- Email: [email protected]
Biological considerations of tendon graft incorporation within the bone tunnel
Biological Considerations of Tendon Graft Incorporation Within the Bone Tunnel Boris A. Zelle, MD,* Christian Lattermann, MD,* Anikar Chhabra, MD, MS,* Freddie H. Fu, MD, DSc (Hon), DPs (Hon),* and Johnny Huard, PhD† Incorporation of the tendon graft within the bone tunnel is crucial for a successful outcome after anterior cruciate ligament reconstruction. Previous studies demonstrated that the incorporation of the tendon graft may take up to several months. Thus, it is of concern whether the early tendon– bone interface is strong enough to tolerate accelerated early postoperative rehabilitation. Biological solutions to enhance tendon healing within the bone tunnel may allow further advances in anterior cruciate ligament surgery. Previous studies emphasized the important role of bone growth factors. In animal models, successful stimulation of graft incorporation has been achieved by the application of bone morphogenetic protein-2. Moreover, periosteal enveloping of the tendon graft has shown to improve the healing response of the tendon graft within the bone tunnel, and preliminary clinical results appear promising. Further advances may be achieved by the use of tissueengineered ligaments. Successful tissue engineering of ligaments requires optimized structural scaffolds, tissue specific cells, biological stimulation by growth factors, and mechanical stimulation by cyclic stretching. Oper Tech Orthop 15:36-42 © 2005 Elsevier Inc. All rights reserved. KEYWORDS gene therapy, growth factor, tissue engineering, ligament replacement
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ntegration of anterior cruciate ligament grafts into bone tunnels is critical for the success of anterior cruciate ligament (ACL) reconstruction. Depending on the type of the graft, it may take up to several months until the tendon– bone interface is regenerated after ACL reconstruction.1-4 With soft-tissue grafts, fixation occurs predominantly with fibrous tissue with collagen fibers interspersed along the load axis.1-3,5-8 Connective tissue has been shown to surround the graft up to 12 months after implantation.1,2,9 Moreover, tunnel expansion, most likely caused by micro-motion and synovial fluid extravasation in the tunnel, remains a concern, in particular with soft-tissue grafts.10 Thus, it is of concern whether the early tendon– bone interface is strong enough to tolerate accelerated early postoperative rehabilitation.10 For this reason, most rehabilitation programs after ACL reconstruction require a protected mobilization of the operated
*Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA. †Growth and Development Laboratory, Children’s Hospital of Pittsburgh, Pittsburgh, PA. Address reprint requests to Boris A. Zelle, MD, University of Pittsburgh, Ferguson Laboratory for Orthopaedic Research, Presbyterian University Hospital C-313, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail: [email protected]
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knee to allow for adequate graft incorporation. Therefore, a detailed understanding of the biological situation of the tendon graft within the bone tunnel appears important for knee surgeons. The specific aims of this article will be to review (1) the histology of tendon graft incorporation, (2) biological approaches to enhance the healing stimulation of the tendonto-bone insertion site, (3) the concepts of ligament bioengineering.
Histology of Graft Incorporation It is well accepted that the bone plug of bone–patella tendon– bone graft becomes anchored to the bone wall by appositional bone formation and that the grafted bone becomes necrotic as new bone forms.5,8 Several recent studies have demonstrated the time course of this bone-to-bone healing. Tomita and coworkers evaluated the histologic response using adult beagle dogs at 3, 6, and 12 weeks.11 At 3 weeks, one side of the graft was in contact with the bony wall, whereas the opposite side gap between the plug and the tunnel wall was filled with granulation tissue. The bone plug was necrotic as demonstrated by empty lacunae. No changes were noted in the fibrocartilage layers at the tendon– bone junction. By 6 weeks, on the side of bony contact, the newly formed bone
Tendon graft incorporation within the bone tunnel
Growth Factor Concentration
was evident and attached at the graft-tunnel interface, whereas on the opposite side, the granulation tissue remained. Sharpey’s fibers were present in the granulation tissue around the interosseous tendon portion of the graft. In addition, at the tendon bone interface, there was a decrease in the number and quantity of cartilaginous cells. By 12 weeks, newly formed bone obscured the tunnel graft interface at the side of bony contact. In the granulation tissue, collagen fibers are more densely packed. At the tendon– bone interface, no significant histological changes were present.11 These histologic observations were confirmed by Kawakami and coworkers in rabbits.5 It is generally accepted that by 16 to 20 weeks, complete bony incorporation of the graft into the tunnel is present.6,7 Soft-tissue (bone-free) grafts undergo a different healing process that was characterized by Rodeo and coworkers in mongrel dogs.3 At 2 weeks, the area between the tendon– bone interface comprises highly vascular and cellular fibrous tissue. The tissue is poorly organized, and no collagen continuity is present. At 4 weeks, a thin seam of new bone is present lining the tunnel. The tendon in the tunnel remains viable with normal fibroblasts. There is a more abundant extracellular matrix and the presence of collagen fiber continuity at the bone–fibrous tissue interface. By 8 weeks, the tendon– bone interface was more organized, with more collagen fibers spanning the junction pulling in the direction of tension from the graft to the tunnel. Remodeling of the trabecular bone around the tendon was also present. At 12 weeks, there was increased organization and maturation of the fibrous tissue, with continuity of collagen fibers aligned along the direction of the pull of the muscle tendon unit between the tunnel and the graft. By 26 weeks, maturation of continuity of collagen fibers between the graft and tunnel was near complete, and there was significant remodeling of the trabecular bone surrounding the tendon. This progression of healing and collagen formation is consistent with the morphology of Sharpey’s fiber development attaching soft tissue to bone, emphasizing the differences between BPTB and soft tissue graft incorporation.3 These finding and time course are confirmed by several animal studies.5,12,13 However, recent evidence suggests that tendon to bone healing is bone-specific. Grassman and coworkers demonstrated that incorporation and remodeling is more extensive and occurs at a faster rate in cancellous filled femoral bone when compared with the marrow dominated tibial bone.14 Tendon healing in a bone tunnel is influenced by mechanical stress. As demonstrated by Yamakado and coworkers in rabbits, tensile stress (at the anterior aspect of the bone tunnel) enhances and promotes the tendon-bone interface healing process.15 However, compressive stresses (at the posterior side of the graft) promote chondroid formation, and eventually woven bone, rather than Sharpey’s fibers. These stress influences on regions of tendon to bone healing needs to be further clarified. Liu and coworkers traced this healing with immunohistochemistry to localize the different types of collagen in rabbits.16 Initially, types I, II, and III are present at the bone–tendon interface. By 6 weeks, type III collagen fibers, representing Sharpey’s
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Viral Injection
Growth Factor Injection (Protein)
Application
Time
Figure 1 Growth factor concentration in musculoskeletal tissue. After injection of the pure growth factor protein, the concentration in the tissue reaches a maximum and returns instantly to the baseline level. Gene therapy results in a continuous growth factor concentration in the target tissue over a longer time period.
fibers, spanned the interface. Although a definitive fibrocartilage region did not form, there was localization of type II collagen at the interface. As type II and III collagen decrease over time, Type I collagen increases as more woven bone develops.16
Augmentation Techniques Improvements in soft tissue graft healing to bone are critical to allow more aggressive and earlier rehabilitation and an earlier return to sports and activities. Thus, biological solutions to stimulate the graft incorporation within the bone tunnel appear to be a promising approach. Several recent studies in animals of various augmentation techniques show promise.
Growth Factor Stimulation Growth factors are proteins that have the ability to stimulate cell proliferation, cell migration, and cell differentiation. The efficacy of growth factor stimulation has been studied in various musculoskeletal tissues, such as muscle, cartilage, meniscus, ligament, tendon, and bone.17 The use of most growth factors, however, is limited by their short biological halflives.18,19 Therefore, gene transfer techniques frequently are used to test the efficacy of growth factor stimulation. Gene therapy is based on the modification of cellular genetic information. Therefore, the genes encoding for growth factors are transferred into the local cells using viral or nonviral vectors. Thus, the genetic information of the local cells is modified so that growth factors are continuously released, resulting in a continuous stimulation of the tissue (Fig. 1). Various studies have demonstrated the efficacy of growth factor stimulation for bone tissue and for tendon tissue. After ACL reconstruction, however, the tendon graft is incorporated within a bony tunnel (Fig. 2). This incorporation of tendon tissue within a bone tunnel represents a unique biological situation and few studies have evaluated the efficacy of growth factor stimula-
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Figure 2 Schematic drawing of a reconstructed ACL. The schematic drawing demonstrates the tendon graft within the bone tunnel of the distal femur. Postoperative graft incorporation occurs at the tendon– bone interface.
tion to enhance the healing response of the tendon graft within the bony tunnel. Using a rabbit hallucis longus and calcaneus process model (Fig. 3), Lattermann and coworkers were able to show that transfection of the tendon-to-bone insertion site with a marker gene is a feasible technique and results in a prolonged gene expression.20 Further experimental investigations in rabbits demonstrated that gene delivery of bone morphogenetic protein-2 (BMP-2) to the tendon-to-bone insertion site in the reconstructed ACL enhances the healing response in the bone tunnel and the ultimate load failure of the construct.21 Rodeo and coworkers reported on the use of recom-
binant human BMP-2 for tendon healing in a bone tunnel using an extraarticular dog model.22 These authors demonstrated histologic and biomechanical evidence of earlier graft incorporation in those grafts treated with BMP-2 when compared with normal controls. Weiler and coworkers investigated the efficacy of platelet-derived growth factor-BB (PDGF-BB) on graft incorporation using an ACL model in sheep.23 These authors used coated sutures as growth factor delivery system for the PDGF-BB and compared the outcomes with normal controls. The application of PDGF-BB resulted in higher biomechanical strength, higher vascular density, and a higher collagen fibril amount. These experimental data suggest that growth factor stimulation of the tendon-to-bone insertion site is a promising treatment opportunity with important clinical implications. Therefore, current research projects in our laboratories continue to focus on the biological stimulation of tendon healing in the bone tunnel, using a rabbit hallucis longus and calcaneus process model as described above.
Enveloping of Tendon Graft with Periosteum Numerous studies have demonstrated the ability of periosteum to induce new chondroid and bone formation.24-26 Periosteum comprises multipotent stem cells to differentiate to osteogenic and chondrogenic tissues. Thus, enveloping tendon grafts with periosteum appears to be an interesting approach for biologic augmentation of the tendon graft in the bone tunnel. Using the above described rabbit hallucis longus and calcaneus process model, Youn and coworkers were able to show that periosteal augmentation of a soft-tissue graft in the bone tunnel increases the ultimate load failure of
Figure 3 Schematic drawing of a rabbit model to investigate the tendon-to-bone healing.20 The hallucis longus tendon is detached from the Achilles tendon. A bone canal is drilled into the calcaneus and the hallucis longus tendon is pulled into the bone canal.
Tendon graft incorporation within the bone tunnel
Ligament Tissue Engineering The technical aspects of autograft and allograft ACL replacements have undergone improvements over the last 10 years and yield an overall success rate of about 90%.31-34 Despite the ability to adapt to the mechanical demands both autografts and allografts do not reach the structural identity of either the original tendon tissue or the native ACL. In addition, autografts are associated with donor site morbidity in up to 40% of all patients.35-37 On the other hand, allograft transplants have a risk of disease transmission and possible immune reactions. One possible way to improve this situation may be to engineer a neoligament under ex vivo conditions that can be preconditioned for the host environment. A preconditioning would mimic the biomechanical, biological and the cellular recipient environment before implantation and may be able to reduce or eliminate the typical process of tissue degradation and necrosis that current allografts and autografts undergo. Novel tissue engineering strategies attempt to achieve this ambitious goal and have recently been introduced on an experimental level. The concept of tissue engineering is based on the understanding that the engineering of a functional tissue requires the complex interaction of four basic principles. This basic paradigm includes: a structural scaffold, tissue specific cells, biological modulators such as growth factors and cytokines, and mechanical modulators (Fig. 4). Any engineered tissue requires a structural scaffold providing a basic architecture for the different cellular and extracellular components of the engineered tissue. Another key component is the presence of cells that are able to populate the scaffold and modify the scaffold according to the mechanical and biological conditions. To create the desired material properties of the engineered tissue the cells need to be manipulated such that they modify the extracellular environment within the scaffold accordingly. This manipulation can be done with intrinsic factors such as growth factors, cytokines and nutritional supplements. In addition, extrinsic, mechanical factors will be required to define the later environment to which the engineered tissue will have to adapt to.
Tissue Engineered Ligament (TEL)
Physical Stimuli
Biological Stimuli
TELL Cells
the tendon– bone interface at 6 weeks after surgery.27 Similar results were reported for periosteal soft-tissue graft augmentation with other extraarticular rabbit models.28,29 Interestingly, the periosteal enveloping technique also has been used successfully in clinical trials. Thus, Chen and coworkers reported satisfactory clinical outcomes in a patient series undergoing arthroscopic ACL reconstruction with periosteumenveloping hamstring tendon graft.30 These authors suggested that periosteal augmentation may limit tunnel enlargement and improves the healing response of the graft. However, these promising results need to be confirmed in further comparative trials.
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Structural Scaffold Figure 4 The basic principals of ligament tissue engineering. The structural scaffold needs to be cultured with cells. Biological stimulation, such as growth factor stimulation, and physical stimulation, such as cyclic stretching, is a crucial component.
ligament and tendon healing, either to augment ligaments and tendons or as ligament/tendon prostheses.38-42 Kato described a carbodiimide and a glutaraldehyde cross-linked collagen fiber prosthesis to bridge gaps in the Achilles tendon of rabbits.41 They identified different resorption properties for the carbodiimide and the glutaraldehyde cross-linked scaffolds and observed that the scaffold material gets repopulated with cells and transforms into a fibrous scar with similar, but not identical ligament and tendon characteristics. The long-term follow up from the Kato study revealed that the scaffolds were replaced by a fibrous scar that did not regenerate either a neotendon or ligament after 52 weeks. Dunn and coworkers reported on the use of acidcrosslinked collagen prostheses with a tensile strength of 60 Mpa.40 The collagen fibers of this prosthesis were between 20 and 60 nm, similar in size to human ACL fibers. The glutaraldehyde-treated prosthesis was tested in several animal models and showed biodegradation as well as the ability to increase the healing response over the untreated group. However, the overall biomechanical properties were unsatisfactory. Badylak and coworkers used a clinically approved tissue scaffold derived from the small intestine submucosa (SIS) of pigs.43,44 They applied SIS to a dog model for Achilles tendon repair and the overall strength of the construct was higher at 12 weeks than the suture-repaired control.44 Further testing in an ACL replacement model in goats indicated that the SIS undergoes a marked reduction in tensile strength at 3 months as opposed to the patellar tendon transplant where a gradual increase in strength occurs. After 12 months, the ultimate tensile strength was similar for both techniques.43
Cells for Ligament Tissue Engineering Scaffolds To meet the challenge of engineering a ligament, various bioresorbable scaffold materials have been investigated for
Cells are the integral nonstructural component of every tissue-engineered tissue. They are responsible for the remodeling and adaptation of the scaffold. There are 2 fundamentally
40 different populations of cells that can be used for ligament tissue engineering. One cell type is undifferentiated stem cells that can be differentiated into ligament fibroblasts or myofibroblasts. The other cell type is primary ligament fibroblasts that can be obtained directly from human ligament tissue. The undifferentiated mesenchymal stem cells (MSCs) have the potential to differentiate into differentiated cells of different tissues such as osteoblasts, chondroblasts, lipoblasts and also fibroblasts. MSCs can be obtained from the bone marrow, fat or even muscle. Once harvested, MSCs can be expanded in culture until they are being stimulated to differentiate. The relatively easy harvesting procedure (ie, bone marrow biopsy, muscle biopsy) and the ability to determine the line of differentiation by the means of controlled culture media makes mesenchymal stem cells an excellent tool for the use of tissue engineering. The disadvantage of MSCs, however, is that they need to differentiate into ligament fibroblasts. The conditions necessary for this differentiation are currently not known. Primary ligament fibroblasts can be obtained directly form harvested ligaments or ligament remnants. The harvested ligament tissue undergoes an enzymatic digestion process yielding primary ligament fibroblasts. These primary ligament fibroblasts are fully differentiated cells that have a limited ability to divide. The advantage of primary fibroblasts is that these cells may have a memory for the extracellular and environmental conditions inside a native ligament. However, the harvest of these ligament fibroblasts requires the harvest of ligament tissue followed by a time sensitive culture process until these cells become available for implantation. This poses a significant technical challenge to the use of primary fibrobroblasts that may be more readily applicable in an experimental than in a clinical situation.
Growth Factors As described previously, growth factors are proteins that can be used to stimulate cell proliferation, cell migration, and cell differentiation in musculoskeletal tissues. Growth factors with the specific ability to improve tendon healing and proliferation include basic fibroblast growth factor, growth differentiation factor 5 and 6 (GDF-5, GDF-6), insuline-like growth factor, PDGF, and transforming growth factor-.45-56 These factors play an important role in tissue engineering of tendons and ligaments. In particular, GDF-5 (also known as BMP-14 or cartilage-derived morphogenetic protein-1) has been shown to modify ligament healing. Wolfman injected GDF-5 into skeletal muscle and reported formation of a tendinous structure.55 Aspenberg added GDF-5 to a rabbit Achilles tendon defect and observed enhanced superior healing.45 Similar results could be reported with an adenoviral vector delivering the GDF-5 gene.47 GDF-5 is implicated in tenocyte lineage progression.55 GDF-5 may therefore be an efficient growth factor with the potential to accelerate ligament remodeling and increase the healing response. Therefore, we are currently testing the efficacy of GDF-5 in our biodynamic bioreactor system.
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Biomechanical Stimulation in Bioreactors Previous studies demonstrated that cyclic stretching stimulates the ligament development.38,39,42,57 Thus, much of the recent work on ligament engineering has focused on the design and the use of bioreactor systems in which the growing ligament can be exposed to cyclic stretching. A bioreactor provides a biochemical and biomechanical environment for tissue growth. A favorable bioreactor design for ligament tissue engineering may allow biochemical stimulation through growth factor delivery as well as physical stimulation through cyclic stretching (Fig. 5). Langelier and coworkers integrated biomechanical preconditioning of an engineered graft with integration of fibroblasts into a collagen scaffold.42 Using a static and cyclic stretching apparatus, they grew a bone–ACL– bone construct that incorporated human ACL fibroblasts. The construct was maintained in culture for 3 months. These authors reported that the crimp pattern of the collagen fibers and the cell density were comparable with the human ACL. Moreover, the integrated ACL fibroblasts produced extracellular matrix and collagen within the scaffold. However, the biomechanical properties of the tissue engineered ligament were not reported. Altman and coworkers further investigated the effect of physical graft stimulation in the bioreactor.39 They assessed bone marrow derived stem cells in a collagen matrix that was stretched dynamically and rotated inside a bioreactor. The effect of continuous stretching and torque on the orientation of the collagen fibers and the morphology of the seeded stem cells were assessed. A 10% strain rate and a 25% torque produced regular orientation of the fibroblasts and enhanced expression of type I and III collagen, compared with the nonfunctionally treated control group.39 These studies provided encouraging data and emphasized the importance of physical stimulation in bioreactor systems for tissue engineering of ligaments. The goal of our future tissue engineering research will be to engineer a tissue-engineered ligament from an acellular matrix scaffold augmented with human cells and growth factors in a bioreactor. Current efforts in our laboratory focus on the optimized design of a biodynamic bioreactor. The design of an optimized biodynamic bioreactor must consider various requirements, such as sterile containment of tissue culture, sufficient nutrient supply and oxygenation, dynamic stretching of the ligament, satisfactory visibility of the tissue culture, and easy access to tissue culture. Figure 5 shows the technical drawing of a currently used bioreactor in our laboratory. The ligament is attached to the base plate distally and to the actuator proximally through special clamps. Cyclic stretching of the graft is applied from a moving unit with a motor through the actuator. The bioreactor chamber is surrounding the ligament. Despite promising experimental data on ligament engineering, these techniques have not yet been translated into clinical practice. Future advances can be expected by the identification of the ideal composition of the culture medium, identification of the ideal levels of mechanical stimuli, identification of the optimal cell population and optimal
Tendon graft incorporation within the bone tunnel
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Figure 5 Principals of ligament tissue engineering using a biodynamic bioreactor. The structural scaffolds need to be cultured with cells. Continuous tissue culture fluid can be delivered to the bioreactor chamber. Physical stimulation by cyclic stretching of the tissue engineered ligament is crucial.
number of cells to be used, and new techniques to induce vascularization of the tissue engineered ligament.
References 1. Blickenstaff KR, Grana WA, Egle D: Analysis of a semitendinosus autograft in a rabbit model. Am J Sports Med 25:554-559, 1997 2. Grana WA, Egle DM, Mahnken R, et al: An analysis of autograft fixation after anterior cruciate ligament reconstruction in a rabbit model. Am J Sports Med 22:344-351, 1994 3. Rodeo SA, Arnoczky SP, Torzilli PA, et al: Tendon-healing in a bone tunnel. A biomechanical and histological study in the dog. J Bone Joint Surg Am 75:1795-1803, 1993 4. Shino K, Horibe S: Experimental ligament reconstruction by allogeneic tendon graft in a canine model. Acta Orthop Belg 57:44-53, 1991 (suppl 2) 5. Kawakami H, Shino K, Hamada M, et al: Graft healing in a bone tunnel: bone-attached graft with screw fixation versus bone-free graft with extra-articular suture fixation. Knee Surg Sports Traumatol Arthrosc 12:384-390, 2004 6. Weiler A, Hoffmann RF, Bail HJ, et al: Tendon healing in a bone tunnel. Part II: Histologic analysis after biodegradable interference fit fixation in a model of anterior cruciate ligament reconstruction in sheep. Arthroscopy 18:124-135, 2002 7. Weiler A, Peine R, Pashmineh-Azar A, et al: Tendon healing in a bone tunnel. Part I: Biomechanical results after biodegradable interference fit fixation in a model of anterior cruciate ligament reconstruction in sheep. Arthroscopy 18:113-123, 2002 8. Yoshiya S, Nagano M, Kurosaka M, et al: Graft healing in the bone tunnel in anterior cruciate ligament reconstruction. Clin Orthop 376: 278-286, 2000 9. Panni AS, Milano G, Lucania L, et al: Graft healing after anterior cruciate ligament reconstruction in rabbits. Clin Orthop 343:203-212, 1997
10. L’Insalata JC, Klatt B, Fu FH, et al: Tunnel expansion following anterior cruciate ligament reconstruction: A comparison of hamstring and patellar tendon autografts. Knee Surg Sports Traumatol Arthrosc 5:234238, 1997 11. Tomita F, Yasuda K, Mikami S, et al: Comparisons of intraosseous graft healing between the doubled flexor tendon graft and the bone-patellar tendon-bone graft in anterior cruciate ligament reconstruction. Arthroscopy 17:461-476, 2001 12. Goradia VK, Rochat MC, Grana WA, et al: Tendon-to-bone healing of a semitendinosus tendon autograft used for ACL reconstruction in a sheep model. Am J Knee Surg 13:143-151, 2000 13. Arnoczky SP: Biology of ACL reconstructions: What happens to the graft? Instr Course Lect 45:229-233, 1996 14. Grassman SR, McDonald DB, Thornton GM, et al: Early healing processes of free tendon grafts within bone tunnels is bone-specific: A morphological study in a rabbit model. Knee 9:21-26, 2002 15. Yamakado K, Kitaoka K, Yamada H, et al: The influence of mechanical stress on graft healing in a bone tunnel. Arthroscopy 18:82-90, 2002 16. Liu SH, Panossian V, al-Shaikh R, et al: Morphology and matrix composition during early tendon to bone healing. Clin Orthop 339:253260, 1997 17. Huard J, Li Y, Peng H, et al: Gene therapy and tissue engineering for sports medicine. J Gene Med 5:93-108, 2003 18. Evans C, Robbins PD: Possible orthopaedic applications of gene therapy. J Bone Joint Surg Am 77:1103-1114, 1995 19. Robbins PD, Ghivizzani S: Viral vectors for gene therapy. Pharmacol Ther 80:35-47, 1998 20. Lattermann C, Zelle BA, Whalen JD, et al: Gene transfer to the tendonbone insertion site. Knee Surg Sports Traumatol Arthrosc 12:510-515, 2004 21. Martinek V, Lattermann C, Usas A, et al: Enhancement of tendon-bone integration of anterior cruciate ligament grafts with bone morphoge-
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ntegration of anterior cruciate ligament grafts into bone tunnels is critical for the success of anterior cruciate ligament (ACL) reconstruction. Depending on the type of the graft, it may take up to several months until the tendon– bone interface is regenerated after ACL reconstruction.1-4 With soft-tissue grafts, fixation occurs predominantly with fibrous tissue with collagen fibers interspersed along the load axis.1-3,5-8 Connective tissue has been shown to surround the graft up to 12 months after implantation.1,2,9 Moreover, tunnel expansion, most likely caused by micro-motion and synovial fluid extravasation in the tunnel, remains a concern, in particular with soft-tissue grafts.10 Thus, it is of concern whether the early tendon– bone interface is strong enough to tolerate accelerated early postoperative rehabilitation.10 For this reason, most rehabilitation programs after ACL reconstruction require a protected mobilization of the operated
*Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA. †Growth and Development Laboratory, Children’s Hospital of Pittsburgh, Pittsburgh, PA. Address reprint requests to Boris A. Zelle, MD, University of Pittsburgh, Ferguson Laboratory for Orthopaedic Research, Presbyterian University Hospital C-313, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail: [email protected]
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1048-6666/05/$-see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1053/j.oto.2004.11.014
knee to allow for adequate graft incorporation. Therefore, a detailed understanding of the biological situation of the tendon graft within the bone tunnel appears important for knee surgeons. The specific aims of this article will be to review (1) the histology of tendon graft incorporation, (2) biological approaches to enhance the healing stimulation of the tendonto-bone insertion site, (3) the concepts of ligament bioengineering.
Histology of Graft Incorporation It is well accepted that the bone plug of bone–patella tendon– bone graft becomes anchored to the bone wall by appositional bone formation and that the grafted bone becomes necrotic as new bone forms.5,8 Several recent studies have demonstrated the time course of this bone-to-bone healing. Tomita and coworkers evaluated the histologic response using adult beagle dogs at 3, 6, and 12 weeks.11 At 3 weeks, one side of the graft was in contact with the bony wall, whereas the opposite side gap between the plug and the tunnel wall was filled with granulation tissue. The bone plug was necrotic as demonstrated by empty lacunae. No changes were noted in the fibrocartilage layers at the tendon– bone junction. By 6 weeks, on the side of bony contact, the newly formed bone
Tendon graft incorporation within the bone tunnel
Growth Factor Concentration
was evident and attached at the graft-tunnel interface, whereas on the opposite side, the granulation tissue remained. Sharpey’s fibers were present in the granulation tissue around the interosseous tendon portion of the graft. In addition, at the tendon bone interface, there was a decrease in the number and quantity of cartilaginous cells. By 12 weeks, newly formed bone obscured the tunnel graft interface at the side of bony contact. In the granulation tissue, collagen fibers are more densely packed. At the tendon– bone interface, no significant histological changes were present.11 These histologic observations were confirmed by Kawakami and coworkers in rabbits.5 It is generally accepted that by 16 to 20 weeks, complete bony incorporation of the graft into the tunnel is present.6,7 Soft-tissue (bone-free) grafts undergo a different healing process that was characterized by Rodeo and coworkers in mongrel dogs.3 At 2 weeks, the area between the tendon– bone interface comprises highly vascular and cellular fibrous tissue. The tissue is poorly organized, and no collagen continuity is present. At 4 weeks, a thin seam of new bone is present lining the tunnel. The tendon in the tunnel remains viable with normal fibroblasts. There is a more abundant extracellular matrix and the presence of collagen fiber continuity at the bone–fibrous tissue interface. By 8 weeks, the tendon– bone interface was more organized, with more collagen fibers spanning the junction pulling in the direction of tension from the graft to the tunnel. Remodeling of the trabecular bone around the tendon was also present. At 12 weeks, there was increased organization and maturation of the fibrous tissue, with continuity of collagen fibers aligned along the direction of the pull of the muscle tendon unit between the tunnel and the graft. By 26 weeks, maturation of continuity of collagen fibers between the graft and tunnel was near complete, and there was significant remodeling of the trabecular bone surrounding the tendon. This progression of healing and collagen formation is consistent with the morphology of Sharpey’s fiber development attaching soft tissue to bone, emphasizing the differences between BPTB and soft tissue graft incorporation.3 These finding and time course are confirmed by several animal studies.5,12,13 However, recent evidence suggests that tendon to bone healing is bone-specific. Grassman and coworkers demonstrated that incorporation and remodeling is more extensive and occurs at a faster rate in cancellous filled femoral bone when compared with the marrow dominated tibial bone.14 Tendon healing in a bone tunnel is influenced by mechanical stress. As demonstrated by Yamakado and coworkers in rabbits, tensile stress (at the anterior aspect of the bone tunnel) enhances and promotes the tendon-bone interface healing process.15 However, compressive stresses (at the posterior side of the graft) promote chondroid formation, and eventually woven bone, rather than Sharpey’s fibers. These stress influences on regions of tendon to bone healing needs to be further clarified. Liu and coworkers traced this healing with immunohistochemistry to localize the different types of collagen in rabbits.16 Initially, types I, II, and III are present at the bone–tendon interface. By 6 weeks, type III collagen fibers, representing Sharpey’s
37
Viral Injection
Growth Factor Injection (Protein)
Application
Time
Figure 1 Growth factor concentration in musculoskeletal tissue. After injection of the pure growth factor protein, the concentration in the tissue reaches a maximum and returns instantly to the baseline level. Gene therapy results in a continuous growth factor concentration in the target tissue over a longer time period.
fibers, spanned the interface. Although a definitive fibrocartilage region did not form, there was localization of type II collagen at the interface. As type II and III collagen decrease over time, Type I collagen increases as more woven bone develops.16
Augmentation Techniques Improvements in soft tissue graft healing to bone are critical to allow more aggressive and earlier rehabilitation and an earlier return to sports and activities. Thus, biological solutions to stimulate the graft incorporation within the bone tunnel appear to be a promising approach. Several recent studies in animals of various augmentation techniques show promise.
Growth Factor Stimulation Growth factors are proteins that have the ability to stimulate cell proliferation, cell migration, and cell differentiation. The efficacy of growth factor stimulation has been studied in various musculoskeletal tissues, such as muscle, cartilage, meniscus, ligament, tendon, and bone.17 The use of most growth factors, however, is limited by their short biological halflives.18,19 Therefore, gene transfer techniques frequently are used to test the efficacy of growth factor stimulation. Gene therapy is based on the modification of cellular genetic information. Therefore, the genes encoding for growth factors are transferred into the local cells using viral or nonviral vectors. Thus, the genetic information of the local cells is modified so that growth factors are continuously released, resulting in a continuous stimulation of the tissue (Fig. 1). Various studies have demonstrated the efficacy of growth factor stimulation for bone tissue and for tendon tissue. After ACL reconstruction, however, the tendon graft is incorporated within a bony tunnel (Fig. 2). This incorporation of tendon tissue within a bone tunnel represents a unique biological situation and few studies have evaluated the efficacy of growth factor stimula-
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Figure 2 Schematic drawing of a reconstructed ACL. The schematic drawing demonstrates the tendon graft within the bone tunnel of the distal femur. Postoperative graft incorporation occurs at the tendon– bone interface.
tion to enhance the healing response of the tendon graft within the bony tunnel. Using a rabbit hallucis longus and calcaneus process model (Fig. 3), Lattermann and coworkers were able to show that transfection of the tendon-to-bone insertion site with a marker gene is a feasible technique and results in a prolonged gene expression.20 Further experimental investigations in rabbits demonstrated that gene delivery of bone morphogenetic protein-2 (BMP-2) to the tendon-to-bone insertion site in the reconstructed ACL enhances the healing response in the bone tunnel and the ultimate load failure of the construct.21 Rodeo and coworkers reported on the use of recom-
binant human BMP-2 for tendon healing in a bone tunnel using an extraarticular dog model.22 These authors demonstrated histologic and biomechanical evidence of earlier graft incorporation in those grafts treated with BMP-2 when compared with normal controls. Weiler and coworkers investigated the efficacy of platelet-derived growth factor-BB (PDGF-BB) on graft incorporation using an ACL model in sheep.23 These authors used coated sutures as growth factor delivery system for the PDGF-BB and compared the outcomes with normal controls. The application of PDGF-BB resulted in higher biomechanical strength, higher vascular density, and a higher collagen fibril amount. These experimental data suggest that growth factor stimulation of the tendon-to-bone insertion site is a promising treatment opportunity with important clinical implications. Therefore, current research projects in our laboratories continue to focus on the biological stimulation of tendon healing in the bone tunnel, using a rabbit hallucis longus and calcaneus process model as described above.
Enveloping of Tendon Graft with Periosteum Numerous studies have demonstrated the ability of periosteum to induce new chondroid and bone formation.24-26 Periosteum comprises multipotent stem cells to differentiate to osteogenic and chondrogenic tissues. Thus, enveloping tendon grafts with periosteum appears to be an interesting approach for biologic augmentation of the tendon graft in the bone tunnel. Using the above described rabbit hallucis longus and calcaneus process model, Youn and coworkers were able to show that periosteal augmentation of a soft-tissue graft in the bone tunnel increases the ultimate load failure of
Figure 3 Schematic drawing of a rabbit model to investigate the tendon-to-bone healing.20 The hallucis longus tendon is detached from the Achilles tendon. A bone canal is drilled into the calcaneus and the hallucis longus tendon is pulled into the bone canal.
Tendon graft incorporation within the bone tunnel
Ligament Tissue Engineering The technical aspects of autograft and allograft ACL replacements have undergone improvements over the last 10 years and yield an overall success rate of about 90%.31-34 Despite the ability to adapt to the mechanical demands both autografts and allografts do not reach the structural identity of either the original tendon tissue or the native ACL. In addition, autografts are associated with donor site morbidity in up to 40% of all patients.35-37 On the other hand, allograft transplants have a risk of disease transmission and possible immune reactions. One possible way to improve this situation may be to engineer a neoligament under ex vivo conditions that can be preconditioned for the host environment. A preconditioning would mimic the biomechanical, biological and the cellular recipient environment before implantation and may be able to reduce or eliminate the typical process of tissue degradation and necrosis that current allografts and autografts undergo. Novel tissue engineering strategies attempt to achieve this ambitious goal and have recently been introduced on an experimental level. The concept of tissue engineering is based on the understanding that the engineering of a functional tissue requires the complex interaction of four basic principles. This basic paradigm includes: a structural scaffold, tissue specific cells, biological modulators such as growth factors and cytokines, and mechanical modulators (Fig. 4). Any engineered tissue requires a structural scaffold providing a basic architecture for the different cellular and extracellular components of the engineered tissue. Another key component is the presence of cells that are able to populate the scaffold and modify the scaffold according to the mechanical and biological conditions. To create the desired material properties of the engineered tissue the cells need to be manipulated such that they modify the extracellular environment within the scaffold accordingly. This manipulation can be done with intrinsic factors such as growth factors, cytokines and nutritional supplements. In addition, extrinsic, mechanical factors will be required to define the later environment to which the engineered tissue will have to adapt to.
Tissue Engineered Ligament (TEL)
Physical Stimuli
Biological Stimuli
TELL Cells
the tendon– bone interface at 6 weeks after surgery.27 Similar results were reported for periosteal soft-tissue graft augmentation with other extraarticular rabbit models.28,29 Interestingly, the periosteal enveloping technique also has been used successfully in clinical trials. Thus, Chen and coworkers reported satisfactory clinical outcomes in a patient series undergoing arthroscopic ACL reconstruction with periosteumenveloping hamstring tendon graft.30 These authors suggested that periosteal augmentation may limit tunnel enlargement and improves the healing response of the graft. However, these promising results need to be confirmed in further comparative trials.
39
Structural Scaffold Figure 4 The basic principals of ligament tissue engineering. The structural scaffold needs to be cultured with cells. Biological stimulation, such as growth factor stimulation, and physical stimulation, such as cyclic stretching, is a crucial component.
ligament and tendon healing, either to augment ligaments and tendons or as ligament/tendon prostheses.38-42 Kato described a carbodiimide and a glutaraldehyde cross-linked collagen fiber prosthesis to bridge gaps in the Achilles tendon of rabbits.41 They identified different resorption properties for the carbodiimide and the glutaraldehyde cross-linked scaffolds and observed that the scaffold material gets repopulated with cells and transforms into a fibrous scar with similar, but not identical ligament and tendon characteristics. The long-term follow up from the Kato study revealed that the scaffolds were replaced by a fibrous scar that did not regenerate either a neotendon or ligament after 52 weeks. Dunn and coworkers reported on the use of acidcrosslinked collagen prostheses with a tensile strength of 60 Mpa.40 The collagen fibers of this prosthesis were between 20 and 60 nm, similar in size to human ACL fibers. The glutaraldehyde-treated prosthesis was tested in several animal models and showed biodegradation as well as the ability to increase the healing response over the untreated group. However, the overall biomechanical properties were unsatisfactory. Badylak and coworkers used a clinically approved tissue scaffold derived from the small intestine submucosa (SIS) of pigs.43,44 They applied SIS to a dog model for Achilles tendon repair and the overall strength of the construct was higher at 12 weeks than the suture-repaired control.44 Further testing in an ACL replacement model in goats indicated that the SIS undergoes a marked reduction in tensile strength at 3 months as opposed to the patellar tendon transplant where a gradual increase in strength occurs. After 12 months, the ultimate tensile strength was similar for both techniques.43
Cells for Ligament Tissue Engineering Scaffolds To meet the challenge of engineering a ligament, various bioresorbable scaffold materials have been investigated for
Cells are the integral nonstructural component of every tissue-engineered tissue. They are responsible for the remodeling and adaptation of the scaffold. There are 2 fundamentally
40 different populations of cells that can be used for ligament tissue engineering. One cell type is undifferentiated stem cells that can be differentiated into ligament fibroblasts or myofibroblasts. The other cell type is primary ligament fibroblasts that can be obtained directly from human ligament tissue. The undifferentiated mesenchymal stem cells (MSCs) have the potential to differentiate into differentiated cells of different tissues such as osteoblasts, chondroblasts, lipoblasts and also fibroblasts. MSCs can be obtained from the bone marrow, fat or even muscle. Once harvested, MSCs can be expanded in culture until they are being stimulated to differentiate. The relatively easy harvesting procedure (ie, bone marrow biopsy, muscle biopsy) and the ability to determine the line of differentiation by the means of controlled culture media makes mesenchymal stem cells an excellent tool for the use of tissue engineering. The disadvantage of MSCs, however, is that they need to differentiate into ligament fibroblasts. The conditions necessary for this differentiation are currently not known. Primary ligament fibroblasts can be obtained directly form harvested ligaments or ligament remnants. The harvested ligament tissue undergoes an enzymatic digestion process yielding primary ligament fibroblasts. These primary ligament fibroblasts are fully differentiated cells that have a limited ability to divide. The advantage of primary fibroblasts is that these cells may have a memory for the extracellular and environmental conditions inside a native ligament. However, the harvest of these ligament fibroblasts requires the harvest of ligament tissue followed by a time sensitive culture process until these cells become available for implantation. This poses a significant technical challenge to the use of primary fibrobroblasts that may be more readily applicable in an experimental than in a clinical situation.
Growth Factors As described previously, growth factors are proteins that can be used to stimulate cell proliferation, cell migration, and cell differentiation in musculoskeletal tissues. Growth factors with the specific ability to improve tendon healing and proliferation include basic fibroblast growth factor, growth differentiation factor 5 and 6 (GDF-5, GDF-6), insuline-like growth factor, PDGF, and transforming growth factor-.45-56 These factors play an important role in tissue engineering of tendons and ligaments. In particular, GDF-5 (also known as BMP-14 or cartilage-derived morphogenetic protein-1) has been shown to modify ligament healing. Wolfman injected GDF-5 into skeletal muscle and reported formation of a tendinous structure.55 Aspenberg added GDF-5 to a rabbit Achilles tendon defect and observed enhanced superior healing.45 Similar results could be reported with an adenoviral vector delivering the GDF-5 gene.47 GDF-5 is implicated in tenocyte lineage progression.55 GDF-5 may therefore be an efficient growth factor with the potential to accelerate ligament remodeling and increase the healing response. Therefore, we are currently testing the efficacy of GDF-5 in our biodynamic bioreactor system.
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Biomechanical Stimulation in Bioreactors Previous studies demonstrated that cyclic stretching stimulates the ligament development.38,39,42,57 Thus, much of the recent work on ligament engineering has focused on the design and the use of bioreactor systems in which the growing ligament can be exposed to cyclic stretching. A bioreactor provides a biochemical and biomechanical environment for tissue growth. A favorable bioreactor design for ligament tissue engineering may allow biochemical stimulation through growth factor delivery as well as physical stimulation through cyclic stretching (Fig. 5). Langelier and coworkers integrated biomechanical preconditioning of an engineered graft with integration of fibroblasts into a collagen scaffold.42 Using a static and cyclic stretching apparatus, they grew a bone–ACL– bone construct that incorporated human ACL fibroblasts. The construct was maintained in culture for 3 months. These authors reported that the crimp pattern of the collagen fibers and the cell density were comparable with the human ACL. Moreover, the integrated ACL fibroblasts produced extracellular matrix and collagen within the scaffold. However, the biomechanical properties of the tissue engineered ligament were not reported. Altman and coworkers further investigated the effect of physical graft stimulation in the bioreactor.39 They assessed bone marrow derived stem cells in a collagen matrix that was stretched dynamically and rotated inside a bioreactor. The effect of continuous stretching and torque on the orientation of the collagen fibers and the morphology of the seeded stem cells were assessed. A 10% strain rate and a 25% torque produced regular orientation of the fibroblasts and enhanced expression of type I and III collagen, compared with the nonfunctionally treated control group.39 These studies provided encouraging data and emphasized the importance of physical stimulation in bioreactor systems for tissue engineering of ligaments. The goal of our future tissue engineering research will be to engineer a tissue-engineered ligament from an acellular matrix scaffold augmented with human cells and growth factors in a bioreactor. Current efforts in our laboratory focus on the optimized design of a biodynamic bioreactor. The design of an optimized biodynamic bioreactor must consider various requirements, such as sterile containment of tissue culture, sufficient nutrient supply and oxygenation, dynamic stretching of the ligament, satisfactory visibility of the tissue culture, and easy access to tissue culture. Figure 5 shows the technical drawing of a currently used bioreactor in our laboratory. The ligament is attached to the base plate distally and to the actuator proximally through special clamps. Cyclic stretching of the graft is applied from a moving unit with a motor through the actuator. The bioreactor chamber is surrounding the ligament. Despite promising experimental data on ligament engineering, these techniques have not yet been translated into clinical practice. Future advances can be expected by the identification of the ideal composition of the culture medium, identification of the ideal levels of mechanical stimuli, identification of the optimal cell population and optimal
Tendon graft incorporation within the bone tunnel
41
Figure 5 Principals of ligament tissue engineering using a biodynamic bioreactor. The structural scaffolds need to be cultured with cells. Continuous tissue culture fluid can be delivered to the bioreactor chamber. Physical stimulation by cyclic stretching of the tissue engineered ligament is crucial.
number of cells to be used, and new techniques to induce vascularization of the tissue engineered ligament.
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