeks [111]. The mechanical properties of the remaining PT tissues also deteriorated compared with sham controls after 24 weeks [115].

APPLICATION OF FUNCTIONAL TISSUE ENGINEERING

1185

After implantation, the autograft becomes inflamed and necrotic, leading to a decrease in graft stiffness and strength [116]. The graft undergoes revascularization and repopulation with fibroblasts followed by remodeling with restructure of the collagen fibers and proteoglycans [117]. Bone block healing within the femoral and tibial tunnels is complete by 6 weeks, whereas graft healing is incomplete, causing consistent failure to occur as the tendon is being pulled out from the tibial tunnel [118]. Over time, however, the femuregraftetibia complex shows improvement with complete incorporation even though its structural properties fail to be restored to levels of the intact femureACLetibia complex (FATC) even after 12 months [119e121]. Thus, accelerating graft incorporation and healing that lead to an earlier return to normal and sports activities has become a goal of FTE efforts. We will discuss the state of events later in this chapter. In addition to the graft selection, a number of important surgical decisions include tunnel placement, graft tension, and fixation. A substantial amount of research has focused on these variables in both animal and human models [78,81e83,122]. All of these parameters can lead to various degrees of graft tunnel motion that may affect graft integration and graft healing. Ultimately, these factors have an impact on postoperative rehabilitation and recovery as well as when to return to normal activities and sports.

Multiple Ligamentous Injuries in the Knee Combined ACL and MCL injuries frequently occur. The ideal treatment is still controversial. Some surgeons elect to reconstruct the ACL surgically without addressing the MCL whereas others advocate reconstruction of the ACL with repair of the MCL. Regardless of the treatment modality, clinical and basic science studies continue to show that the outcome of this injury is worse than for an isolated MCL injury but not as clear compared with an isolated ACL injury. Our research center has elucidated the effects of ACL deficiency on the healing of the injured MCL using canine, rabbit, and goat models [81e83,92,123,124] and recommended that only ACL reconstruction be performed. Furthermore, repairing the MCL in combination with ACL reconstruction resulted in reduced valgus laxity and improved the structural properties of the FMTC but only in the very early stages. In the longer term, all positive effects diminished [123,124]. To date, surgeons prefer to perform ACL reconstruction after a combined ACLeMCL injury. Using a larger animal model, i.e., the goat stifle joint, the function of the knee and quality of the healing MCL after a combined ACLeMCL injury treated with ACL reconstruction was examined [81e83]. It was found that the valgus rotation of the stifle joint was twice that for an isolated MCL injury. Moreover, the structural properties of the FMTC and tangent modulus of the MCL substance were substantially lower than those for the isolated MCL injury [8,81e83]. These results further demonstrate a clear need for new treatment strategies to enhance ligament healing after multiple ligament injuries.

APPLICATION OF FUNCTIONAL TISSUE ENGINEERING FTE emphasizes the importance of biomechanical considerations in the design and development of cell and matrix-based implants for soft and hard tissue healing. Ligaments and tendons are accustomed to being mechanically challenged; therefore, tissue engineered constructs used to replace these tissues after injury or disease must meet these demands. By combining the fields of molecular biology, biochemistry, and biomechanics, novel therapeutic approaches (e.g., growth factors, gene transfer/gene therapy, cell therapy, and biological scaffolds) offer new potential for better treatment. The following is a brief review of available approaches to enhance ligament and tendon healing.

Growth Factors The application of exogenous growth factors is based on the premise that they can promote healing that will lead to a biologically and biomechanically superior healed ligament substance. Many in vitro and in vivo studies have tried to examine their roles and determine appropriate strategies for their use. In Vitro Studies Cell culture or tissue explant methodologies have been the major study designs. By adding exogenous growth factors, responses including cell proliferation, synthesis of ECM proteins such as collagen, proteoglycans, tissue

1186

67. FUNCTIONAL TISSUE ENGINEERING OF LIGAMENT AND TENDON INJURIES

remodeling enzymes, and cell migration or chemotaxis were measured and compared [125e131]. Early studies in our research center measured the effects of eight different growth factors on the MCL and ACL fibroblast culture [123,132e134]. For cell proliferation, PDGF-BB, epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were found to have a significant effect on cell proliferation and caused greater proliferation in MCL fibroblasts versus ACL fibroblasts [134]. The proliferation of MCL and ACL fibroblasts from skeletally immature rabbits actually increased by 7.6 times in response to EGF and 5.6 times in response to bFGF [123]. In skeletally mature rabbits, the insulin-like growth factor and bFGF had significant effects on fibroblast proliferation in both cell types, but the difference was much less pronounced [134]. Other authors had shown the effect of TGF-b1 on ACL fibroblast proliferation to be dose-dependent and smaller doses could act synergistically with PDGF [135]. However, higher concentrations of TGF-b1 inhibited the stimulatory effect of PDGF. These findings suggest complex interactions of growth factors and their potential role in enhancing the proliferation of ligament fibroblasts. In terms of protein synthesis, TGF-b1 was most effective in collagen synthesis. In MCL and ACL fibroblasts, the level of increase was 160% over controls; most of this increase was for type I collagen [133]. These data suggest that TGF-b1 may improve ligament healing by increasing matrix synthesis during the proliferative and remodeling phases and confirm what was found by other investigators [135]. In vitro models, however, are limited in the extent that they cannot reproduce the complex interplay of signals affected by growth factors in the intricate process of ligament or tendon healing where there is a highly integrated biochemical network of cell signaling events with intrinsic stimulatory and inhibitory feedback loops. Thus, in vivo studies are needed to examine the interaction of biology and biomechanics and the degree to which the healed ligament or tendon substance could restore the biomechanical properties to those for the native tissue. In Vivo Studies Based on in vitro studies, EGF and PDGF-BB have the greatest effect on ligament fibroblast proliferation whereas TGF-b1 better promotes ECM production. These growth factors were then applied at different dosages, in isolation and in combination, for an MCL injury in the in vivo rabbit model. It was found that only a higher dose of PDGF-BB improved the structural properties of the FMTC [75,76]. A lower dose of PDGF-BB had little effect, which demonstrated that the effects of PDGF-BB were dose-dependent. However, the mechanical properties of the ligament substance remained unchanged from untreated controls, which demonstrated that the improved structural properties resulted from the formation of a larger quantity of tissue (instead of improved tissue quality). Other investigators showed that higher doses of PDGF could improve the structural properties of the healed ligament [136]. However, administration of PDGF for more than 24 h after the injury markedly decreased its efficacy. One possible approach to improving the in vivo application of growth factors and cytokines could be to combine it with gene transfer technology to extend their effectiveness with time. Adenoviral bone morphogenetic protein-2 (AdBMP-2) delivered to the boneetendon interface using a gene transfer technique has been shown to improve the integration of semitendinosus tendon grafts in rabbits [137]. The stiffness (29.0  7.1 versus 16.7  8.3 N/mm) and ultimate load (108.8  50.8 versus 45.0  18.0) were also significantly increased in specimens with AdBMP-2 compared with untreated controls. Based on these studies, an optimal therapy of introducing growth factors to injury sites is still an open question. The timing of application, mode of delivery, dosage, inclusion of scaffolds and/or cells, and interactions among these variables remain major hurdles. As an example, the delivery of stem cells transduced to express BMP-2 had adverse effects on rotator cuff repair [138]. As a result, investigations have been devoted to therapies such as platelet-rich plasma (PRP) and ECM bioscaffolds, which contain a number of bioactive factors [84,139]. In addition, it is well-recognized that stem cell delivery could modulate the healing response via trophic factors produced by the cells and not by engraftment of those cells [140]. Thus, these therapies could offer highly translational approaches to growth factor delivery.

Gene Therapy Gene therapy offers an exciting approach to improving ligament and tendon healing. Foreign nucleic acid gene transfer can be introduced into cells to alter protein synthesis or induce the expression of therapeutic proteins. Modern gene therapy relies on mammalian viruses and cationic liposomes as delivery vectors, and both have been developed to deliver genes into host tissue via direct (in vivo injection) and indirect methods (in vitro transduction).

APPLICATION OF FUNCTIONAL TISSUE ENGINEERING

1187

In our research center, we sought to determine whether genes could be transduced into MCL and ACL fibroblasts and whether ligament injury affected gene transfer and expression [141]. Both direct and indirect methods using adenovirus and BAG retrovirus, respectively, were employed. It was found that both techniques resulted in expression of the LacZ marker gene by fibroblasts from intact as well as injured ligaments. Gene expression lasted longer (6 weeks) with the direct method compared with the indirect technique (3 weeks). Fibroblasts from injured ligaments showed transduction in both the wound site and the ligament substance. There was no difference in the duration of gene expression by fibroblasts from intact and injured ligaments, which suggested that injury does not affect gene transfer or expression [141]. Antisense gene therapy that could block the transcription or translation of specific genes that are excessively expressed within healing tissue has also been studied. By binding antisense oligodeoxynucleotides (ODN) to target DNA, the efficacy of using ODNs to regulate the overproduction of collagens III and V was studied [35,142]. Normal human patellar tendon fibroblasts were transfected with antisense collagen III or V ODNs by mixing with lipofectamine. The uptake of ODNs was detected as early as 1 h and as late as 3 days after delivery. The relative expression of collagen V messenger RNA (mRNA) was reduced to 67.8%  5.1% of missense levels. Also, reverse transcriptaseepolymerase chain reaction results showed that the inhibitory effects of the collagen III antisense ODNs were most dominant at day 1, because the type III collagen mRNA level was 38.9%  19.6% of missense controls. At days 3 and 7, differences could no longer be observed. These results suggested that antisense gene therapy can be a potential FTE approach to enhance the quality of healing ligaments and tendons. Despite these promising results, several obstacles impede the practical implementation of gene transfer as a biological intervention for ligament healing. The immune reaction against these antigens decreases the expression of the introduced gene [143]. In addition, retroviral infection of fibroblasts often leads to shut-off of the promoter region, which adversely affects expression of the incorporated gene [144]. Thus, delivering ODNs to the appropriate target and reproducibility of the results remain great but exciting challenges. The literature has shown other techniques such as the use of lentivirus for gene transfer. For example, bFGF was transfected into bone marrowederived cells (BMDCs) and then implanted into the Achilles tendon [145]. However, these investigators did not observe an enhanced healing response from the injection of stem cells. Also, lentiviruse tumor necrosis factor-a (TNF-a)eRNA interferenceeexpressing TNF-a small interfering RNA was injected in the rotator cuff tendon in a rat model [146]. Thus, the inflammatory response was attenuated by the gene therapy after a chemical insult. Novel strategies including the search for more effective and less immunogenic vectors, modification of promoters to ensure gene expression after incorporation, and temporary and self-limiting gene expression regulation tailored to the changing environment continue to evolve in gene transfer to aid in ligament healing. Technologies such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/CRISPR-associated protein 9 could become a new strategy. As the complex steps involved in gene expression and regulation are further elucidated, the potential therapeutic efficacy of gene transfer is likely to find clinical application.

Cell Therapy Cell therapy is another potential approach to enhancing ligament and tendon healing because the use of autogenous cells would minimize the immune response. Studies have focused mainly on the application of mesenchymal stem cells (MSCs), specifically BMDCs and synovial tissue-derived fibroblasts into the healing site [147,148]. BMDCs have been shown to have an important role in wound healing [149e151] and can be obtained in higher numbers with relative ease [152]. BMDCs combined with PRP were used to treat rabbit ACL after reconstruction, which resulted in improved structural properties after 8 weeks of healing [153]. The treated tissues with combined PRP and BMDCs had a more mature boneeligament interface than those in the untreated controls and PRP-only group. Another study attempted transplantation of allogeneic tendon-derived stem cells to rat PT and observed improvement in healing at 16 weeks after injury [154]. Alternatively, fibroblasts, myoblasts, and bone marrow cells have been transplanted into injured ligaments after the induction of marker genes or stimulation by growth factors in vitro. However, issues remain regarding cell therapy. The number of MSCs from bone marrow are relatively low and decrease after transplantation. Thus, it is essential to develop novel in vitro techniques to expand MSCs without altering their differentiation potential. Furthermore, other methods, i.e., bioscaffolds, may be designed to prevent the loss of these cells and to sustain the positive effects.

1188

67. FUNCTIONAL TISSUE ENGINEERING OF LIGAMENT AND TENDON INJURIES

HEALING OF LIGAMENTS AND TENDONS The Use of Scaffolds An empirical method to improve the healing of ligaments and tendons is to use scaffolds to augment the injured part or bridge defects. An ideal scaffold should provide a suitable biological environment for cells to migrate into it and then proliferate, an appropriate mechanical cue to guide the formation of the newly synthesized ECM, and a built-in property to degrade slowly such that the newly formed matrix could take over in bearing the mechanical loads. Both synthetic and naturally occurring biological scaffolds have been used in FTE of ligament and tendon healing [155e158]. Major advantages of using synthetic polymers as scaffolds are their ease of fabrication and reproducibility. A structure can be created that mimics the structure of a ligament or tendon, and appropriate bioactive factors (i.e., growth factors) can be incorporated into the scaffold during manufacturing. However, because the performance of synthetic grafts has been found to be less than satisfactory [159,160], the focus has been placed on the supplementary use of cells, including BMDCs and fibroblasts [161,162]. Alternatively, a number of naturally occurring biological scaffolds, e.g., bovine pericardium (Integra Life Science), human dermal collagen (Alloderm), and porcine small intestinal submucosa (SIS), have been used after proper decellularization and sterilization. Among these scaffolds, SIS products have been approved by US Food and Drug Administration and are widely used in the field. Because SIS contains a large amount of collagen (over 90%) and has a preferred collagen alignment [163], it has the potential of acting as a contact guide to promote cells to deposit new matrix in a more aligned manner [164e167]. As a result, the mechanical and viscoelastic properties of the neotissue are improved. When used, preferably as a noncross-linked biodegradable scaffold, SIS degrades gradually when implanted in vivo. The pace of the degradation not only allows gradual replacement of the scaffold with the newly synthesized host matrix, it controls the timely release of degradation products that are biologically active and beneficial to healing [167a,168,170]. Moreover, SIS entraps a certain amount of bioactive agents (growth factors, fibronectin, and so on) that can be released during degradation to enhance healing [171e173].

Medial Collateral Ligament and Patellar Tendon Healing With Extracellular Matrix (Small Intestinal Submucosa) Multidisciplinary studies were performed to determine the effect of SIS treatment on the healing of an injured MCL (with a 6-mm gap) using a rabbit model [61,174]. Compared with the untreated group, SIS treatment resulted in improved histomorphological appearance, mechanical properties, and biochemical compositions of the healing MC; they were closer to those of normal ligament in both the short and long term (12 and 26 weeks) (Fig. 67.5). Specifically, SIS treatment further restored the heterogeneous distribution of collagen fibrils, because both large and small fibrils could be found compared with the persistently homogeneous small collagen fibrils in the untreated groups (Fig. 67.6). Accordingly, the CSA of the healing ligament in the SIS-treated group concomitantly decreased by 28%, indicating improved tissue quality. Indeed, improved mechanical properties of the healing MCL were 40

SIS-treated

35 Non-treated

Stress (MPa)

30 25 20 15 10 5 0 0

FIGURE 67.5

5

10 Strain (%)

15

20

Typical stressestrain curves for small intestinal submucosa (SIS)-treated and untreated groups at 12 weeks after injury.

HEALING OF LIGAMENTS AND TENDONS

1189

FIGURE 67.6 Transmission electron micrographs (70,000) of collagen fibrils in (A) sham-operated medial collateral ligament (MCL) (I), small intestinal submucosa (SIS)-treated MCL (II), and untreated MCL (III) at 26 weeks after injury. Arrow indicates the appearance of large fibrils between cells in the SIS-treated MCL. (B) Transmission electron microscopy appearance of both large and small fibrils (heterogeneity) in the pericellular area in the SIS-treated MCL (I) and untreated MCL (II). Arrows indicates the large fibrils surrounding a cell process. F, fibroblast.

found, because the tangent modulus increased by 33% and stress at failure increased by 49% [174]. These positive findings in morphology and biomechanics were mechanistically associated with better collagen organization as well as the regulation of collagen subtypes by a number of small leucine-rich proteoglycans, decorin, lumican, and biglycan, and so on, to reduce the collagen type VeI ratio [174a]. We extended the use of SIS for PT healing. After the central third of the tendon was harvested for ACL reconstruction, an SIS scaffold was introduced to aid its healing and limit the formation of adhesion (and permit motion) between the healing PT and the underlying fat pad (Fig. 67.7). The maintenance of stress and motion of the patellaefemoral joint motion would keep the homeostasis of the remaining PT [63-65,72], thus limiting problems associated with poor healing, including excessive hypertrophy of the healing PT with poor quality. When the SIS was applied to the anterior and posterior of a central-third PT defect (3 mm wide) in rabbits, the healing defect had a more organized matrix with a large number of spindle-shaped cells at 12 weeks after surgery compared with the untreated group, which had only patches of collagen with a sparse distribution of cells [174b]. The CSA increased by 61% in the SIS-treated group; concomitantly, the BPTB complex showed 38% higher stiffness and 58% higher ultimate load. These results clearly demonstrated that SIS treatment could accelerate PT healing.

Anterior Cruciate Ligament Healing With the advances in FTE, alternative approaches are being revisited to healing the injured ACL. Investigators have employed various biological methods such as PRP, ECM bioscaffolds, and so on to incite and accelerate ACL healing. Meanwhile, investigators also recognize that the rate of ACL healing is slow, and thus mechanical augmentation could be advantageous to maintain knee stability to facilitate the biological processes. The following overview covers both biological and mechanical augmentation for ACL healing as well as the combination of biological and mechanical strategies.

1190

67. FUNCTIONAL TISSUE ENGINEERING OF LIGAMENT AND TENDON INJURIES

FIGURE 67.7

Schematic of possible mechanism of action of small intestinal submucosa (SIS) on medial collateral ligament healing response. XSA, cross-sectional area.

Biological Augmentation There has been renewed interest in healing soft tissues with limited healing potential, such as the ACL, using biological augmentation. For example, Steadman and coworkers developed a microfracture technique that involves making very small holes in the bone at the femoral insertion site to encourage hematoma formation and provide an enriched environment for ACL healing [175,176]. This approach was used successfully to treat patients aged more than 40 years. In the laboratory, Murray and coworkers used collagen-PRP as a bioscaffold to heal a fully transected and sutured ACL in a porcine model [177,178]. At 4 weeks, the stiffness and load at yield and at failure were more than two times those of the suture-repaired control group [178]. In our research center, we used ECM bioscaffold to heal a surgically transected ACL in a goat model [84]. We combined SIS with SIS-hydrogel to heal a surgically transected ACL after primary repair. The SIS was derived from genetically modified Gala1-3Galbedeficient pigs to reduce the potential immunological reaction in humans. The SIS sheet wrapped around the defect was used to contain the healing response at the injury site and guide tissue growth, whereas the SIS hydrogel was used to fill the defect and accelerate tissue formation by means of its desirable chemoattractant and angiogenic factors. With the new treatment, the transected ACL was found to heal with continuous neotissue by 12 weeks. Its CSA and shape were similar to those of the sham-operated intact ACL and were significantly more robust than the suture repaired (control) group. Morphologically, its collagen fibers were aligned with spindle-shaped fibroblasts. Functionally, the SIS-treated ACL had reduced anteroposterior knee instability compared with the control group whereas the in situ forces were similar to intact ACL. Furthermore, the structural properties of the FATC when tested in uniaxial tension showed that tensile stiffness was 2.5 times higher than suture repair controls and actually reached approximately 50% of the intact FATC. A separate series of studies were performed by Nguyen and coworkers [179,180]. First attempts were made to suture the transected ACL and wrap it with SIS in a sheet form. Histologically, the collagen fibers were more dense and more compactly arranged than in the suture repair control group [180]. Afterward, an in vivo evaluation of this approach was performed and the CSA was found to be 50% of the intact ACL whereas stiffness for the SIS group was 35% higher than in the control group [179]. Studies have also shown, the feasibility of enhancing ACL graft integration after reconstruction using a triphasic scaffold using fibroblast and osteoblasts were seeded on a section of the scaffold that mimicked the native environment of the ligament insertion and bone, respectively [181,182]. Collectively these approaches demonstrate that ECM bioscaffolds have substantive potentials for improving treatment for injured ligaments and tendons. Mechanical Augmentation Mechanical augmentation has been shown to stabilize the knee with an injured ACL, which could aid in ACL healing. In a porcine model, Fleming and coworkers used sutures passing from bone to bone to reduce anterior joint laxity [183]. Our research center had also systematically used sutures (number 2 FiberWire sutures) for mechanical augmentation. In a goat model, we first determined the best locations of the bone tunnels for the sutures; i.e., the anterior footprint of the femoral origin and medial aspect of the tibial footprint on stifle joint stability after ACL transection [184]. In these positions, the anterior tibial translation was within 3 mm of the intact joint and the in situ force was similar to that of the intact ACL. A follow-up study showed the relative contribution of the soft tissues in resisting the anterior tibial load. Under the 67-N anterior tibial load, the ACL provided the dominant support in the intact

HEALING OF LIGAMENTS AND TENDONS

1191

joint. In the case of ACL deficiency, the MCL and medial meniscus carried significant loads, reaching up to 36% and 53% of the intact ACL, respectively (at 30 degrees flexion). After suture repair of the ACL, the in situ force was 81% of the intact ACL whereas that for the augmentation sutures was 103% [185]. Thus, it could be concluded that suture augmentation provided good initial joint stability and concomitantly lowered the loads on the MCL as well as the medial meniscus. Finally, an in vivo study was performed to examine whether suture augmentation could heal a surgically transected ACL in a goat model. After 12 weeks, the anteroposterior tibial translation of the stifle joint for the suture augmentation group was about 20% lower than that of the suture repair group (control) whereas the in situ force in the healing ACL was more than 50% higher. Morphologically, the ACL was found to heal with good neotissue formation. In terms of the structural properties of the FATC, the linear stiffness was 75% greater than that of the suture repair control. It was shown that suture augmentation had provided the needed stability to the stifle joint for intrinsic healing of the ACL to take place. Combined Biological and Mechanical Augmentation With evidence that biological and mechanical augmentation could individually enhance ACL healing, it begs the question whether their combination would work in synergy to enhance the ACL healing process further. It was hypothesized that mechanical augmentation would stabilize the knee immediately after surgery and enhance biological augmentation to stimulate and accelerate the ACL healing further. For the purpose of mechanical augmentation, a novel Magnesium (Mg)-based ring was designed to bridge the two torn ends of the ACL and serve as an internal splint for mechanical augmentation (Fig. 67.8). Furthermore, the Mg ring could simultaneously load the healing ACL as well as its insertion sites to prevent disuse atrophy. The function of the Mg ring-repaired ACL was first evaluated in an in vitro study using a goat model. After the application of a Mg-based ring, the anterior tibial translation of the repaired ACL was reduced by 60e70% from the ACL-deficient state and was within approximately 3 mm of that of the intact stifle joint [186,187]. Mg-ring repair could also restore the in situ forces of the repaired ACL to within 5 N of the levels of the intact ACL. These promising in vitro results suggest that the Mg-based ring is a good device for mechanical augmentation. Then, the Mg-ring was used combined with ECM bioscaffolds in an in vivo animal study [186,187]. It was hypothesized that the Mg-based ring would degrade as the ACL healing progressed and its mechanical function would be replaced by the healing ACL, because Mg is biodegradable and bioresorbable [188]. After surgical transection, the Mg-based ring was sutured to connect the stumps of the ACL followed by wrapping the transection site with an ECM sheet and injection with ECM hydrogel (Fig. 67.8) [186,187]. After 6 weeks, the device had degraded 40%

FIGURE 67.8 Schematic diagram showing the application of the Mg-based ring device to bridge the transected anterior cruciate ligament. Fixation sutures through the femoral bone tunnels are fixed using a surgical button whereas those through the tibial bone tunnels are fixed using a double-spiked plate and fixation post.

1192

67. FUNCTIONAL TISSUE ENGINEERING OF LIGAMENT AND TENDON INJURIES

and translucent healing tissue was seen [186,187]. By 12 weeks, the function of the stifle joint was determined by the roboticeUFS testing system under 67 N anteroposterior load, the anteroposterior tibial translation of the Mg ringrepaired group was about 33% less than that of the suture repair group. The in situ forces carried by the Mg ring repair group were approximately twice those of the suture repair group. Similarly, the structural properties of the FATC (stiffness and ultimate load) for the Mg ring repair group were three times greater than the suture repair group [186,187]. These results also compared favorably to those after ACL reconstruction in the same animal model, because the stiffness was 1.5 times higher than that after ACL reconstruction in a goat model at 12 weeks [186,187,189]. Similarly, the ultimate load was 1.8 times higher. Thus, it is clear that the combined use of biological and mechanical augmentation therapy worked in synergy to accelerate ACL healing. Future work will include a long-term (26-week) study to determine whether these benefits persist.

SUMMARY AND FUTURE DIRECTIONS In this review, biomechanical and biological problems facing repair and healing of ligament and tendon injuries were discussed. There have been tremendous improvements to clinical treatment paradigms based on studies that established a fundamental understanding of healing after ligament or tendon injury and the benefits of controlled mobilization. Nevertheless, many issues remain. For ligaments and tendons that display healing potential after injury, major challenges are recovery of normal ultrastructural appearance, biochemical composition, and mechanical properties. Specifically, important steps to be taken are increasing the fibril diameters of healing tissues by limiting the production of type V collagen and decorin and improving the alignment of healing tissue by guiding the organization of newly produced matrix. By manipulating the healing response at the molecular and cellular levels and guiding tissue formation, the following FTE approaches may offer the potential to restore the properties of healing tissue to normal levels. We are particularly interested in bioscaffolds such as ECM. Applied to a healing ligament or tendon in vivo, it serves as a substrate that provides contact guidance for cells to form more aligned collagen fibers with a concomitant improvement in mechanical and viscoelastic properties compared with untreated controls. Furthermore, the chemoattractant degradation products and bioactive agents of SIS could enhance the rate of healing [168], allowing better maintenance of stress and motion-dependent homeostasis. More excitingly, the SIS can be modified in vitro by seeding BMDCs on the scaffold and applying cyclic stretching to increase its alignment. Hence, when applied in vivo, the tissue engineered scaffold could serve to accelerate the initiation of the healing process by improving the production and orientation of collagen, which ultimately will help to make a better neoligament or tendon. On the other hand, for ligaments and tendons that do not heal after injury and require surgical reconstruction using replacement grafts (e.g., ACL reconstruction), the major challenge is to promote a remodeling response so that the graft maintains sufficient stiffness and strength to provide functional stability of the joint. Most important is enhancing the rate of integration of tendonebone interfaces during early graft incorporation, which may permit an earlier and more aggressive postoperative rehabilitation [190]. These complex issues may require a combination of approaches including gene and cell therapies as well as biologic scaffolds. Indeed, grafts treated with AdBMP-2 have shown some potential [137] in both canine and rabbit models. In addition, other biological tissues such as periosteum have been used to enhance the interface between tendon and bone, with some success [190]. All of these results suggest an exciting potential for clinical application. Indeed, FTE has generated many exciting developments. For example, there is an exciting class of biodegradable metallic scaffolds, namely, porous magnesium or magnesium oxide, that has the advantage of initial stiffness to provide needed stability for the ligament to heal while performing its function. The degradation rate of these “smart” scaffolds could also be controlled as they are replaced by the neotissue. Furthermore, protein coating of these biodegradable metallic scaffolds could be performed for better tissue integration and controlled release of growth factors and cytokines to sustain tissue healing as well as guide tissue regeneration. In particular, Mg has been used combined with ECM bioscaffolds to heal the ACL, and it has the potential to serve as an alternative to surgical reconstruction. To translate knowledge gained about a particular gene, protein, or cell to a clinical application will require expertise from many disciplines to work in seamlessly. One roles of biomedical engineers within this framework would be to link interactions of the functions of molecules to cells, cells to tissues, tissues to organs, and organs to body. When biologists, biomedical engineers, clinicians, and experts from other disciplines work together, this results in better therapies that lead to injured ligaments and tendons healing with properties closer to those of normal ligaments and tendons. Efforts of such a team-based approach to the new developments of FTE will bring a bright future to

REFERENCES

1193

the outcome of healing of ligaments and tendon injuries. With the development of precision medicine based on “big data” and cellular reprogramming on the horizon, it is easy to imagine that these FTE techniques for healing ligaments and tendons can be tailored to be patient specific to gain a more positive, long-term outcome.

References [1] Beaty J. Knee and leg: soft tissue trauma. In: Arendt EA, editor. OKU orthopaedic knowledge update. Rosemont, IL: American Academy of Orthopaedic Surgeons; 1999. p. xix, 442. [2] Frank C, Woo SL-Y, Amiel D, Harwood F, Gomez M, Akeson W. Medial collateral ligament healing. A multidisciplinary assessment in rabbits. Am J Sports Med 1983;11(6):379e89. [3] Indelicato PA. Non-operative treatment of complete tears of the medial collateral ligament of the knee. J Bone Joint Surg Am 1983;65(3): 323e9. [4] Jokl P, Kaplan N, Stovell P, Keggi K. Non-operative treatment of severe injuries to the medial and anterior cruciate ligaments of the knee. J Bone Joint Surg Am 1984;66(5):741e4. [5] Woo SL-Y, Gomez MA, Sites TJ, Newton PO, Orlando CA, Akeson WH. The biomechanical and morphological changes in the medial collateral ligament of the rabbit after immobilization and remobilization. J Bone Joint Surg Am 1987;69(8):1200e11. [6] Woo SL-Y, Inoue M, McGurk-Burleson E, Gomez MA. Treatment of the medial collateral ligament injury. II: structure and function of canine knees in response to differing treatment regimens. Am J Sports Med 1987;15(1):22e9. [7] Kannus P. Long-term results of conservatively treated medial collateral ligament injuries of the knee joint. Clin Orthop Relat Res 1988;(226): 103e12. [8] Scheffler SU, Clineff TD, Papageorgiou CD, Debski RE, Benjamin C, Woo SL-Y. Structure and function of the healing medial collateral ligament in a goat model. Ann Biomed Eng 2001;29(2):173e80. [9] Adachi E, Hayashi T. In vitro formation of hybrid fibrils of type V collagen and type I collagen. Limited growth of type I collagen into thick fibrils by type V collagen. Connect Tissue Res 1986;14(4):257e66. [10] Birk DE. Type V collagen: heterotypic type I/V collagen interactions in the regulation of fibril assembly. Micron 2001;32(3):223e37. [11] Birk DE, Fitch JM, Babiarz JP, Doane KJ, Linsenmayer TF. Collagen fibrillogenesis in vitro: interaction of types I and V collagen regulates fibril diameter. J Cell Sci 1990;95(Pt 4):649e57. [12] Frank C, McDonald D, Bray D, Bray R, Rangayyan R, Chimich D, Shrive N. Collagen fibril diameters in the healing adult rabbit medial collateral ligament. Connect Tissue Res 1992;27(4):251e63. [13] Frank C, McDonald D, Shrive N. Collagen fibril diameters in the rabbit medial collateral ligament scar: a longer term assessment. Connect Tissue Res 1997;36(3):261e9. [14] Hart DA, Nakamura N, Marchuk L, Hiraoka H, Boorman R, Kaneda Y, Shrive NG, Frank CB. Complexity of determining cause and effect in vivo after antisense gene therapy. Clin Orthop 2000;379(Suppl.):S242e51. [15] Hart RA, Woo SL-Y, Newton PO. Ultrastructural morphometry of anterior cruciate and medial collateral ligaments: an experimental study in rabbits. J Orthop Res 1992;10(1):96e103. [16] Marchant JK, Hahn RA, Linsenmayer TF, Birk DE. Reduction of type V collagen using a dominant-negative strategy alters the regulation of fibrillogenesis and results in the loss of corneal-specific fibril morphology. J Cell Biol 1996;135(5):1415e26. [17] Nakamura N, Hart DA, Boorman RS, Kaneda Y, Shrive NG, Marchuk LL, Shino K, Ochi T, Frank CB. Decorin antisense gene therapy improves functional healing of early rabbit ligament scar with enhanced collagen fibrillogenesis in vivo. J Orthop Res 2000;18(4):517e23. [18] Niyibizi C, Kavalkovich K, Yamaji T, Woo SL-Y. Type V collagen is increased during rabbit medial collateral ligament healing. Knee Surg Sports Traumatol Arthrosc 2000;8(5):281e5. [19] Weiss JA, Woo SL-Y, Ohland KJ, Horibe S, Newton PO. Evaluation of a new injury model to study medial collateral ligament healing: primary repair versus nonoperative treatment. J Orthop Res 1991;9(4):516e28. [20] Coupens SD, Yates CK, Sheldon C, Ward C. Magnetic resonance imaging evaluation of the patellar tendon after use of its central one-third for anterior cruciate ligament reconstruction. Am J Sports Med 1992;20(3):332e5. [21] Svensson M, Kartus J, Christensen LR, Movin T, Papadogiannakis N, Karlsson J. A long-term serial histological evaluation of the patellar tendon in humans after harvesting its central third. Knee Surg Sports Traumatol Arthrosc 2005;13(5):398e404. [22] Freedman KB, D’Amato MJ, Nedeff DD, Kaz A, Bach Jr BR. Arthroscopic anterior cruciate ligament reconstruction: a metaanalysis comparing patellar tendon and hamstring tendon autografts. Am J Sports Med 2003;31(1):2e11. [23] Aune AK, Holm I, Risberg MA, Jensen HK, Steen H. Four-strand hamstring tendon autograft compared with patellar tendon-bone autograft for anterior cruciate ligament reconstruction. A randomized study with two-year follow-up. Am J Sports Med 2001;29(6):722e8. [24] Marder RA. Arthroscopic-assisted reconstruction of the anterior cruciate ligament. West J Med 1991;155(2):172. [25] Clatworthy MG, Annear P, Bulow JU, Bartlett RJ. Tunnel widening in anterior cruciate ligament reconstruction: a prospective evaluation of hamstring and patella tendon grafts. Knee Surg Sports Traumatol Arthrosc 1999;7(3):138e45. [26] Feller JA, Webster KE. A randomized comparison of patellar tendon and hamstring tendon anterior cruciate ligament reconstruction. Am J Sports Med 2003;31(4):564e73. [27] Jansson KA, Harilainen A, Sandelin J, Karjalainen PT, Aronen HJ, Tallroth K. Bone tunnel enlargement after anterior cruciate ligament reconstruction with the hamstring autograft and endobutton fixation technique. A clinical, radiographic and magnetic resonance imaging study with 2 years follow-up. Knee Surg Sports Traumatol Arthrosc 1999;7(5):290e5. [28] Nebelung W, Becker R, Merkel M, Ropke M. Bone tunnel enlargement after anterior cruciate ligament reconstruction with semitendinosus tendon using Endobutton fixation on the femoral side. Arthroscopy 1998;14(8):810e5. [29] Pinczewski LA, Lyman J, Salmon LJ, Russell VJ, Roe J, Linklater J. A 10-year comparison of anterior cruciate ligament reconstructions with hamstring tendon and patellar tendon autograft - a controlled, prospective trial. Am J Sports Med 2007;35(4):564e74.

1194

67. FUNCTIONAL TISSUE ENGINEERING OF LIGAMENT AND TENDON INJURIES

[30] Salmon LJ, Russell VJ, Refshauge K, Kader D, Connolly C, Linklater J, Pinczewski LA. Long-term outcome of endoscopic anterior cruciate ligament reconstruction with patellar tendon autograft - minimum 13-year review. Am J Sports Med 2006;34(5):721e32. [31] von Porat A, Roos EM, Roos H. High prevalence of osteoarthritis 14 years after an anterior cruciate ligament tear in male soccer players: a study of radiographic and patient relevant outcomes. Ann Rheum Dis 2004;63(3):269e73. [32] Badylak SF, Tullius R, Kokini K, Shelbourne KD, Klootwyk T, Voytik SL, Kraine MR, Simmons C. The use of xenogeneic small intestinal submucosa as a biomaterial for Achilles tendon repair in a dog model. J Biomed Mater Res 1995;29(8):977e85. [33] Hildebrand KA, Frank CB. Scar formation and ligament healing. Can J Surg 1998;41(6):425e9. [34] Huang D, Chang TR, Aggarwal A, Lee RC, Ehrlich HP. Mechanisms and dynamics of mechanical strengthening in ligament-equivalent fibroblast-populated collagen matrices. Ann Biomed Eng 1993;21(3):289e305. [35] Shimomura T, Jia F, Niyibizi C, Woo SL-Y. Antisense oligonucleotides reduce synthesis of procollagen alpha1 (V) chain in human patellar tendon fibroblasts: potential application in healing ligaments and tendons. Connect Tissue Res 2003;44(3e4):167e72. [36] Spindler KP, Dawson JM, Stahlman GC, Davidson JM, Nanney LB. Collagen expression and biomechanical response to human recombinant transforming growth factor beta (rhTGF-beta2) in the healing rabbit MCL. J Orthop Res 2002;20(2):318e24. [37] Woo SL-Y, Hildebrand K, Watanabe N, Fenwick JA, Papageorgiou CD, Wang JH. Tissue engineering of ligament and tendon healing. Clin Orthop Relat Res 1999;(367 Suppl.):S312e23. [38] Bray RC, Rangayyan RM, Frank CB. Normal and healing ligament vascularity: a quantitative histological assessment in the adult rabbit medial collateral ligament. J Anat 1996;188(Pt 1):87e95. [39] Lo IK, Ou Y, Rattner JP, Hart DA, Marchuk LL, Frank CB, Rattner JB. The cellular networks of normal ovine medial collateral and anterior cruciate ligaments are not accurately recapitulated in scar tissue. J Anat 2002;200(Pt 3):283e96. [40] Manske PR. Flexor tendon healing. J Hand Surg [Br] 1988;13(3):237e45. [41] Hildebrand KA, Frank CB, Hart DA. Gene intervention in ligament and tendon: current status, challenges, future directions. Gene Ther 2004; 11(4):368e78. [42] Woo SL-Y, An K-N, Frank C, Livesay G, Ma C, Zeminski J. Anatomy, biology and biomechanics of tendon and ligaments. In: Einhorn TA, Simon SR, American Academy of Orthopaedic Surgeons, editors. Orthopaedic basic science: biology and biomechanics of the musculoskeletal system. Rosemont, Ill: American Academy of Orthopaedic Surgeons; 2000. p. 581e616. [43] Birk DE, Trelstad RL. Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts. J Cell Biol 1984;99(6):2024e33. [44] Maffulli N. Rupture of the achilles tendon. J Bone Joint Surg Am 1999;81(7):1019e36. [45] Amiel D, Frank C, Harwood F, Fronek J, Akeson W. Tendons and ligaments: a morphological and biochemical comparison. J Orthop Res 1984;1(3):257e65. [46] Thornton GM, Boorman RS, Shrive NG, Frank CB. Medial collateral ligament autografts have increased creep response for at least two years and early immobilization makes this worse. J Orthop Res 2002;20(2):346e52. [47] Dyer RF, Enna CD. Ultrastructural features of adult human tendon. Cell Tissue Res 1976;168(2):247e59. [48] Eyden B, Tzaphlidou M. Structural variations of collagen in normal and pathological tissues: role of electron microscopy. Micron 2001;32(3): 287e300. [49] Goh JC, Ouyang HW, Teoh SH, Chan CK, Lee EH. Tissue-engineering approach to the repair and regeneration of tendons and ligaments. Tissue Eng 2003;9(Suppl. 1):S31e44. [50] Oakes BW. Collagen ultrastructure in the normal ACL and in ACL graft. In: The anterior cruciate ligament. Current and future concepts; 1993. p. 209e17. [51] Ottani V, Raspanti M, Ruggeri A. Collagen structure and functional implications. Micron 2001;32(3):251e60. [52] Liu SH, Yang RS, al-Shaikh R, Lane JM. Collagen in tendon, ligament, and bone healing. A current review. Clin Orthop Relat Res 1995;318: 265e78. [53] Birk DE, Mayne R. Localization of collagen types I, III and V during tendon development. Changes in collagen types I and III are correlated with changes in fibril diameter. Eur J Cell Biol 1997;72(4):352e61. [54] Linsenmayer TF, Gibney E, Igoe F, Gordon MK, Fitch JM, Fessler LI, Birk DE. Type V collagen: molecular structure and fibrillar organization of the chicken alpha 1(V) NH2-terminal domain, a putative regulator of corneal fibrillogenesis. J Cell Biol 1993;121(5):1181e9. [55] Niyibizi C, Visconti CS, Kavalkovich K, Woo SL-Y. Collagens in an adult bovine medial collateral ligament: immunofluorescence localization by confocal microscopy reveals that type XIV collagen predominates at the ligament-bone junction. Matrix Biol 1995;14(9):743e51. [56] Fukuta S, Oyama M, Kavalkovich K, Fu FH, Niyibizi C. Identification of types II, IX and X collagens at the insertion site of the bovine achilles tendon. Matrix Biol 1998;17(1):65e73. [57] Niyibizi C, Sagarrigo Visconti C, Gibson G, Kavalkovich K. Identification and immunolocalization of type X collagen at the ligament-bone interface. Biochem Biophys Res Commun 1996;222(2):584e9. [58] Sagarriga Visconti C, Kavalkovich K, Wu J, Niyibizi C. Biochemical analysis of collagens at the ligament-bone interface reveals presence of cartilage-specific collagens. Arch Biochem Biophys 1996;328(1):135e42. [59] Cooper RR, Misol S. Tendon and ligament insertion. A light and electron microscopic study. J Bone Joint Surg Am 1970;52(1):1e20. [60] Matyas JR, Anton MG, Shrive NG, Frank CB. Stress governs tissue phenotype at the femoral insertion of the rabbit MCL. J Biomech 1995; 28(2):147e57. [61] Woo SL-Y, Abramowitch SD, Kilger R, Liang R. Biomechanics of knee ligaments: injury, healing, and repair. J Biomech 2006;39(1):1e20. [62] Fung YC, Perrone N, Anliker M, University of California San Diego, United States Office of Naval Research. Biomechanics, its foundations and objectives. Englewood Cliffs, N.J.: Prentice-Hall; 1972. [63] Woo SL-Y, Gelberman RH, Cobb NG, Amiel D, Lothringer K, Akeson WH. The importance of controlled passive mobilization on flexor tendon healing. A biomechanical study. Acta Orthop Scand 1981;52(6):615e22. [64] Woo SL-Y, Gomez MA, Akeson WH. The time and history-dependent viscoelastic properties of the canine medical collateral ligament. J Biomech Eng 1981;103(4):293e8. [65] Woo SL-Y, Gomez MA, Amiel D, Ritter MA, Gelberman RH, Akeson WH. The effects of exercise on the biomechanical and biochemical properties of swine digital flexor tendons. J Biomech Eng 1981;103(1):51e6.

REFERENCES

1195

[66] Lee TQ, Woo SL-Y. A new method for determining cross-sectional shape and area of soft tissues. J Biomech Eng 1988;110(2):110e4. [67] Woo SL-Y, Gomez MA, Seguchi Y, Endo CM, Akeson WH. Measurement of mechanical properties of ligament substance from a boneligament-bone preparation. J Orthop Res 1983;1(1):22e9. [68] Johnson GA, Tramaglini DM, Levine RE, Ohno K, Choi NY, Woo SL-Y. Tensile and viscoelastic properties of human patellar tendon. J Orthop Res 1994;12(6):796e803. [69] Woo SL-Y, Hollis JM, Adams DJ, Lyon RM, Takai S. Tensile properties of the human femur-anterior cruciate ligament-tibia complex. The effects of specimen age and orientation. Am J Sports Med 1991;19(3):217e25. [70] Woo SL-Y, Orlando CA, Camp JF, Akeson WH. Effects of postmortem storage by freezing on ligament tensile behavior. J Biomech 1986;19(5): 399e404. [71] Woo SL-Y, Orlando CA, Gomez MA, Frank CB, Akeson WH. Tensile properties of the medial collateral ligament as a function of age. J Orthop Res 1986;4(2):133e41. [72] Woo SL-Y, Gomez MA, Woo YK, Akeson WH. Mechanical properties of tendons and ligaments. II. The relationships of immobilization and exercise on tissue remodeling. Biorheology 1982;19(3):397e408. [73] Inoue M, McGurk-Burleson E, Hollis JM, Woo SL-Y. Treatment of the medial collateral ligament injury. I: the importance of anterior cruciate ligament on the varus-valgus knee laxity. Am J Sports Med 1987;15(1):15e21. [74] Livesay GA, Rudy TW, Woo SL-Y, Runco TJ, Sakane M, Li G, Fu FH. Evaluation of the effect of joint constraints on the in situ force distribution in the anterior cruciate ligament. J Orthop Res 1997;15(2):278e84. [75] Woo SL-Y, Fox RJ, Sakane M, Livesay GA, Rudy TW, Fu FH. Biomechanics of the ACL: measurements of in situ force in the ACL and knee kinematics. Knee 1998;5(4):267e88. [76] Woo SL-Y, Smith DW, Hildebrand KA, Zeminski JA, Johnson LA. Engineering the healing of the rabbit medial collateral ligament. Med Biol Eng Comput 1998;36(3):359e64. [77] Woo SL-Y, Kanamori A, Zeminski J, Yagi M, Papageorgiou C, Fu FH. The effectiveness of reconstruction of the anterior cruciate ligament with hamstrings and patellar tendon. A cadaveric study comparing anterior tibial and rotational loads. J Bone Joint Surg Am 2002;84A(6):907e14. [78] Yagi M, Wong EK, Kanamori A, Debski RE, Fu FH, Woo SL-Y. Biomechanical analysis of an anatomic anterior cruciate ligament reconstruction. Am J Sports Med 2002;30(5):660e6. [79] Zamarra G, Fisher MB, Woo SL-Y, Cerulli G. Biomechanical evaluation of using one hamstrings tendon for ACL reconstruction: a human cadaveric study. Knee Surg Sports Traumatol Arthrosc 2010;18(1):11e9. [80] Sasaki N, Farraro KF, Kim KE, Woo SL-Y. Biomechanical evaluation of the quadriceps tendon autograft for anterior cruciate ligament reconstruction a cadaveric study. Am J Sports Med 2014;42(3):723e30. [81] Abramowitch SD, Papageorgiou CD, Debski RE, Clineff TD, Woo SL-Y. A biomechanical and histological evaluation of the structure and function of the healing medial collateral ligament in a goat model. Knee Surg Sports Traumatol Arthrosc 2003;11(3):155e62. [82] Abramowitch SD, Papageorgiou CD, Withrow JD, Gilbert TW, Woo SL-Y. The effect of initial graft tension on the biomechanical properties of a healing ACL replacement graft: a study in goats. J Orthop Res 2003;21(4):708e15. [83] Abramowitch SD, Yagi M, Tsuda E, Woo SL-Y. The healing medial collateral ligament following a combined anterior cruciate and medial collateral ligament injuryea biomechanical study in a goat model. J Orthop Res 2003;21(6):1124e30. [84] Fisher MB, Liang R, Jung H-J, Kim KE, Zamarra G, Almarza AJ, McMahon PJ, Woo SL-Y. Potential of healing a transected anterior cruciate ligament with genetically modified extracellular matrix bioscaffolds in a goat model. Knee Surg Sports Traumatol Arthrosc 2012;20(7): 1357e65. [85] Frank CB, Bray RC, Hart DA, Shirve NG, Loitz BJ, Matyas JR, Wilson JE. Soft tissue healing. In: Fu FH, Harner CD, Vince Kelly G, editors. Knee surgery. Baltimore, MD: Williams & Wilkins; 1994. 2 v. (xvii, 1595 pp.): 189e229. [86] Murphy PG, Loitz BJ, Frank CB, Hart DA. Influence of exogenous growth factors on the synthesis and secretion of collagen types I and III by explants of normal and healing rabbit ligaments. Biochem Cell Biol 1994;72(9e10):403e9. [87] Doillon CJ, Dunn MG, Bender E, Silver FH. Collagen fiber formation in repair tissue: development of strength and toughness. Coll Relat Res 1985;5(6):481e92. [88] Parry D, Barnes G, Craig A. A comparision of the size distribution of collagen fibrils in connective tissues as a function of age and a possible relation between fibril size distribution and mechanical properties. Proc R Soc Lond (Biol) 1978;203:305e21. [89] Clayton ML, Miles JS, Abdulla M. Experimental investigations of ligamentous healing. Clin Orthop Relat Res 1968;61:146e53. [90] O’Donoghue DH, Frank GR, Jeter GL, Johnson W, Zeiders JW, Kenyon R. Repair and reconstruction of the anterior cruciate ligament in dogs. Factors influencing long-term results. J Bone Joint Surg Am 1971;53(4):710e8. [91] Tipton CM, James SL, Mergner W, Tcheng TK. Influence of exercise on strength of medial collateral knee ligaments of dogs. Am J Physiol 1970;218(3):894e902. [92] Woo SL-Y, Young EP, Ohland KJ, Marcin JP, Horibe S, Lin HC. The effects of transection of the anterior cruciate ligament on healing of the medial collateral ligament. A biomechanical study of the knee in dogs. J Bone Joint Surg Am 1990;72(3):382e92. [93] Inoue M, Woo SL-Y, Gomez MA, Amiel D, Ohland KJ, Kitabayashi LR. Effects of surgical treatment and immobilization on the healing of the medial collateral ligament: a long-term multidisciplinary study. Connect Tissue Res 1990;25(1):13e26. [94] Indelicato P. Isolated medial collateral ligament injuries in the knee. J Am Acad Orthop Surg 1995;3(1):9e14. [95] Reider B, Sathy MR, Talkington J, Blyznak N, Kollias S. Treatment of isolated medial collateral ligament injuries in athletes with early functional rehabilitation. A five-year follow-up study. Am J Sports Med 1994;22(4):470e7. [96] Aglietti P, Buzzi R, D’Andria S, Zaccherotti G. Arthroscopic anterior cruciate ligament reconstruction with patellar tendon. Arthroscopy 1992;8(4):510e6. [97] Cooper DE, Deng XH, Burstein AL, Warren RF. The strength of the central third patellar tendon graft. A biomechanical study. Am J Sports Med 1993;21(6):818e23. discussion 823e814. [98] Jones KG. Reconstruction of the anterior cruciate ligament using the central one-third of the patellar ligament. J Bone Joint Surg Am 1970; 52(4):838e9.

1196

67. FUNCTIONAL TISSUE ENGINEERING OF LIGAMENT AND TENDON INJURIES

[99] Kurosaka M, Yoshiya S, Andrish JT. A biomechanical comparison of different surgical techniques of graft fixation in anterior cruciate ligament reconstruction. Am J Sports Med 1987;15(3):225e9. [100] Lambert KL. Vascularized patellar tendon graft with rigid internal fixation for anterior cruciate ligament insufficiency. Clin Orthop Relat Res 1983;(172):85e9. [101] Noyes FR, Butler DL, Grood ES, Zernicke RF, Hefzy MS. Biomechanical analysis of human ligament grafts used in knee-ligament repairs and reconstructions. J Bone Joint Surg Am 1984;66(3):344e52. [102] Cerullo G, Puddu G, Gianni E, Damiani A, Pigozzi F. Anterior cruciate ligament patellar tendon reconstruction: it is probably better to leave the tendon defect open! Knee Surg Sports Traumatol Arthrosc 1995;3(1):14e7. [103] Nixon RG, SeGall GK, Sax SL, Cain TE, Tullos HS. Reconstitution of the patellar tendon donor site after graft harvest. Clin Orthop Relat Res 1995;317:162e71. [104] Rubinstein Jr RA, Shelbourne KD, VanMeter CD, McCarroll JC, Rettig AC. Isolated autogenous bone-patellar tendon-bone graft site morbidity. Am J Sports Med 1994;22(3):324e7. [105] Breitfuss H, Frohlich R, Povacz P, Resch H, Wicker A. The tendon defect after anterior cruciate ligament reconstruction using the midthird patellar tendonea problem for the patellofemoral joint? Knee Surg Sports Traumatol Arthrosc 1996;3(4):194e8. [106] Kartus J, Magnusson L, Stener S, Brandsson S, Eriksson BI, Karlsson J. Complications following arthroscopic anterior cruciate ligament reconstruction. A 2-5-year follow-up of 604 patients with special emphasis on anterior knee pain. Knee Surg Sports Traumatol Arthrosc 1999;7(1):2e8. [107] Paulos LE, Rosenberg TD, Drawbert J, Manning J, Abbott P. Infrapatellar contracture syndrome. An unrecognized cause of knee stiffness with patella entrapment and patella infera. Am J Sports Med 1987;15(4):331e41. [108] Sachs RA, Daniel DM, Stone ML, Garfein RF. Patellofemoral problems after anterior cruciate ligament reconstruction. Am J Sports Med 1989; 17(6):760e5. [109] Shelbourne KD, Wilckens JH, Mollabashy A, DeCarlo M. Arthrofibrosis in acute anterior cruciate ligament reconstruction. The effect of timing of reconstruction and rehabilitation. Am J Sports Med 1991;19(4):332e6. [110] Tibone JE, Antich TJ. A biomechanical analysis of anterior cruciate ligament reconstruction with the patellar tendon. A two year followup. Am J Sports Med 1988;16(4):332e5. [111] Awad HA, Boivin GP, Dressler MR, Smith FN, Young RG, Butler DL. Repair of patellar tendon injuries using a cell-collagen composite. J Orthop Res 2003;21(3):420e31. [112] Beynnon BD, Proffer D, Drez Jr DJ, Stankewich CJ, Johnson RJ. Biomechanical assessment of the healing response of the rabbit patellar tendon after removal of its central third. Am J Sports Med 1995;23(4):452e7. [113] Kamps BS, Linder LH, DeCamp CE, Haut RC. The influence of immobilization versus exercise on scar formation in the rabbit patellar tendon after excision of the central third. Am J Sports Med 1994;22(6):803e11. [114] Linder LH, Sukin DL, Burks RT, Haut RC. Biomechanical and histological properties of the canine patellar tendon after removal of its medial third. Am J Sports Med 1994;22(1):136e42. [115] Tohyama H, Yasuda K, Kitamura Y, Yamamoto E, Hayashi K. The changes in mechanical properties of regenerated and residual tissues in the patellar tendon after removal of its central portion. Clin Biomech 2003;18(8):765e72. [116] Tohyama H, Yasuda K. The effects of stress enhancement on the extracellular matrix and fibroblasts in the patellar tendon. J Biomech 2000; 33(5):559e65. [117] Arnoczky SP, Tarvin GB, Marshall JL. Anterior cruciate ligament replacement using patellar tendon. An evaluation of graft revascularization in the dog. J Bone Joint Surg Am 1982;64(2):217e24. [118] Papageorgiou CD, Ma CB, Abramowitch SD, Clineff TD, Woo SL-Y. A multidisciplinary study of the healing of an intraarticular anterior cruciate ligament graft in a goat model. Am J Sports Med 2001;29(5):620e6. [119] Ballock RT, Woo SL-Y, Lyon RM, Hollis JM, Akeson WH. Use of patellar tendon autograft for anterior cruciate ligament reconstruction in the rabbit: a long-term histologic and biomechanical study. J Orthop Res 1989;7(4):474e85. [120] Butler DL, Grood ES, Noyes FR, Olmstead ML, Hohn RB, Arnoczky SP, Siegel MG. Mechanical properties of primate vascularized vs. nonvascularized patellar tendon grafts; changes over time. J Orthop Res 1989;7(1):68e79. [121] Clancy Jr WG, Narechania RG, Rosenberg TD, Gmeiner JG, Wisnefske DD, Lange TA. Anterior and posterior cruciate ligament reconstruction in rhesus monkeys. J Bone Joint Surg Am 1981;63(8):1270e84. [122] Loh JC, Fukuda Y, Tsuda E, Steadman RJ, Fu FH, Woo SL-Y. Knee stability and graft function following anterior cruciate ligament reconstruction: comparison between 11 o’clock and 10 o’clock femoral tunnel placement. 2002 Richard O’Connor Award Paper. Arthroscopy 2003;19(3):297e304. [123] Ohno K, Pomaybo AS, Schmidt CC, Levine RE, Ohland KJ, Woo SL-Y. Healing of the medial collateral ligament after a combined medial collateral and anterior cruciate ligament injury and reconstruction of the anterior cruciate ligament: comparison of repair and nonrepair of medial collateral ligament tears in rabbits. J Orthop Res 1995;13(3):442e9. [124] Yamaji T, Levine RE, Woo SL-Y, Niyibizi C, Kavalkovich KW, Weaver-Green CM. Medial collateral ligament healing one year after a concurrent medial collateral ligament and anterior cruciate ligament injury: an interdisciplinary study in rabbits. J Orthop Res 1996;14(2):223e7. [125] Duffy Jr FJ, Seiler JG, Gelberman RH, Hergrueter CA. Growth factors and canine flexor tendon healing: initial studies in uninjured and repair models. J Hand Surg [Am] 1995;20(4):645e9. [126] Gelberman RH. The effect of adipose-derived stem cells on the inflammatory stage of flexor tendon repair. J Hand Surg 2016;41(9):S3e4. [127] Panossian V, Liu SH, Lane JM, Finerman GA. Fibroblast growth factor and epidermal growth factor receptors in ligament healing. Clin Orthop Relat Res 1997;(342):173e80. [128] Saether EE, Chamberlain CS, Aktas E, Leiferman EM, Brickson SL, Vanderby R. Primed mesenchymal stem cells alter and improve rat medial collateral ligament healing. Stem Cell Rev 2016;12(1):42e53. [129] Sciore P, Boykiw R, Hart DA. Semiquantitative reverse transcription-polymerase chain reaction analysis of mRNA for growth factors and growth factor receptors from normal and healing rabbit medial collateral ligament tissue. J Orthop Res 1998;16(4):429e37. [130] Steenfos HH. Growth factors and wound healing. Scand J Plast ReConstr Surg Hand Surg 1994;28(2):95e105.

REFERENCES

1197

[131] Thomopoulos S, Kim HM, Das R, Silva MJ, Sakiyama-Elbert S, Amiel D, Gelberman RH. The effects of exogenous basic fibroblast growth factor on intrasynovial flexor tendon healing in a canine model. J Bone Joint Surg Am 2010;92(13):2285e93. [132] Deie M, Marui T, Allen CR, Hildebrand KA, Georgescu HI, Niyibizi C, Woo SL-Y. The effects of age on rabbit MCL fibroblast matrix synthesis in response to TGF-beta 1 or EGF. Mech Ageing Dev 1997;97(2):121e30. [133] Marui T, Niyibizi C, Georgescu HI, Cao M, Kavalkovich KW, Levine RE, Woo SL-Y. Effect of growth factors on matrix synthesis by ligament fibroblasts. J Orthop Res 1997;15(1):18e23. [134] Scherping Jr SC, Schmidt CC, Georgescu HI, Kwoh CK, Evans CH, Woo SL-Y. Effect of growth factors on the proliferation of ligament fibroblasts from skeletally mature rabbits. Connect Tissue Res 1997;36(1):1e8. [135] Desrosiers EA, Methot S, Yahia L, Rivard CH. Responses of ligamentous fibroblasts to mechanical stimulation. Ann Chir 1995;49(8):768e74. [136] Batten ML, Hansen JC, Dahners LE. Influence of dosage and timing of application of platelet-derived growth factor on early healing of the rat medial collateral ligament. J Orthop Res 1996;14(5):736e41. [137] Martinek V, Latterman C, Usas A, Abramowitch S, Woo SL-Y, Fu FH, Huard J. Enhancement of tendon-bone integration of anterior cruciate ligament grafts with bone morphogenetic protein-2 gene transfer: a histological and biomechanical study. J Bone Joint Surg Am 2002;84-A(7): 1123e31. [138] Lipner JH. Development of nanofiber scaffolds with controllable structure and mineral content for tendon-to-bone repair. 2015. [139] Murray MM, Fleming BC. Biology of anterior cruciate ligament injury and repair: Kappa delta ann doner vaughn award paper 2013. J Orthop Res 2013;31(10):1501e6. [140] Caplan AI, Mason C, Reeve B. The 3Rs of cell therapy. Stem Cells Transl Med 2016. https://doi.org/10.5966/sctm.2016-0180. [141] Hildebrand KA, Deie M, Allen CR, Smith DW, Georgescu HI, Evans CH, Robbins PD, Woo SL-Y. Early expression of marker genes in the rabbit medial collateral and anterior cruciate ligaments: the use of different viral vectors and the effects of injury. J Orthop Res 1999;17(1): 37e42. [142] Woo SL-Y, Jia F, Zou L, Gabriel MT. Functional tissue engineering for ligament healing: potential of antisense gene therapy. Ann Biomed Eng 2004;32(3):342e51. [143] Tripathy SK, Black HB, Goldwasser E, Leiden JM. Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors. Nat Med 1996;2(5):545e50. [144] Krall WJ, Challita PM, Perlmutter LS, Skelton DC, Kohn DB. Cells expressing human glucocerebrosidase from a retroviral vector repopulate macrophages and central nervous system microglia after murine bone marrow transplantation. Blood 1994;83(9):2737e48. [145] Kraus TM, Imhoff FB, Reinert J, Wexel G, Wolf A, Hirsch D, Hofmann A, Stockle U, Buchmann S, Tischer T, Imhoff AB, Milz S, Anton M, Vogt S. Stem cells and bFGF in tendon healing: effects of lentiviral gene transfer and long-term follow-up in a rat Achilles tendon defect model. BMC Musculoskelet Disord 2016;17:148. [146] Wang Y, Li Q, Wei X, Xu J, Chen Q, Song S, Lu Z, Wang Z. Targeted knockout of TNF-alpha by injection of lentivirus-mediated siRNA into the subacromial bursa for the treatment of subacromial bursitis in rats. Mol Med Rep 2015;12(3):4389e95. [147] Watanabe N, Woo SL-Y, Papageorgiou C, Celechovsky C, Takai S. Fate of donor bone marrow cells in medial collateral ligament after simulated autologous transplantation. Microsc Res Tech 2002;58(1):39e44. [148] Young RG, Butler DL, Weber W, Caplan AI, Gordon SL, Fink DJ. Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair. J Orthop Res 1998;16(4):406e13. [149] Badiavas EV, Abedi M, Butmarc J, Falanga V, Quesenberry P. Participation of bone marrow derived cells in cutaneous wound healing. J Cell Physiol 2003;196(2):245e50. [150] Galiano RD, Tepper OM, Pelo CR, Bhatt KA, Callaghan M, Bastidas N, Bunting S, Steinmetz HG, Gurtner GC. Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells. Am J Pathol 2004;164(6):1935e47. [151] Mathews V, Hanson PT, Ford E, Fujita J, Polonsky KS, Graubert TA. Recruitment of bone marrow-derived endothelial cells to sites of pancreatic beta-cell injury. Diabetes 2004;53(1):91e8. [152] Juncosa-Melvin N, Boivin GP, Galloway MT, Gooch C, West JR, Sklenka AM, Butler DL. Effects of cell-to-collagen ratio in mesenchymal stem cell-seeded implants on tendon repair biomechanics and histology. Tissue Eng 2005;11(3e4):448e57. [153] Teng C, Zhou C, Xu D, Bi F. Combination of platelet-rich plasma and bone marrow mesenchymal stem cells enhances tendon-bone healing in a rabbit model of anterior cruciate ligament reconstruction. J Orthop Surg Res 2016;11(1):96. [154] Lui PP, Kong SK, Lau PM, Wong YM, Lee YW, Tan C, Wong OT. Immunogenicity and escape mechanisms of allogeneic tendon-derived stem cells. Tissue Eng 2014;20(21e22):3010e20. [155] Aragona J, Parsons JR, Alexander H, Weiss AB. Medial collateral ligament replacement with a partially absorbable tissue scaffold. Am J Sports Med 1983;11(4):228e33. [156] Badylak S, Arnoczky S, Plouhar P, Haut R, Mendenhall V, Clarke R, Horvath C. Naturally occurring extracellular matrix as a scaffold for musculoskeletal repair. Clin Orthop Relat Res 1999;367(Suppl.):S333e43. [157] Bourke SL, Kohn J, Dunn MG. Preliminary development of a novel resorbable synthetic polymer fiber scaffold for anterior cruciate ligament reconstruction. Tissue Eng 2004;10(1e2):43e52. [158] Dunn MG, Tria AJ, Kato YP, Bechler JR, Ochner RS, Zawadsky JP, Silver FH. Anterior cruciate ligament reconstruction using a composite collagenous prosthesis. A biomechanical and histologic study in rabbits. Am J Sports Med 1992;20(5):507e15. [159] Bellincampi LD, Closkey RF, Prasad R, Zawadsky JP, Dunn MG. Viability of fibroblast-seeded ligament analogs after autogenous implantation. J Orthop Res 1998;16(4):414e20. [160] Guidoin MF, Marois Y, Bejui J, Poddevin N, King MW, Guidoin R. Analysis of retrieved polymer fiber based replacements for the ACL. Biomaterials 2000;21(23):2461e74. [161] Cao Y, Vacanti JP, Ma X, Paige KT, Upton J, Chowanski Z, Schloo B, Langer R, Vacanti CA. Generation of neo-tendon using synthetic polymers seeded with tenocytes. Transplant Proc 1994;26(6):3390e2. [162] Lin VS, Lee MC, O’Neal S, McKean J, Sung KL. Ligament tissue engineering using synthetic biodegradable fiber scaffolds. Tissue Eng 1999; 5(5):443e52.

1198

67. FUNCTIONAL TISSUE ENGINEERING OF LIGAMENT AND TENDON INJURIES

[163] Sacks MS, Gloeckner DC. Quantification of the fiber architecture and biaxial mechanical behavior of porcine intestinal submucosa. J Biomed Mater Res 1999;46(1):1e10. [164] Brunette DM. Fibroblasts on micromachined substrata orient hierarchically to grooves of different dimensions. Exp Cell Res 1986;164(1): 11e26. [165] Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Micropatterned surfaces for control of cell shape, position, and function. Biotechnol Prog 1998;14(3):356e63. [166] Clark P, Connolly P, Curtis AS, Dow JA, Wilkinson CD. Topographical control of cell behaviour: II. Multiple grooved substrata. Development 1990;108(4):635e44. [167] Walboomers XF, Croes HJ, Ginsel LA, Jansen JA. Contact guidance of rat fibroblasts on various implant materials. J Biomed Mater Res 1999; 47(2):204e12. [167a] Badylak S, Freytes DO, Gilbert TW. Reprint of: Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomater 2015;23(Suppl.):S17e26. [168] Li F, Li W, Johnson S, Ingram D, Yoder M, Badylak S. Low-molecular-weight peptides derived from extracellular matrix as chemoattractants for primary endothelial cells. Endothelium 2004;11(3e4):199e206. [169] Badylak SF, Park K, Peppas N, McCabe G, Yoder M. Marrow-derived cells populate scaffolds composed of xenogeneic extracellular matrix. Exp Hematol 2001;29(11):1310e8. [170] Zantop T, Gilbert TW, Yoder M, Badylak S. Extracellular matrix scaffolds attract bone marrow- derived cells in a mouse model of Achilles tendon reconstruction. J Orthop Res 2005;24(6):1299e309. [171] Hodde J, Record R, Tullius R, Badylak S. Fibronectin peptides mediate HMEC adhesion to porcine-derived extracellular matrix. Biomaterials 2002;23(8):1841e8. [172] McPherson TB, Liang H, Record RD, Badylak SF. Galalpha(1,3)Gal epitope in porcine small intestinal submucosa. Tissue Eng 2000;6(3): 233e9. [173] Voytik-Harbin SL, Brightman AO, Kraine MR, Waisner B, Badylak SF. Identification of extractable growth factors from small intestinal submucosa. J Cell Biochem 1997;67(4):478e91. [174] Liang R, Woo SL-Y, Takakura Y, Moon DK, Jia FY, Abramowitch S. Long-term effects of porcine small intestine submucosa on the healing of medial collateral ligament: a functional tissue engineering study. J Orthop Res 2006;24(4):811e9. [174a] Liang R, Woo SL-Y, Nguyen TD, Liu PC, Almarza A. Effects of a bioscaffold on collagen fibrillogenesis in healing medial collateral ligament in rabbits. J Orthop Res 2008;26:1098e104. [174b] Karaoglu S, Fisher MB, Woo SL-Y, Fu YC, Liang R, Abramowitch SD. Use of a bioscaffold to improve healing of a patellar tendon defect after graft harvest for ACL reconstruction: a study in rabbits. J Orthop Res 2008;26:255e63. [175] Steadman JR, Cameron ML, Briggs KK, Rodkey WG. Healing-response treatment for ACL injuries. Orthop Technol Rev 2002;3(3). [176] Steadman JR, Rodkey WG. Role of primary anterior cruciate ligament repair with or without augmentation. Clin Sports Med 1993;12(4): 685e95. [177] Joshi SM, Mastrangelo AN, Magarian EM, Fleming BC, Murray MM. Collagen-platelet composite enhances biomechanical and histologic healing of the porcine anterior cruciate ligament. Am J Sports Med 2009;37(12):2401e10. [178] Murray MM, Spindler KP, Abreu E, Muller JA, Nedder A, Kelly M, Frino J, Zurakowski D, Valenza M, Snyder BD. Collagen-platelet rich plasma hydrogel enhances primary repair of the porcine anterior cruciate ligament. J Orthop Res 2007;25(1):81e91. [179] Nguyen DT, Dellbru¨gge S, Tak PP, Woo SL-Y, Blankevoort L, van Dijk NC. Histological characteristics of ligament healing after bio-enhanced repair of the transected goat ACL. J Exp Orthop 2015;2(1):1. [180] Nguyen DT, Geel J, Schulze M, Raschke MJ, Woo SL-Y, van Dijk CN, Blankevoort L. Healing of the goat anterior cruciate ligament after a new suture repair technique and bioscaffold treatment. Tissue Eng 2013;19(19e20):2292e9. [181] Mosher CZ, Spalazzi JP, Lu HH. Stratified scaffold design for engineering composite tissues. Methods 2015;84:99e102. [182] Spalazzi JP, Doty SB, Levine WN, Lu HH. Co-culture of human fibroblasts and osteoblasts on a tri-phasic scaffold for interface tissue engineering. Chicago: Orthopaedic Research Society; 2006. [183] Fleming BC, Carey JL, Spindler KP, Murray MM. Can suture repair of ACL transection restore normal anteroposterior laxity of the knee? An ex vivo study. J Orthop Res 2008;26(11):1500e5. [184] Fisher MB, Jung HJ, McMahon PJ, Woo SL-Y. Evaluation of bone tunnel placement for suture augmentation of an injured anterior cruciate ligament: effects on joint stability in a goat model. J Orthop Res 2010;28(10):1373e9. [185] Fisher MB, Jung HJ, McMahon PJ, Woo SL-Y. Suture augmentation following ACL injury to restore the function of the ACL, MCL, and medial meniscus in the goat stifle joint. J Biomech 2011;44(8):1530e5. [186] Farraro, Pastrone A, Mau JR, Woo SL-Y. A novel magnesium ring device can enhance anterior cruciate ligament healing. In: Orthopaedic Research Society annual meeting. Orlando, Florida; 2016. [187] Farraro KF, Sasaki N, Woo SL-Y, Kim KE, Tei MM, Speziali A, McMahon PJ. Magnesium ring device to restore function of a transected anterior cruciate ligament in the goat stifle joint. J Orthop Res 2016;34(11). [188] Farraro KF, Kim KE, Woo SL-Y, Flowers JR, McCullough MB. Revolutionizing orthopaedic biomaterials: the potential of biodegradable and bioresorbable magnesium-based materials for functional tissue engineering. J Biomech 2014;47(9):1979e86. [189] Ng GY, Oakes BW, Deacon OW, McLean ID, Lampard D. Biomechanics of patellar tendon autograft for reconstruction of the anterior cruciate ligament in the goat: three-year study. J Orthop Res 1995;13(4):602e8. [190] Chen CH, Chen WJ, Shih CH. Enveloping of periosteum on the hamstring tendon graft in anterior cruciate ligament reconstruction. Arthroscopy 2002;18(5):27E.