Recent Advancements in Ligament Replacement

Recent Patents on Biomedical Engineering 2011, 4, 000-000

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Recent Advancements in Ligament Replacement Emmanuel C. Ekwueme1, Albert L. Kwansa2, Kevin Sharif3, Saadiq F. El-Amin3 and Joseph W. Freeman1,* 1

Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854, USA; 2School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, VA, USA; 3Division of Orthopaedic Surgery, Southern Illinois University School of Medicine, Springfield, IL Received: June 09, 2011

Revised: August 15, 2011

Accepted: August 23, 2011

Abstract: The anterior cruciate ligament (ACL) is important for knee stability and kinematics. It is also the most commonly injured ligament of the knee and due to its poor healing potential, severe damage warrants surgical intervention including complete replacement. Therefore, investigators have begun to pursue new techniques and devices for the repair, regeneration, and replacement of the ACL. These options involve the use of mechanically functional grafts that are designed to increase implant stability in order to withstand normal mechanical loads (while promoting ligament development in some cases). This article presents background on the ACL and its replacement, novel replacement approaches utilizing a variety of materials, and recent patent coverage.

Keywords: Anterior cruciate ligament (ACL), silk, poly (L-lactic acid) (PLLA), xenograft, scaffold, polymer, tissue engineering. INTRODUCTION TO THE ACL The anterior cruciate ligament (ACL) is the major intraarticular ligament of the knee and the most commonly injured ligament of the four ligaments of the knee with over 250,000 patients per year diagnosed with ACL disruptions [1, 2]. It supports and stabilizes the knee and limits anterior translation of the tibia. The ACL crosses over within the knee joint from the femur to the tibia in the lateral-to-medial and posterior-to-anterior directions Fig. (1) [3]. ACL injuries are a growing problem; one study that included 17,397 patients with 19,530 sport injuries over a 10-year period noted that 37% of the patients had knee injuries. The ACL was damaged in 45.4% of these cases and 33.9% of them required surgery [4]. Each year in the United States there are between 100,000 and 250,000 ACL injuries, or 1 in 3,000 in the general population; approximately 50,000 ligament reconstructions are performed annually [5-7]. A number of repair techniques are currently available, and the success rates for long-term clinical outcome are 85-90% [8-11]. The ACL is a dense, organized, rope-like tissue composed of types I, III, and V collagen, elastin, proteoglycans, water, and cells. Ligaments have a hierarchical structure with increasing levels of organization; the levels are collagen molecules, microfibrils, subfibrils, fibrils, fibril bundles, fibers, and fascicles. These are all arranged parallel to the ligament long axis [12]. The collagen fibrils also have a periodic change in direction called a crimp pattern Fig. (2). In ACL, this crimp pattern repeats every 45–60 nm [13, 14]. The fascicles contain collagen fibrils, proteoglycans, and elastin. The ligament is surrounded by an epiligament sheath [15]. The ACL is also twisted approximately 180° from the *Address correspondence to this author at the Department of Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ 08854, USA; Tel:/ Fax: ??????????????; E-mail: [email protected] 1874-7647/11 $100.00+.00

femoral attachment site to the tibial attachment site and has antromedial and posterolateral bands [14].

Fig. (1). (a) Anatomical drawings of a right knee from the anterior perspective. (b) Medial views of the same knee in extension and (c) flexion. ACL, anterior cruciate ligament; PCL, posterior cruciate ligament; LC, lateral condyle of the femur; ICR, intercondylar region of the tibia; AM, anteromedial bundle of the ACL; and PL, posterolateral bundle of the ACL. (From Freeman 2011, with permission [3]).

Ligaments have unique mechanical behavior due in part to the crimp pattern of the collagen fibers. The presence of this crimp pattern allows ligaments to increase in length under low strains without straining the collagen molecules and plastically deforming the collagen fibers Fig. (3). This allows the tissue to respond to maintained stress and still recover (up to a certain amount of strain). The stress-strain behavior of ligaments can be divided into 3 sections as seen in Fig. (3). The first section is the toe region. It is characterized by a low slope caused by the © 2011 Bentham Science Publishers

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straightening of the crimp pattern in type I collagen in response to mechanical traction. The second section of the slope is linear, corresponding to the stretching of the collagen fibers in the direction of the tensile stress that is applied to the tissue. The last section, the yield region, is identified by a decrease in slope and represents defibrillation and failure of the ligament [14, 17-19].

Ekwueme et al.

with autografts, such as initial mechanical strength and promotion of cell and tissue growth [6, 21, 22]. They also do not require a second surgical incision for the patient and there is no limit to their supply. However, allografts may be associated with potential disease transmission, infection, and complications from host immunogenic response. Sterilizing these grafts can also drastically decrease their mechanical properties [5,18,20,21]. Synthetic devices, although having shown promise as viable ligament replacements, are currently rarely used in clinical surgery.

Fig. (2). Polarized light micrograph illustrating the crimp pattern seen in a rabbit Achilles tendon (From Silver 2003, with permission [16]).

Fig. (4). Double-bundle bone-patellar tendon-bone autograft (From Pujol 2009, with permission [23]).

For the athlete, an ACL injury represents a careerdefining event. An injured ACL causes rotational and translational instability at the femoral-tibial articulation. This impedes athlete’s ability to make sudden cutting movements. This subsequently hinders the ability to perform at a high level. Furthermore longer-term studies show that individuals with an ACL deficient knee have accelerated chondral wear and increased incidence of meniscal tears [24].

Fig. (3). Example of the stress-strain behavior of a ligament; the toe, linear, and yield regions are noted. The toe region represents the straightening of the collagen crimp pattern and lateral contraction of the collagen fibrils. In the linear region, the force is translated into strain of the collagen molecules. In the yield region, there is defibrillation and ligament failure.

INTRODUCTION TO ACL REPLACEMENT Present options for ACL replacement include autografts, allografts, and synthetic devices. Autografts [tissue from the patient) are considered to be the gold standard in treating ACL injuries Fig. (4). Autografts possess appropriate initial mechanical strength and promote cell proliferation and new tissue growth. There is no risk of rejection associated with autografts since the tissue comes from the patient. However, autografts also have disadvantages. Autografts are limited in availability and require an additional surgical incision for tissue harvest, which may cause donor site morbidity [6, 20]. Allografts (tissues from cadavers) share some advantages

Once the decision between the patient and clinician are made to pursue ACL reconstruction, the type of graft must be discussed. For the clinicians several factors fall in to play in regards to graft selection. These factors are type of graft, long-term viability of the graft, morbidity of harvesting the graft, and expeditious return to activity. Graft failure and subsequent ACL revision surgery is a major concern for clinicians. Not only is revision surgery technically challenging, studies have shown that revision surgery has poorer outcomes than primary reconstruction [25]. In order to avoid graft failure graft designs should consider the following. First, the graph material needs to have adequate strength. The native ACL has a tensile strength of 1800-2196 N to failure.[26] Any graft material must meet or surpass this benchmark. Graft incorporation and fixation to native tissue is also a very important aspect for successful reconstruction. Some graft constructs that rely on suturing to screw posts exhibit the “bungee cord effect” increasing the length and elasticity of the whole unit, thus potentially inferring with graft to bone tunnel healing [27, 28]. Logically this can lead to early graft failure. The use of autograph is associated with donor site morbidity. Harvesting of certain grafts necessitate the need of

Recent Advancements in Ligament Tissue Engineering

more than one incision. Some studies show higher incidence of residual knee pain [29]. Residual knee pain has the potential to slow rehabilitation and return to previous activity. Therefore a tissue engineered graft would help alleviate this concern. The ultimate goal for the ACL injured patient is to return to activity. Problems that exist with allo/autografts that utilize bone-to-soft tissue healing are slower and more incomplete healing when compared to bone- to- bone healing.[3032] A graft that would exhibit accelerated healing rate would be very attractive and promising. This would have the potential to reduce graft fixation failure while allowing for early aggressive rehabilitation and return to play. Synthetic materials have been used in ligament replacements. These include non-degradable synthetic materials such as polyethylene terephthalate [Leeds-Keio ligament), carbon fibers, polytetrafluoroethylene (Gore-Tex), and polypropylene (Kennedy Ligament Augmentation Device) [3344]. These synthetic ligament replacements have been conditionally approved by the FDA for testing and augmentation but are not recommended for primary ACL repair [12]. Two of these devices, the Leeds-Keio ligament and the Kennedy Ligament Augmentation Device, are braided polymer structures [14, 40-42]. Non-degradable synthetic grafts can be divided into three categories: permanent replacements, augmentation devices, and scaffolds. Permanent replacements must supply the function of the ligaments that they replace and are not amendable to tissue ingrowth. As a result, they are susceptible to long-term mechanical failure due to creep and fatigue. Augmentation devices are used to protect autografts and allografts from high loads during the early postoperative period when they are at their weakest [20, 44]. These devices may shield the biological graft from stress, which leads to poor long-term neoligament formation. Devices in the last category, scaffolds, are designed with a porous structure to allow tissue ingrowth; they also are designed to transfer loads to the new tissue. Although these synthetic devices initially supply the function of the ligaments that they replace or protect the ligament that they augment, these devices fail over time because they cannot duplicate the mechanical behavior of the native ligament [20, 44]. Repeated elongation of these devices leads to permanent deformation at the points of stress. Contact with the sharp edges of the bone tunnel causes abrasions, which weaken the implant and create debris that can cause synovitis in the joint. Woven prostheses face the additional problems of axial splitting, low tissue infiltration, low extensibility, and abrasive wear [20, 44]. Synthetic augmentation devices may bear most of the stress placed on the repaired ligament, which leads to stress shielding and a reduction in strength of the remaining tissue and any new tissue. Devices that are designed for tissue ingrowth, such as the Leeds-Keio ligament remain implanted while the new tissue grows. This leads to the production of new tissue with reduced strength because the implant bears most of the applied load. In both cases, if the implant fails the remaining tissue does not have the strength to withstand the load. The presence of these scaffolds leads to poor longterm neoligament formation.

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In light of these limitations a number of researchers have created new options for ACL replacement; these include permanent replacements and options for tissue engineered replacement and augmentation. A successful ACL device must be designed to successfully support the mechanical loads experienced in the knee. In addition, tissue engineered replacements should also support cell and tissue growth while slowly degrading over time allowing the new tissue to slowly bear the load without risk of sudden rupture. Each of these design criteria are extremely important in the development of a successful tissue engineered construct. A variety of new devices have been developed that fulfill each of these criteria; they vary in their structure and base materials. This paper will review some of the more recent tissue engineered replacement devices in each of these areas. NEW OPTIONS FOR ACL REPLACEMENT Metallic Devices Although there are currently no metallic ligament replacement devices recommended for use by the FDA, there has been investigation into the use of metals and textiles as potential ligament replacement devices. These devices are not typically designed for tissue engineering due to the lack of sufficient degradability and differences in strength between the implant and soft tissue that it is meant to replace. Subsequently most metallic devices in ligament replacement or repair involve the attachment of a graft to anchoring or insertion sites in the bone. Justin et al. have patented a ligament fixation device [45]. The patent describes a device and method for fixing an anterior cruciate ligament graft in a bone tunnel in a patient's distal femur. Tuke has patented a jig for use in locating the epicondylar axis [46]. This is useful in the placement of an ACL replacement along with a kit to be used for implanting the replacement. An additional fixation device has been designed by Whittaker [47] and Mingozzi has recently patented anchors for tendons used in the reconstruction of ligament [48]. One metallic device for actual replacement was developed by Cimino in U.S. Pat. No 7905918 Fig. (5) [49]. Cimino describes an improved elastic replacement ligament composed of braided metallic wires. Cimino applies the braided design of metallic wires commonly used for several medical purposes including tubular braid designs for reinforcing and stiffening catheter walls. The device employs a titanium or titanium alloy material shown to be biocompatible for applications such as bone screws and bone fixation equipment. In order to make the inherent stiffness appropriate for use as a replacement ligament that requires both the axial elastic properties and bending flexibility of a natural ligament, the titanium wires are organized in a braided configuration. The individual metallic wires have a diameter varying between 0.0005 inches and 0.005 inches. The method for the fabrication of the replacement ligament includes organizing the metallic wires into strands of more than one [in parallel, twisted, or braided together] and then braiding the strands such that the resulting ligament possesses the desired strength and stiffness properties.


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ment of load bearing materials; this makes them excellent materials for ACL replacements [2, 50, 53]. One tissue-engineered device for ACL replacement developed by Altman et al. is a twisted fibrous matrix composed of silk fibers [2, 50, 53, 55]. This fibrous silk matrix is a hierarchical structure consisting of bundles of silk fibers wound into strands; these strands are wound together into cords and wound together to form the matrix. Each layer of the hierarchy is wound in a different direction [2, 50, 53]. The device is assembled into a 6-cord matrix. A single cord is shown in Fig. (7).

Fig. (5). Portion of an elastic metallic replacement ligament composed of metallic wires arranged into three strands (From U.S. Pat. No 7905918 to Cimino, with permission [49]).

Natural Polymers Among the materials used in biodegradable tissueengineered grafts are natural polymers such as type I collagen and silk [2, 33, 50-52]. Some of the advantages of collagen are the capability of altering resorption rate and mechanical properties of scaffolds through crosslinking and its low antigenicity. These scaffolds experience an early decrease in mechanical strength followed by tissue remodeling between 10 and 20 weeks with a strength gain similar to autografts. Fibrous proteins such as silk or collagen are composed mainly of specific amino acid sequences repeated throughout the primary structure, this creates homogeneity in the protein’s secondary structure [2, 50, 51, 53]. While most silks display -sheet conformations, type I collagen is a triple helix composed of three left-handed polyproline II helices intertwined in a right-handed configuration. In vivo, type I collagen assembles into fibrils in quarter-staggered arrays of molecules [54]. The structure of collagen is shown in Fig. (6). The rigid, extended structure of these proteins gives them the mechanical properties necessary for the replace-

Fig. (7). A single silk fibroin cord composed of 540 individual fibers. Six cords are assembled to form the device matrix (From Altman 2003, with permission [53]).

Studies have shown that these scaffolds are not cytotoxic and are conducive to the proliferation of cells as seen in tests with bone marrow stromal cells (BMSCs) [2, 50, 51]. In addition, these scaffolds have mechanical properties similar to ACL. The scaffolds have maximum loads of over 2 kN, strains at failure of approximately 39%, and elastic moduli of over 350 N/mm [50]. These values are similar to those seen in tests with native ACL. The scaffolds also demonstrate the three phase mechanical behavior seen in ligament and tendon. The scaffolds display a toe region (low stress per unit strain) followed by a linear region (high stress per unit strain) [50]; natural ligament is characterized by this behavior [14, 16]. This characteristic curve is important for the prevention of scaffold damage due to fatigue and creep. In other studies, Altman et al. have increased matrix biocompatibility and regenerative ability by coating the surface with RGD sequences [51]; this greatly increase cellular attach-

Fig. (6). Representation of collagen molecules in a quarter-staggered array. (Top) Banding pattern of the repeat sequences in the overlap and gap regions. (Bottom) The overlap and gap regions located in a group of self assembled collagen molecules (From Freeman 2004, with permission [54]).


ment, cellular proliferation, and extracellular matrix production by BMSCs [51]. As a recent natural polymer based device, U.S. Pat. No 7901455 to Koob describes methods of production and compositions for a flexible implantable ligament and tendon prosthesis composed of nordihydroguaiaretic acid (NDGA)treated collagen fibers attached together by a suture [56]. The collagen fibers are cross-linked by NDGA, a di-catechol comprised of two o-catechols at the ends of a short alkane, 3,4-dimethylbutane (2,3-bis(3,4-dihydroxyphenylmethyl)butane). Koob and Hernandez developed a process to incorporate the di-catechol into collagen I fibers and oxidizing the constituent catechols to form polymerizing quinone functionalities. This process produced fibers having comparable material properties to native tendon [57]. Results suggest that an NDGA-cross-linked collagen fiber ligament implant could serve as a viable ligament replacement device. U.S. Pat. No 7335230 to Goulet describes methods of production for a tissue engineered connective tissue substitute [58]. Goulet et al developed an ACL substitute composed entirely of a type I collagen matrix and anchored with two bone plugs. The scaffolds were shown to promote ACL fibroblast growth, migration, and collagen synthesis in vitro and in vivo. [59, 60] Synthetic Polymers Many artificial biodegradable polymers have been investigated for ACL repair including poly glycolic acid (PGA), poly lactic acid (PLA), their copolymers, poly desaminotyrosyl-tyrosine ethyl carbonate (poly (DTE carbonate)), and poly caprolactone (PCL) [12, 61]. Synthetic biodegradable polymers have several benefits in tissue engineered scaffolds. There is no limit to the supply of grafts (as opposed to autografts) and there is no risk of disease. Cellular compatibility, degradability, and functional strength are ACL reconstruction requirements that further the benefits of synthetic polymer devices [62]. The mechanical properties of these devices may also be controlled by altering the degree of polymer crystallinity, changing the polymer molecular weight, or changing the ratio of each polymer in the copolymer. Challenges of synthetic grafts include higher complication and failure rates when compared to autografts and allografts [63]. Laurencin and colleagues have developed a three-dimensional (3-D), cell seeded, degradable, braided poly L-lactic acid (PLLA) scaffold [6, 12, 37, 61, 64, 65]. PLLA, is FDA approved for a variety of clinical applications including sutures. It does not elicit a permanent foreign body reaction and degrades gradually (allowing it to be replaced by natural tissue] [12]. Unlike autografts, use of these devices carries no risk of disease transmission and there is no limit to the number of these devices due to their synthetic source. In comparison, these scaffolds can also be sterilized easier than grafts made from natural materials without sacrificing the mechanical properties of the device. The lack of structural reinforcement and arrangement of fibers in parallel in the direction of stress associated with natural polymers may lead to long-term failure due to fatigue, creep, and abrasive wear [12].


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Unlike the synthetic polymers used in earlier ACL replacements (such as the Leeds-Keio ligament and the Kennedy Ligament Augmentation Device), the fatigue properties of PLLA are not a problem. This is because the polymer degrades over time in vivo and the scaffold is eventually replaced by natural tissue. In a degradation study, PLLA fibers showed only a slight change in mechanical properties over an 8-week period in media [6]. The scaffolds produced using this 3-D braiding technique have initial mechanical properties comparable to the ACL. The braiding method provides wear and rupture resistance to the scaffolds [6, 12]. The 3-D braiding technique causes the fibers to be woven throughout the entire thickness of the braid; this gives the braid strength and reinforces the structure, thus preventing total scaffold failure if some of the fibers are damaged. Past ligament prostheses made of flexible composites composed of fibers that have been woven or braided into structures have had disappointing long-term outcomes [39, 43]. Many of these scaffolds were limited by poor tissue integration, poor abrasion resistance, and structural fatigue [39, 43]. In addition, the weaving process creates a network of integrated pores of regulated size. This network of pores aids in the movement of nutrients throughout the scaffolds and removal of waste from the cells [64]. Both of these properties enhance cell proliferation and tissue ingrowth. The presence of pore interconnectivity throughout the entire implant also increases the overall surface area for cell attachment and allows for tissue ingrowth throughout the entire scaffold [12, 64]. This braided scaffold also has a fibrous, hierarchical structure. The scaffold is composed of microfibers that are similar in diameter to collagen fibers in natural ligament. The microfibers are grouped together to form fibers. The fibers are arranged into bundles and braided throughout the entire thickness of the scaffold [6, 12, 37, 64]. The braids are split into three sections: femoral tunnel attachment site (bony attachment end), ligament region (intra-articular zone), and tibial tunnel attachment site (bony attachment end) Fig. (8). The angle of the fibers at the attachment sites is higher than the angle of the intra-articular zone. The differences in fiber orientation create differences in pore sizes of the regions [6, 12, 64]. This is important for cellular proliferation and the growth of tissue in each region. Studies have shown that a minimum pore diameter of 150 μm is necessary for bone ingrowth and 200–250 μm for soft tissue ingrowth [66, 67]. The pore sizes of the different sections reflect these tissues preferences order to encourage ingrowth of the appropriate tissue in the appropriate area as well as capillary supply. After implantation in New Zealand white rabbit studies, the device, when seeded with cells displayed the ability to regenerate new ligament tissue with oriented, mature collagen fibers [64]. Scaffolds seeded with primary ACL cells prior to implantation displayed a decreased fibrous capsule, blood vessels, and the presence of mature, oriented collagen fibers 12 weeks after the surgery. The collagen fibers were also able to infiltrate the full thickness of the graft [64].


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Fig. (8). 3-D braided ligament device showing the macrostructure of the intra-articular and bony attachment sections (From Laurencin 2005, with permission [12]).

Biological Tissues Concerns with devices produced from biological tissues are based on the tissue source. The main concerns of use of this graft source in humans include potential transmission of disease to the patient, possible unfavorable immunogenic response, and bacterial infection [12, 68]. As mentioned earlier, it is also difficult to sterilize these devices without decreasing their mechanical properties. On the other hand, their use does not require a second surgery for tissue harvest. There is no limit to the supply of the graft tissue for these devices and they have the appropriate initial mechanical strength (depending on the source of tissue) [12]. Devices based on biological tissues also promote cell proliferation and new tissue growth. Xenografts (tissues from animals) have been studied as an option for ACL repair. Xenografts share some advantages and disadvantages with allografts. They may also carry the additional risk of transferring a disease that is normally seen in an animal to their human hosts as well as the risk of rejection. Despite these risks, Stone et al. have shown that treated xenografts may be a viable option for ACL repair [12, 6972] Fig. (9). In their studies, Stone and colleagues have used chemically modified grafts from cloned pigs as ACL replacements. These studies show the successful use of immunochemically modified, chemically crosslinked porcine grafts for ACL reconstruction [12, 70, 71, 73]. To prevent rejection of the graft, the -gal epitopes were enzymatically removed from the grafts. The interaction between the natural anti-Gal antibody and -gal epitopes has been an obstacle in the use of xenotransplantation with porcine tissues [55, 74]. This obstacle has been eliminated through the cloning of pigs lacking -gal epitopes [55, 65, 73]. In addition, the ACL grafts used in these studies have been pulse lavaged to remove cellular components and crosslinked with 0.10% gluteraldehyde for 12 hours; this treatment was followed by glycine endcapping to block un-

reacted gluteraldehyde molecules and sterilization by electron beam irradiation at 17.8 kGy [55, 71].

Fig. (9). Porcine patellar tendon xenograft (From Stone 2007, with permission [(55]).

In an in vivo study, the treated porcine grafts were implanted into rhesus monkeys [55]. Twenty animals were reconstructed with treated grafts for 2, 6 and 12 months; 3 monkeys were used at the 2 month time point, 5 monkeys were used at the 6 month time point and another 5 monkeys were used at the 12 month time point. The controls were 1 untreated porcine allograft and 1 rhesus allograft at 2 months along with 5 rhesus allografts at 12 months. The grafts and corresponding intact ligaments from the other leg were biomechanically tested at 6 months (3 grafts) and 12 months (10 grafts); a histological examination followed the mechanical tests. Treated porcine bone–patellar tendon–bone grafts and fresh frozen rhesus bone–patellar tendon–bone devices were cut into bone-tendon constructs. The devices for implantation were 30-mm long by 4-mm wide (tendon material) grafts with bone plug ends of 5 mm diameter by 7 mm in length. The treated implants promoted the regeneration of new ligament tissue [55]. There were signs of graft remodeling extending from the graft periphery to the graft center. After 12 months the porcine grafts displayed comparable ultimate load, yield load, stiffness and ultimate displacement. The strength of the treated grafts increased from 43% to 58% between 6 and 12 months.


Unfortunately, the grafts also demonstrated lower values for ultimate strength, yield strength, ultimate strain, and modulus. Though these final values require improvement, the values based on load and displacement demonstrate that this graft system can become a viable option for ACL replacement. Along with mechanical tests the animals were tested for the rejection of the treated porcine grafts [75]. Blood samples were taken prior to surgery and at days 10, 14, 21, 28, 42, 56 as well as at 3, 6, 9, and 12 months and analyzed for anti-Gal and anti–non-Gal antibodies such as antibodies to proteins present in the porcine grafts. Serum immunoglobulin (Ig] anti-Gal IgG and IgM activity was determined by ELISA. There was a greater increase in anti-Gal titers (>200%) in the monkey engrafted with untreated porcine graft when compared to anti-Gal titers from the monkey implanted with the treated graft (95% lower than the untreated) within 2 weeks following implantation. The response to the untreated grafts is an indicator of acute rejection and can lead to graft destruction and resorption. It is hypothesized that the small increase in anti-Gal titers in monkeys with the treated grafts may be due to an immune response to -gal epitopes on the porcine bone marrow cells in cancellous bone interstices of the bone-ligament-bone graft. The antiGal titers reached resolved preimplantation values by 8 to 12 weeks after implantation. In a different study, the porcine grafts were implanted into human subjects for ACL replacement [55). Western blotting analysis and ELISA showed that the subjects produced anti-non-gal antibodies against multiple pig xenoproteins. Their level of production peaked from two to 6 months; all antibodies were no longer produced after two years with no antibodies were produced against human ligament proteins. After two years five of the six patients in the study showed had no problem with the function of the porcine graft. Results from this proof of concept study show exciting promise for the use of xenografts as viable ligament replacements. The U.S. Pat. No 7513910 to Buskirk describes methods of production and compositions for a novel dermis-derived graft as a potential ligament replacement [76]. After surgical implantation, the implant undergoes cellular remodeling to produce a new replacement ligament. The processed dermis tissue is rolled into a cylindrical shape with bone blocks positioned at opposite ends. The bone blocks are derived from cortical, cancellous, cortico-cancellous, or demineralized bone from allograft or xenograft sources. The dermis is first decellularized and retains the structural functionality of the basement membrane. Given that the main component of the processed dermis is collagen I, increased implant mechanical strength can be achieved by further processing techniques including chemical crosslinking and dehydrothermal treatment. The implant ends are calcified by incorporating a calcium hydroxide solution into the processing method. In a small animal study, results showed an increased level of bone deposition that is related to the initial levels of calcification. This suggests that the calcification of the dermis implants led to better incorporation in the bone.


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CURRENT & FURTHER RESEARCH AND DEVELOPMENT The ACL is a complex, highly ordered tissue with mechanical properties that are important for normal knee kinematics. To maintain knee function after injury, repair devices must be able to bear the appropriate amount of load and display similar mechanical properties in the short term while promoting the growth of new mature ligament to bear the load in the long term. The devices listed above represent some of the advancements that have been made in ACL replacement. They display the range of options available from anchoring devices and permanent replacements to tissue engineered scaffolds. The structures can be made from metals, natural polymers, synthetic polymers, and treated tissues. They are all designed to fulfill the needs of a functional replacement, these include structural stability and appropriate mechanical strength. The tissue engineered scaffolds displayed the promotion of cell and tissue growth, and the ability to slowly degrade and allow the new tissue to bear the load. Although this blend of biological activity and mechanical stability make these devices excellent options for ACL replacement, further research is required to translate these technologies from the bench to the bedside. DISCLOSURE This manuscript is an extended and updated version of the paper: Joseph W. Freeman and Albert L. Kwansa. Recent Advancements in Ligament Tissue Engineering: The Use of Various Techniques and Materials for ACL Repai, Recent Patents on Biomedical Engineering, Vol. 1, pp.18-23, 2008.

REFERENCES [1]

[2] [3]

[4] [5]

[6]

[7] [8]

[9] [10] [11]

Noyes FR, Grood ES. The strength of the anterior cruciate ligament in humans and Rhesus monkeys. The Journal of bone and joint surgery. 1976;58 (8):1074-82. Vunjak-Novakovic G, Altman G, Horan R, Kaplan DL. Tissue engineering of ligaments. Annual review of biomedical engineering. 2004;6:131-56. Freeman J, Walters V, Kwansa A. Ligaments, biomaterials, and tissue engineering opportunities. In: Hollinger J, editor. An Introduction to Biomaterials, Second Edition: Taylor & Francis; 2011. Majewski M, Susanne H, Klaus S. Epidemiology of athletic knee injuries: A 10-year study. The Knee. 2006;13 (3):184-8. Cameron ML, Mizung Y, Cosgarea AJ. Diagnosing and managing anterior cruciate ligament injuries. J Musculoskeletal Med. 2000; 17:7. Cooper JA, Lu HH, Ko FK, Freeman JW, Laurencin CT. Fiberbased tissue-engineered scaffold for ligament replacement: design considerations and in vitro evaluation. Biomaterials. 2005;26 (13): 1523-32. Miyasaka K, Daniel D, Stone M, Hirshman P. The Incidence of Knee Ligament Injuries in the General Population. American Journal of Knee Surgery. 1991;4:6. Dandy DJ. Historical overview of operations for anterior cruciate ligament rupture. Knee Surg Sports Traumatol Arthrosc. 1996;3 (4):256-61. Eriksson E. How good are the results of ACL reconstruction? Knee Surg Sports Traumatol Arthrosc. 1997;5 (3):137. Fu FH, Musahl V. Review Article: The future of knee ligament surgery. J Orthop Surg (Hong Kong). 2001;9 (2):77-80. Passler HH. The history of the cruciate ligaments: some forgotten (or unknown) facts from Europe. Knee Surg Sports Traumatol Arthrosc. 1993;1 (1):13-6.

8 Recent Patents on Biomedical Engineering, 2011, Vol. 4, No. 3 [12]

[13] [14] [15]

[16] [17]

[18]

[19]

[20]

[21] [22] [23]

[24]

[25] [26]

[27]

[28] [29]

[30] [31]

[32]

[33]

Laurencin CT, Freeman JW. Ligament tissue engineering: an evolutionary materials science approach. Biomaterials. 2005; 26 (36):7530-6. Cabaud HE, Rodkey WG, Feagin JA. Experimental studies of acute anterior cruciate ligament injury and repair. The American journal of sports medicine. 1979;7 (1):18-22. Silver FH. Biomaterials, medical devices, and tissue engineering: an integrated approach. London: Chapman & Hall; 1994. Amiel D, Billings E, Harwood FL. Collagenase activity in anterior cruciate ligament: protective role of the synovial sheath. J Appl Physiol. 1990;69 (3):902-6. Silver FH, Freeman JW, Seehra GP. Collagen self-assembly and the development of tendon mechanical properties. J Biomech. 2003;36 (10):1529-53. Diamant J, Keller A, Baer E, Litt M, Arridge RG. Collagen; ultrastructure and its relation to mechanical properties as a function of ageing. Proceedings of the Royal Society of London Series B, Containing papers of a Biological character. 1972;180 (60):293315. McBride Jr DJ, Hahn RR, Silver FH. Morphological characterization of tendon development during chick embryogenesis: Measurement of birefringence retardation. Int J Biol Macromol. 1988;7 (71). Mosler E, Folkhard W, Knorzer E, Nemetschek-Gansler H, Nemetschek T, Koch MH. Stress-induced molecular rearrangement in tendon collagen. Journal of molecular biology. 1985;182 (4): 589-96. Laurencin CT, Ambrosio AMA, Borden MD, Cooper JA. Tissue engineering: orthopedic applications. In: Yarmush ML, Diller KR, Toner M, editors. Annual review of biomedical engineering. Palo Alto, CA1999. p. 19-46. Laurencin CT, Ambrosio AM, Borden MD, Cooper JA, Jr., editors. Tissue engineering: orthopedic applications1999. Laurencin CT, Ambrosio AM, Borden MD, Cooper JA, Jr. Tissue engineering: orthopedic applications. Annual review of biomedical engineering. 1999;1:19-46. N P, O F, M K, P B, P B. Anatomic double-bundle ACL reconstruction using a bone-patellar tendon-bone autograft: a technical note. Knee Surg Sports Traumatol Arthrosc. 2009;18:43-6. RN T, O T, A K. Analysis of meniscal and chondral lesions accompanying anterior cruciate ligament tears: Relationship with age, time from injury, and level of sport. Knee Surg Sports Traumatol Arthrosc. 2004;12:262-70. MJ B, FA C, JA H. Results of revision anterior cruciate ligament surgery. The American journal of sports medicine. 2007;35 (12): 2057-66. Noyes FR, Butler DJ, Grood ES, Zemicke RF, Hefzy MS. Biomechanical analysis of human ligament grafts used in knee ligament repairs and reconstructions. J Bone and Joint Surg. 1984;66-A:34452. KA J, A H, J S. 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;7:290-5. PE R. Bungee cord effect in hamstring tendon ACL reconstruction. Orthopedics. 2000;23:184. Freedman KB, D'Amato MJ, Nedeff DD, Kaz A, Bach Jr. BR. Arthroscopic Anterior Cruciate Ligament Reconstruction: a metaanalysis comparing patellar tendon and hamstring autografts. Am J Sports Med. 2003;31 (1):2-11. VK G, MC R, WA G, MD R, HS P. Tendon-to-bone healing of a semitendinosus tendon autograft used for ACL reconstruction in a sheep model. . Am J Knee Surg 2000;13 (3):143-51. WA G, DM E, R M, CW G. An analysis of autograft fixation after anterior cruciate ligament reconstruction in a rabbit model. . The American journal of sports medicine. 1994;22 (3):344-51. F T, K Y, S M, T S, S Y, H T. Comparisons of intraosseous graft healing between the doubled flexor tendon graft and the bonepatellar tendon-bone graft in anterior cruciate ligament reconstruction. Arthroscopy. 2001;17 (5):461-76. Dunn MG, Liesch JB, Tiku ML, Zawadsky JP. Development of fibroblast-seeded ligament analogs for ACL reconstruction. Journal of biomedical materials research. 1995;29 (11):1363-71.

Ekwueme et al. [34]

[35] [36]

[37]

[38]

[39] [40]

[41] [42]

[43]

[44] [45] [46] [47] [48] [49] [50]

[51]

[52]

[53] [54]

[55]

[56] [57]

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):2461-74. Jackson DW, Heinrich JT, Simon TM. Biologic and synthetic implants to replace the anterior cruciate ligament. Arthroscopy. 1994;10 (4):442-52. Kock HJ, Sturmer KM, Letsch R, Schmit-Neuerburg KP. Interface and biocompatibility of polyethylene terephthalate knee ligament prostheses. A histological and ultrastructural device retrieval analysis in failed synthetic implants used for surgical repair of anterior cruciate ligaments. Archives of orthopaedic and trauma surgery. 1994;114 (1):1-7. Lu HH, Cooper JA, Jr., Manuel S, Freeman JW, Attawia MA, Ko FK, et al. Anterior cruciate ligament regeneration using braided biodegradable scaffolds: in vitro optimization studies. Biomaterials. 2005;26 (23):4805-16. Amiel D, Billings E, Akeson WH. Ligament structure, chemistry, and physiology. In: Daniel D, Akeson WH, O'Connor J, editors. Knee ligaments: structures, function, injury and repair. New York: Raven Press; 1990. p. 77-91. Arnoczky SP. Anatomy of the anterior cruciate ligament. Clinical orthopaedics and related research. 1983;172 (Jan-Feb):19-25. Bolton CW, Bruchman WC. The GORE-TEX expanded polytetrafluoroethylene prosthetic ligament. An in vitro and in vivo evaluation. Clin Orthop Relat Res. 1985 (196):202-13. Fujikawa K. Prosthetic ligament reconstruction of the knee. In: Friedman MJ, Ferkel RD, editors. Philadelphia: W. B. Sanders Company; 1988. Olson EJ, Kang JD, Fu FH, Georgescu HI, Mason GC, Evans CH. The biochemical and histological effects of artificial ligament wear particles: in vitro and in vivo studies. The American journal of sports medicine. 1988;16 (6):558-70. Silver FH, Tria AJ, Zawadsky JP, Dunn MG. Anterior Cruciate Ligament Replacement: A Review. Journal of long-term effects of medical implants. 1991;1 (2):135-54. Smith BA, Livesay GA, Woo SL. Biology and biomechanics of the anterior cruciate ligament. Clinics in sports medicine. 1993;12 (4): 637-70. Justin DF, Richard F. Wenstrom J, Levy AS. Expanding ligament graft fixation system method. US6887271, 2005. Tuke MA. A jig for use with a ligament prosthesis. EP1741410, 2007. Whittaker GR. Tissue fixation device. US20080051795, 2007. Mingozzi F, Dovesi, A., Aglietti, P. Anchor for tendons used in the reconstruction of a ligament, particularly of the cruciate ligamnet of the knee. EP1813226, 2007. Cimino WW. Elastic metallic replacement ligament. US7905918, 2011. Altman GH, Horan RL, Lu HH, Moreau J, Martin I, Richmond JC, et al. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials. 2002;23 (20):4131-41. Chen J, Altman GH, Karageorgiou V, Horan R, Collette A, Volloch V, et al. Human bone marrow stromal cell and ligament fibroblast responses on RGD-modified silk fibers. J Biomed Mater Res A. 2003;67 (2):559-70. Dunn MG, Tria AJ, Kato YP, Bechler JR, Ochner RS, Zawadsky JP, et al. Anterior cruciate ligament reconstruction using a composite collagenous prosthesis. A biomechanical and histologic study in rabbits. The American journal of sports medicine. 1992;20 (5): 507-15. Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen J, et al. Silk-based biomaterials. Biomaterials. 2003;24 (3):401-16. Freeman J, Silver F. Elastic energy storage in unmineralized and mineralized extracellular matrices (ECMs): A comparison between molecular modeling and experimental measurements. J Theor Bio. 2004;229:371-81. Stone KR, Abdel-Motal UM, Walgenbach AW, Turek TJ, Galili U. Replacement of human anterior cruciate ligaments with pig ligaments: a model for anti-non-gal antibody response in long-term xenotransplantation. Transplantation. 2007;83 (2):211-9. Koob TJ, Pringle D. Tendon or ligament bioprostheses and methods of making same. US7901455, 2007. Koob T, Hernandez D. Material properties of polymerized NDGAcollagen composite fibers: development of biologically based tendon constructs. Biomaterials. 2002;23 (1):203-12.

Recent Advancements in Ligament Tissue Engineering [58] [59]

[60]

[61]

[62]

[63]

[64]

[65]

[66] [67]

Goulet F, Rancourt D, Cloutier R. Connective tissue substitutes, method of preparation and uses thereof. US7335230, 2008. Goulet F, D R, Cloutier R, Tremblay P, A B, Lamontagne J. Torn ACL: a new bioengineered substitute brought from the laboratory to the knee joint. Appl Bionic Biomech. 2004;1 (2):115-21. Robayo LM, Moulin VJ, Tremblay P, Cloutier R, Lamontagne J, Larkin AM, et al. New ligament healing model based on tissueengineered collagen scaffolds. Wound Repair Regen. 2011;19 (1): 38-48. Bourke SL, Kohn J, Dunn MG. Preliminary development of a novel resorbable synthetic polymer fiber scaffold for anterior cruciate ligament reconstruction. Tissue engineering. 2004;10 (1-2): 43-52. Tovar N, Bourke S, Jaffe M, Murthy NS, Kohn J, Gatt C, et al. A comparison of degradable synthetic polymer fibers for anterior cruciate ligament reconstruction. J Biomed Mater Res A. 2010;93 (2):738-47. PMCID: 2845725. Hospodar M, Miller M. Controversies in ACL Reconstruction: Bone-patellar Tendon-bone Anterior Cruciate Ligament Reconstruction Remains the Gold Standard. Sports Med Arthrosc Rev. 2009 (17):242-6. Cooper JA, Jr., Sahota JS, Gorum WJ, 2nd, Carter J, Doty SB, Laurencin CT. Biomimetic tissue-engineered anterior cruciate ligament replacement. Proceedings of the National Academy of Sciences of the United States of America. 2007;104 (9):3049-54. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science (New York, NY. 2002;295 (5557):1089-92. Konikoff JJ, Billings W, Nelson LJ, Hunter JM. Development of a single stage active tendon prosthesis, I: distal end attachment. The Journal of bone and joint surgery. 1974;56:848. Yahia L. Ligaments and ligamentoplasties. Berlin: Springer; 1997.

Recent Patents on Biomedical Engineering, 2011, Vol. 4, No. 3 [68]

[69]

[70]

[71]

[72]

[73]

[74] [75]

[76]

9

Kainer MA, Linden JV, Whaley DN, Holmes HT, Jarvis WR, Jernigan DB, et al. Clostridium infections associated with musculoskeletal-tissue allografts. The New England journal of medicine. 2004;350 (25):2564-71. Stone KR, Ayala G, Goldstein J, Hurst R, Walgenbach A, Galili U. Porcine cartilage transplants in the cynomolgus monkey. III. Transplantation of alpha-galactosidase-treated porcine cartilage. Transplantation. 1998;65 (12):1577-83. Stone KR, Walgenbach AW, Abrams JT, Nelson J, Gillett N, Galili U. Porcine and bovine cartilage transplants in cynomolgus monkey: I. A model for chronic xenograft rejection. Transplantation. 1997; 63 (5):640-5. Stone KR, Walgenbach AW, Turek TJ, Somers DL, Wicomb W, Galili U. Anterior cruciate ligament reconstruction with a porcine xenograft: a serologic, histologic, and biomechanical study in primates. Arthroscopy. 2007;23 (4):411-9. Stone K, Abdel-Motal U, Walgenbach A, Turek T, Galili U. Replacement of Human Anterior Cruciate Ligaments with Pig Ligaments: A Model for Anti-Non-Gal Antibody Response in Long-Term Xenotransplantation. Transplantation. 2007;83:211-9. Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science (New York, NY. 2003;299 (5605):411-4. Galili U. Interaction of the natural anti-Gal antibody with alphagalactosyl epitopes: A major obstacle for xenotransplantation in humans. Immunology Today. 1993;14:3. Stone K, Walgenbach A, Turek T, Somers D, Wicomb W, Galili U. Anterior cruciate ligament reconstruction with a porcine xenograft: A serologic, histologic, and biomechanical study in primates. Arthroscopy. 2007;23 (4):411-9. Buskirk D, Seid CA, Wironen JF, Gross JM, Scurti G. Soft and calcified tissue implants. US7513910, 2004.