Matthew B. Fisher

Role of biomechanics in the understanding of normal, injured, and healing ligaments and tendons

Ligaments and tendons are soft connective tissues which serve essential roles for biomechanical function of the musculoskeletal system by stabilizing and guiding the motion of diarthrodial joints. Nevertheless, these tissues are frequently injured due to repetition and overuse as well as quick cutting motions that involve acceleration and deceleration. These injuries often upset this balance between mobility and stability of the joint which causes damage to other soft tissues manifested as pain and other morbidity, such as osteoarthritis.The healing of ligament and tendon injuries varies from tissue to tissue. Tendinopathies are ubiquitous and can take up to 12 months for the pain to subside before one could return to normal activity. A ruptured medial collateral ligament (MCL) can generally heal spontaneously; however, its remodeling process takes years and its biomechanical properties remain inferior when compared to the normal MCL. It is also known that a midsubstance anterior cruciate ligament (ACL) tear has limited healing capability, and reconstruction by soft tissue grafts has been regularly performed to regain knee function. However, long term follow-up studies have revealed that 20–25% of patients experience unsatisfactory results. Thus, a better understanding of the function of ligaments and tendons, together with knowledge on their healing potential, may help investigators to develop novel strategies to accelerate and improve the healing process of ligaments and tendons.With thousands of new papers published in the last ten years that involve biomechanics of ligaments and tendons, there is an increasing appreciation of this subject area. Such attention has positively impacted clinical practice. On the other hand, biomechanical data are complex in nature, and there is a danger of misinterpreting them. Thus, in these review, we will provide the readers with a brief overview of ligaments and tendons and refer them to appropriate methodologies used to obtain their biomechanical properties. Specifically, we hope the reader will pay attention to how the properties of these tissues can be altered due to various experimental and biologic factors. Following this background material, we will present how biomechanics can be applied to gain an understanding of the mechanisms as well as clinical management of various ligament and tendon ailments. To conclude, new technology, including imaging and robotics as well as functional tissue engineering, that could form novel treatment strategies to enhance healing of ligament and tendon are presented.

The Effects of Multiple Freeze–Thaw Cycles on the Biomechanical Properties of the Human Bone-Patellar Tendon-Bone Allograft

Soft tissue allografts, such as the bone‐patellar tendon‐bone (BPTB) graft, have been frequently used for anterior cruciate ligament (ACL) reconstruction. As allografts are subjected to freezing and thawing for multiple cycles, the objective of this study was to measure the changes of the biomechanical properties of the human BPTB allograft after 4 and 8 freeze–thaw cycles in comparison to a single freeze–thaw cycle. Three BPTB specimens were procured from 21 human donors and divided into three groups: 1, 4, or 8 freeze–thaw cycles. Each freeze–thaw cycle consisted of freezing at −20 ± 10°C for more than 6 h and thawing at 22 ± 3°C for at least 6 h. Tensile testing of the BPTB specimens consisted of loading between 50 N and 250 N for 100 cycles and then loading to failure. Cyclic loading revealed a similar amount of creep (∼0.5 mm) among the three freeze–thaw cycles groups (p = 0.38). The stiffness of the BPTB graft for the 1, 4, and 8 freeze–thaw cycle groups were 244 ± 42 N/mm, 235 ± 39 N/mm, and 231 ± 40 N/mm, respectively (p = 0.43). Similar findings were obtained for the ultimate load of the BPTB graft (p = 0.14) and the tangent modulus of the PT substance (p = 0.41). The results of this study suggest that there would be little measurable effect on the structural properties of the BPTB graft or mechanical properties of the PT tissue substance following 8 freeze–thaw cycles. These BPTB allografts could potentially be re‐frozen without a loss in their biomechanical properties, given appropriate storage and care.

Functional properties of bone marrow-derived MSC-based engineered cartilage are unstable with very long-term in vitro culture

The success of stem cell-based cartilage repair requires that the regenerate tissue reach a stable state. To investigate the long-term stability of tissue engineered cartilage constructs, we assessed the development of compressive mechanical properties of chondrocyte and mesenchymal stem cell (MSC)-laden three dimensional agarose constructs cultured in a well defined chondrogenic in vitro environment through 112 days. Consistent with previous reports, in the presence of TGF-β, chondrocytes outperformed MSCs through day 56, under both free swelling and dynamic culture conditions, with MSC-laden constructs reaching a plateau in mechanical properties between days 28 and 56. Extending cultures through day 112 revealed that MSCs did not simply experience a lag in chondrogenesis, but rather that construct mechanical properties never matched those of chondrocyte-laden constructs. After 56 days, MSC-laden constructs underwent a marked reversal in their growth trajectory, with significant declines in glycosaminoglycan content and mechanical properties. Quantification of viability showed marked differences in cell health between chondrocytes and MSCs throughout the culture period, with MSC-laden construct cell viability falling to very low levels at these extended time points. These results were not dependent on the material environment, as similar findings were observed in a photocrosslinkable hyaluronic acid (HA) hydrogel system that is highly supportive of MSC chondrogenesis. These data suggest that, even within a controlled in vitro environment that is conducive to chondrogenesis, there may be an innate instability in the MSC phenotype that is independent of scaffold composition, and may ultimately limit their application in functional cartilage repair.

Evaluation of Knee Stability with Use of a Robotic System

In our research center, we have developed and utilized a novel robotic/universal force-moment sensor testing system to gain quantitative data on multiple-degree-of-freedom kinematics of the knee simultaneously with data on the in situ forces in normal and repaired soft tissues. In particular, we have investigated the complex interaction of the anteromedial and posterolateral bundles of the anterior cruciate ligament as well as several key biomechanical variables in anterior cruciate ligament reconstruction, such as graft selection and femoral tunnel placement (both of which impact knee stability). For example, both the bone-patellar tendon-bone and quadrupled hamstrings tendon autografts restored anterior stability but were insufficient in gaining rotatory stability. In a follow-up study, we have shown that a more laterally placed graft was beneficial and could improve these outcomes. Such findings led to additional investigation in which the biomechanical advantages of double-bundle anterior cruciate ligament reconstruction were demonstrated. However, a more laterally placed autograft at the femoral insertion of the posterolateral bundle also worked well, especially when the knee was nearly at full extension (a position in which the anterior cruciate ligament is needed most). At present, we are moving forward by obtaining in vivo kinematics data and then repeating those kinematics exactly to obtain new data with use of the robotic/universal force-moment sensor testing system in order to gain further insight regarding the function of the anterior cruciate ligament and anterior cruciate ligament replacement grafts in vivo. In parallel, we are developing a mathematical model of the knee and validating the computational model with experimental data. The combined approach will yield new and relevant information, including the stress and strain distribution in the anterior cruciate ligament and anterior cruciate ligament grafts. This will facilitate a better understanding of the function of the anterior cruciate ligament and a scientifically based design of surgical procedures and postoperative rehabilitation protocols that will lead to better patient outcomes.

Engineering meniscus structure and function via multi-layered mesenchymal stem cell-seeded nanofibrous scaffolds

Despite advances in tissue engineering for the knee meniscus, it remains a challenge to match the complex macroscopic and microscopic structural features of native tissue, including the circumferentially and radially aligned collagen bundles essential for mechanical function. To mimic this structural hierarchy, this study developed multi-lamellar mesenchymal stem cell (MSC)-seeded nanofibrous constructs. Bovine MSCs were seeded onto nanofibrous scaffolds comprised of poly(ε-caprolactone) with fibers aligned in a single direction (0° or 90° to the scaffold long axis) or circumferentially aligned (C). Multi-layer groups (0°/0°/0°, 90°/90°/90°, 0°/90°/0°, 90°/0°/90°, and C/C/C) were created and cultured for a total of 6 weeks under conditions favoring fibrocartilaginous tissue formation. Tensile testing showed that 0° and C single layer constructs had stiffness values several fold higher than 90° constructs. For multi-layer groups, the stiffness of 0°/0°/0° constructs was higher than all other groups, while 90°/90°/90° constructs had the lowest values. Data for collagen content showed a general positive interactive effect for multi-layers relative to single layer constructs, while a positive interaction for stiffness was found only for the C/C/C group. Collagen content and cell infiltration occurred independent of scaffold alignment, and newly formed collagenous matrix followed the scaffold fiber direction. Structural hierarchies within multi-lamellar constructs dictated biomechanical properties, and only the C/C/C constructs with non-orthogonal alignment within layers featured positive mechanical reinforcement as a consequence of the layered construction. These multi-layer constructs may serve as functional substitutes for the meniscus as well as test beds to understand the complex mechanical principles that enable meniscus function.

Biomaterial-mediated delivery of degradative enzymes to improve meniscus integration and repair.

Endogenous repair of fibrous connective tissues is limited, and there exist few successful strategies to improve healing after injury. As such, new methods that advance repair by promoting cell growth, extracellular matrix (ECM) production, and tissue integration would represent a marked clinical advance. Using the meniscus as a test platform, we sought to develop an enzyme-releasing scaffold that enhances integrative repair. We hypothesized that the high ECM density and low cellularity of native tissue present physical and biological barriers to endogenous healing, and that localized collagenase treatment might expedite cell migration to the wound edge and tissue remodeling. To test this hypothesis, we fabricated a delivery system in which collagenase was stored inside electrospun poly(ethylene oxide) (PEO) nanofibers and released upon hydration. In vitro results showed that partial digestion of the wound interface improved repair by creating a microenvironment that facilitated cell migration, proliferation and matrix deposition. Specifically, treatment with high-dose collagenase led to a 2-fold increase in cell density at the wound margin and a 2-fold increase in integrative tissue compared to untreated controls at 4 weeks (P≤0.05). Furthermore, when composite scaffolds containing both collagenase-releasing and structural fiber fractions were placed inside meniscal tears in vitro, enzyme release acted locally and resulted in a positive cellular response similar to that of global treatment with aqueous collagenase. This innovative approach to targeted enzyme delivery may aid the many patients that exhibit meniscal tears by promoting integration of the defect, thereby circumventing the pathologic consequences of partial meniscus removal, and may find widespread application in the treatment of injuries to a variety of dense connective tissues.

Effects of Mesenchymal Stem Cell and Growth Factor Delivery on Cartilage Repair in a Mini-Pig Model

Objective We have recently shown that mesenchymal stem cells (MSCs) embedded in a hyaluronic acid (HA) hydrogel and exposed to chondrogenic factors (transforming growth factor–β3 [TGF-β3]) produce a cartilage-like tissue in vitro. The current objective was to determine if these same factors could be combined immediately prior to implantation to induce a superior healing response in vivo relative to the hydrogel alone. Design Trochlear chondral defects were created in Yucatan mini-pigs (6 months old). Treatment groups included an HA hydrogel alone and hydrogels containing allogeneic MSCs, TGF-β3, or both. Six weeks after surgery, micro-computed tomography was used to quantitatively assess defect fill and subchondral bone remodeling. The quality of cartilage repair was assessed using the ICRS-II histological scoring system and immunohistochemistry for type II collagen. Results Treatment with TGF-β3 led to a marked increase in positive staining for collagen type II within defects (P < 0.05), while delivery of MSCs did not (P > 0.05). Neither condition had an impact on other histological semiquantitative scores (P > 0.05), and inclusion of MSCs led to significantly less defect fill (P < 0.05). For all measurements, no synergistic interaction was found between TGF-β3 and MSC treatment when they were delivered together (P > 0.05). Conclusions At this early healing time point, treatment with TGF-β3 promoted the formation of collagen type II within the defect, while allogeneic MSCs had little benefit. Combination of TGF-β3 and MSCs at the time of surgery did not produce a synergistic effect. An in vitro precultured construct made of these components may be required to enhance in vivo repair in this model system.

Evaluation of bone tunnel placement for suture augmentation of an injured anterior cruciate ligament: effects on joint stability in a goat model.

Use of novel tissue engineering approaches to heal an injured anterior cruciate ligament (ACL) requires suture repair and/or augmentation to provide joint stability. We evaluated the effects of the location of suture augmentation at the femur and tibia in terms of joint stability using a goat model. Eight goat stifle joints were tested with augmentation sutures placed in two femoral tunnel locations: (1) anterior to, or (2) through the ACL footprint, and two tibial tunnel locations: (1) medial to, or (2) medial and lateral to the footprint. Using a robotic/universal force‐moment sensor testing system, the anterior tibial translation (ATT) and the corresponding in situ force carried by the sutures were obtained at 30°, 60°, and 90° of flexion in response to external loads. No significant differences were found between augmentation groups due to tunnel location in terms of ATT or the in situ forces carried by the sutures at all flexion angles tested. Similar results were found under 5 N m of varus–valgus torque. Under a 67 N anterior tibial load, the ATT was restored to within 3 mm of the intact joint following suture augmentation (p > 0.05). Suture augmentation, when placed close to the ACL insertion, could be helpful in providing initial joint stability to aid ACL healing in the goat model. Published by Wiley Periodicals, Inc. J Orthop Res 28:1373–1379, 2010

Functional Tissue Engineering of Ligament and Tendon Injuries

This chapter reviews the properties of normal and healing ligaments and tendons and discusses the current functional tissue engineering (FTE) methods, which include the use of growth factors, gene delivery, stem cell therapy, and the use of scaffolding as well as external mechanical stimuli, aimed at enhancing tendon and ligament healing. The major function of ligaments and tendons include maintaining the proper anatomical alignment of the skeleton and guiding joint motions. They accomplish this by transmitting forces along their longitudinal axis but their biomechanical properties are measured in uniaxial tension. They demonstrate nonlinear behavior, which is governed by the recruitment of collagen. This allows ligaments to maintain normal joint laxity in response to low loads and also to stiffen dramatically in response to high loads, preventing excessive joint displacements. The events of healing of ligaments and tendons are divided into four overlapping phases: hemorrhage, inflammation, repair (proliferation), and remodeling. Following injury, the hemorrhagic and inflammatory phases occur over the first several days. Minutes after the ligament injury, blood collects and forms a platelet-rich fibrin clot at the injury site. The hemorrhage phase of the injury forms a lattice for many following cellular events. FTE has generated many significant developments; for example, there is a class of biodegradable metallic scaffolds, namely porous magnesium or magnesium oxide, that have the advantage of initial stiffness to provide the needed stability for the ligament to heal while performing its function. The degradation rate of these “smart” scaffolds could also be controlled as they are replaced by the neotissue.

Suture augmentation following ACL injury to restore the function of the ACL, MCL, and medial meniscus in the goat stifle joint

Functional tissue engineering (FTE) approaches have shown promise in healing an injured anterior cruciate ligament (ACL) of the knee. Nevertheless, additional mechanical augmentation is needed to maintain joint stability and appropriate loading of the joint while the ACL heals. The objective of this study was to quantitatively evaluate how mechanical augmentation using sutures restores the joint kinematics as well as the distribution of loading among the ACL, medial collateral ligament, and medial meniscus (MM) in response to externally applied loads. Eight goat stifle joints were tested on a robotic/universal force-moment sensor testing system under two loading conditions: (1) a 67N anterior tibial load (ATL) and (2) a 67N ATL with 100N axial compression. For each joint, four experimental conditions were tested at 30°, 60°, and 90° of flexion: the (1) intact and (2) ACL-deficient joint, as well as following (3) suture repair of the transected ACL, and (4) augmentation using sutures passed from the femur to the tibia. Under the 67N ATL, suture augmentation could restore the anterior tibial translation (ATT) to within 3mm of the intact joint (p>0.05), representing a 54-76% improvement over suture repair (p<0.05). With the additional axial compression, the ATT and in-situ forces of the sutures following suture augmentation remained 2-3 times closer to normal (p<0.05). Also, the in-situ forces in the MM were 58-73% lower (p<0.05). Thus, suture augmentation may be helpful in combination with FTE approaches for ACL healing by providing the needed initial joint stability while lowering the loads on the MM.