Clinical Review

Contribution of biomechanics, orthopaedics and rehabilitation: The past, present and future

S. L-Y. Woo, M. Thomas, S.S. Chan Saw
Musculoskeletal Research Centre, Department of Bioengineering, University of Pittsburgh, Pittsburgh PA, USA

Correspondence to: S. L-Y. Woo, Muscoskeletal Research Centre, Department of Bioengineering Surgery, University of Pittsburgh, E1641 Biomedical Science Tower, 210 Lothrop Street, PO Box 71199, Pittsburgh PA 15213

Introduction Knee ligament Functional tissue engineering

Robotic technology Future directions Acknowledgements References

Biomechanics is a field that has a very long history. From its beginnings in ancient Chinese and Greek literature, the field of orthopaedic biomechanics has grown in the areas of biomechanics of bone, articular cartilage, soft tissues, upper extremities, spine and so on. Bioengineers in collaboration with orthopaedic surgeons have applied biomechanical principles to study clinically relevant problems, improving patient treatment and outcome. In the past 30 years, my colleagues and I have focused our research on the biomechanics of musculoskeletal soft tissues, ligaments and tendons in particular. Therefore, in this review article, the function of the knee ligaments and the associated homeostatic responses secondary to immobilisation and exercise will be described. Research on healing of the medial collateral ligament (MCL) of the knee and possible future approaches in improving the healing of the knee ligaments will be presented. Finally, improvement of the understanding of ligament reconstruction, specifically of the anterior cruciate ligament (ACL), through the use of robotics technology will be included. Throughout the manuscript, specific scientific findings that have guided or changed the clinical management of injury to these soft tissues will be emphasised

INTRODUCTION
Biomechanics is a field that has a very long history. It was described in ancient Chinese and Greek literature as early as 400-500 BC. The foundation of biomechanics, however, was laid during a period between the 1500s and 1700s by renowned contributors, such as da Vinci, Galileo, Borelli, Hooke and Newton.¹ During the 1940s and 1950s, pioneering work of musculoskeletal biomechanics was performed by legends such as Eadweard Muybridge, Arthur Steindler, Verne T. Inman, Henry R. Lissner and A.H. Hirsch. Then, in the 1960s, Al Burstein and colleagues began to teach biomechanical principles to orthopaedic surgeons.² Since then, the field of orthopaedic biomechanics has blossomed and significant works have been published in the areas of biomechanics of bone, articular cartilage, soft tissues, upper extremities, spine, and so on. More sophisticated equipment and analyses have become available to perform better experiments that yielded much better understanding of joint kinematics and tissue function during walking, running and other activities of daily living. Mathematical modelling and improved engineering design of orthopaedic implants have also taken great strides. Bioengineers in collaboration with orthopaedic surgeons have applied biomechanical principles to study clinically relevant problems, improving patient treatment and outcome.

In the past 30 years, my colleagues and I have focused our research on the biomechanics of musculoskeletal soft tissues, ligaments and tendons, in particular. Therefore, in this review article, the function of knee ligaments and the associated homeostatic responses secondary to immobilisation and exercise will be described. Research on healing of the medial collateral ligament (MCL) of the knee will be discussed. In addition, possible future approaches to improving the healing of knee ligaments will be presented. The improvement of the understanding of ligament reconstruction, specifically of the anterior cruciate ligament (ACL), through the use of robotics technology will also be included. Throughout the manuscript, specific scientific findings that have guided or changed the clinical management of injury to these soft tissues will be emphasised.

KNEE LIGAMENT FUNCTION

Anatomy of ACL and MCL
Ligaments are white, shiny, band-like structures that have a highly organised extracellular matrix (ECM) of densely packed collagen fibre bundles that are nearly parallel with the long axis of the ligament. These fibre bundles are composed primarily of Type I collagen. Ligaments are also relatively hypocellular, with fibroblasts interspersed throughout the tissue matrix. Inspection under polarised light microscopy reveals that the fibrils within the bundles exhibit a microstructural form with a sinusoidal wave or crimp pattern.

Insertions of ligaments into the bone are formed through transitional zones in order to minimise stress concentrations. These insertions can be direct or indirect. For direct insertions, there are four distinct zones of transition: ligament, uncalcified fibrocartilage, calcified fibrocartilage and bone (Figure 1a). For indirect insertions, the surface of the ligament connects with the periosteum whereas the deeper layers connect to bone via Sharpey’s fibres (Figure 1b). The MCL of the knee exhibits both types of insertions. Its femoral insertion is direct, whereas the tibial insertion is indirect. In the ACL, however, both the femoral and tibial insertions are direct.³

Figure 1a: Direct ligament insertion

Figure 1b: Indirect ligament insertion

Tissue Homeostasis
Effect of Immobilisation: In the past, immobilisation by plaster cast had often been used as a treatment for musculoskeletal injuries to protect the injured tissue from excessive forces during the early healing period. However, an undesirable consequence of this type of treatment is the change in the structural properties of the ligaments, following immobilisation, that include atrophy and joint stiffness. When a rabbit knee was subjected to nine weeks of immobilisation, the structural properties (stiffness and ultimate load) of the femur-MCL-tibia complex (FMTC) was reduced to one-third that of the control knee.^4,5 This decrease was mainly caused by changes occurring within the insertion sites with some reduction of ligament substance itself (Figure 2).

Figure 2: Effect of immobilisation on structural properties of rabbit FMTC. Adapted from Woo, S.L-Y: Mechanical Properties of Tendons and Ligaments - I. Quasi-static and Nonlinear Viscoelastic Properties. Biorheology, 19:385-396, 1982. Reprinted by permission.

The rate at which the structural properties of the FMTC return to normal following remobilisation is much slower. It took 52 weeks of remobilisation (after only nine weeks of immobilisation) for the tibial insertion site to be re-established histologically. The properties of the MCL substance appeared to recover much quicker, meaning an asynchronous rate of recovery between the MCL tissue and the insertion sites. Thus, one must be careful as the function of the FMTC would continue to be abnormal because of the weakness of the insertion sites.⁶

Effects of Exercise: It has been the belief that exercise can significantly enhance ligament properties.^7-9 However, a longterm study of the effect of exercise on swine FMTC revealed that ultimate tensile strength increased by only 20% after 12 months of running, with minimal changes in the structural properties of the FMTC; significant differences existed only if the maximum separation force was normalised by the animal’s body weight.¹⁰ Further, altering the in situ stress levels experienced by a ligament, elicited some changes in its biomechanical responses.

Homeostatic Response of Ligaments: Therefore, the homeostatic response for ligaments in response to stress and motion must be highly nonlinear (Figure 3). The gains from exercise and increased stress would be moderate, whereas, the penalties from immobilisation and the lack of stress could be enormous. As a result of these studies, plaster casting to immobilise joints following ligamentous injuries has become uncommon. The recommended clinical treatment has been changed to splinting with controlled mobilisation.^{4, 11-15}

Injury and Repair of Ligaments
Incidence of ligament injury: For young and active individuals participating in various sports activities, the ACL and the MCL of the knee are especially susceptible to injury. The frequency of injuries to these ligaments account for as much as 90% of all injuries to the knee ligaments.¹⁶ It is estimated that 150,000-200,000 new ACL injuries and 140,000 new combined ACL and MCL injuries occur in the United States of America annually.^16,17

Further, it has been shown by animal studies that a ruptured MCL can heal spontaneously without surgical treatment and the healed ligament can function quite well. The structural properties of the FMTC are sufficiently recovered within weeks. However, the healed tissue has poor mechanical properties and abnormal biochemistry and ultrastructure.^6,18-23 The knee can function to the level near to those of the sham control because of the large increases in cross-sectional area of the healed MCL.²⁴ Because of the inferior mechanical properties of the tissue, there is a major effort being made to enhance and accelerate MCL healing with various therapeutic strategies.^25-30

On the other hand, most ACL tears do not heal and require surgical reconstruction.^31-34 The results of ACL reconstructions are generally successful and most patients can resume normal activities and return to participating in sports.^35-39 However, both short- and long-term clinical studies have shown that 15-25% of patients have had less than satisfactory results with regards to their ACL reconstruction.^40-46 Therefore, an increased understanding of the complex function of the ACL and knowledge about the function of its replacement are needed before improvement in the clinical outcome may be expected.

MCL Healing: Because the MCL is a frequently torn extra-articular ligament with a great propensity to heal, it has served as an excellent model in which to study the process of ligament healing.^16,47-49 A clinically relevant rabbit model where the MCL of its knee is subjected to a 'mop-end' tear with concomitant damage to the ligament insertion sites at the femur and tibia (confirmed by histology) was developed in our laboratory.¹⁹ Ten days later, a typical vascular inflammatory response was apparent near the MCL insertions and the injured site in the ligament midsubstance had already been united. At six weeks, the healing midsubstance still had an elevated number of plump fibroblasts; the collagen fibre alignment was also irregular. By 12 weeks, the number of fibroblasts had decreased markedly, and there was some improvement in collagen and cell organisation. At the tibial insertion site, the sub-periosteal bone had remodeled due to osteoclastic activity.

Figure 3: Stress and motion dependent homeostatic response of ligaments. Adapted from Woo, S.L-Y: Mechanical Properties of Tendons and Ligaments - I. Quasi-static and Nonlinear Viscoelastic Properties. Biorheology, 19:385-396, 1982. Reprinted by permission.

Figure 4: Structural properties of FMTC in response to mop-end injury. ("Adapted from Woo. S.L-Y.: Die Heilung des Medialen Seitenbands. Sportverletzung Sportschaden, 7:3-16, 1993. Reprinted by permission").

In uniaxial tensile testing six weeks after injury, all experimental FMTCs failed by tibial avulsion, thereby, indicating that the healed midsubstance was stronger than the insertion sites. By 12 weeks, the insertion sites gained strength and half of the tensile failures actually occurred in the MCL midsubstance. At 52 weeks, reossification was evident and the tibial insertion site was well established. Secondary to tensile testing, all failures occurred in the mid-substance of the ligament, reflecting that the structure of the insertion sites had returned to near normal (Figure 4). Based on the failure modes of the FMTC, as well as histological and biomechanical data, it can be stated that the reparative process between the ligament insertions (slower) and the ligament substance (faster) is asynchronous.^3,19

Animal studies were also done to determine if surgical repair of a torn MCL was necessary.^3,19 In half of the animals (the repair group), the torn MCL was repaired with a horizontal mattress suture. In the remaining half of the animals (the non-repair group), the torn ends of the MCL were manually approximated but not repaired. At 10 days, there were observable differences histomorphologically between the repaired and non-repaired MCLs, as there was proliferation of inflammatory cells around the suture material in the MCL substance in the repaired MCLs. By 12 weeks, all MCLs were well healed, whether repaired or not. Also, by that time, the varus-valgus rotation of the knee did not differ statistically. There were also no significant differences in the structural properties of the FMTC or the mechanical properties of the MCL substance between the repair and nonrepair groups. The trend maintained up to 52 weeks. However, the mechanical properties (a measure of tissue quality) of the healing MCL were less than one half that of those for both groups and did not change between 12 and 52 weeks. In addition, the crosssectional area of the healing MCL was 2.5% greater at 52 weeks than the sham control group.²⁴

In more severe knee injuries that involve rupture to both the ACL and MCL, the MCL healing can still take place but will be significantly slower. The absence of the ACL puts additional stress on the healing MCL as it contributes to the restraint of valgus rotations and results in poorer results.^50,51 Studies showed that ACL reconstructions can have a beneficial effect on the healing MCL by reducing in situ forces in the ligament.^52,53 Suture repair of the MCL produced little or no improvements after 12 weeks, when compared to the non-surgically treated MCL.⁵⁴ The treatment of a combined ACL and MCL injury still remains a subject of some debate, but the results of our studies lend support to ACL reconstruction and non-surgical management of the MCL. This method of treatment is the common clinical procedure in practice today. Thus, laboratory results have helped to shift the clinical paradigm.^55-57

In summary, a ruptured MCL can heal spontaneously without surgical intervention. However, while functionally adequate, the healing tissue is abnormal and may remain so for periods of up to two years. In addition, surgical repair appears to have no functional advantage and the quality of the healing MCL is dependent on the extent of injury to other tissues of the knee such as the ACL. As a result of these studies, nonsurgical treatment of Grade 3 MCL injury has become the clinical treatment of choice over surgical repair. In addition, splinting devices used to control excess motion of the knee in order to preserve the function of other tissues, are the methods of choice. Patients have had an earlier return to function and sports activity.

FUNCTIONAL TISSUE ENGINEERING: IMPROVEMENT OF LIGAMENT HEALING
Studies on the healing of ligaments have consistently found the ligament to have a larger quantity of healed tissue mass that has inferior mechanical properties as well as abnormal biochemical constituents. Therefore, new modalities are sought in the hope of improving the properties of healing tissues. Recent advances in the fields of functional tissue engineering (FTE) offer new possibilities that could potentially lead to the modulation of cellular and biochemical mediators in order to improve the quality of the newly formed tissue.

Growth Factors
One obvious approach is the use of growth factors to enhance ligament healing. In vitro studies using rabbit fibroblasts showed that epidermal growth factor (EGF) and platelet derived growth factor-BB (PDGF-BB) significantly increased proliferation of MCL and ACL fibroblasts.^58-62 Meanwhile, transforming growth factor-ß₁ (TGF-ß1) significantly increased matrix synthesis.^63-65 Others have shown that TGF-ß1 can increase proliferation in sheep ACL fibroblasts.⁶⁶ However, when these growth factors were applied in vivo, there were numerous associated difficulties, as well as surprising findings. The addition of TGF-ß₁ failed to lead to any increase in the structural properties of the FMTC, while PDGF-BB only did so at higher dosage levels.²⁵ Changes in the dosage and method of delivery of TGF-ß₁ also did not lead to any improvement in the healing of the injured MCL.⁶⁷ In addition, the responses of healing MCL fibroblasts to growth factors were found to be different than for normal fibroblasts.⁶⁸ As the phenotype of the fibroblasts changed during the healing process with concomitant changes in the milieu of the healing tissue, the application of which growth factor, as well as what time and how much to be applied, became a significan challenge for this approach.²⁵ Much more work will be necessary in order to define the roles growth factors will play in a healing ligament.

Gene Transfer/Gene Therapy: Type III and Type V Collagen
Gene transfer technology is another method used to deliver specific genes and growth factors at an appropriate time to help with the healing of a ligment. The LacZ marker gene has been successfully transferred retrovirally and adenovirally in the normal and injured MCL and normal ACL of rabbits.⁶⁹

Retroviral gene expression was detected between 10 days and three weeks in the ACL of both normal and injured rabbits. Adenoviral gene expression was detected between three and six weeks in the normal and injured MCL and at least six weeks in the ACL. However, gene transfer technology is not without difficulties as the retrovirus can only transfer to dividing cells and the in vivo transduction efficiency is very low. On the other hand, adenoviral gene therapy has known clinical risks.⁷⁰ Further, there are additional factors, such as cytotoxicity and immunological responses that are basically unknown.

Antisense mediated gene inhibition of proteins that are elevated during healing has been offered as an alternative approach to enhance properties of the healing ligament. Antisense oligonucleotides (AS-ODNs) are synthetic oligonucleotides complementary to specific mRNA sequences and are theoretically able to inhibit expression of specific genes by binding to their target mRNA. Introduction of ASdecorin-ODNs to the healing MCL via the haemagglutinating virus of Japan (HVJ)-liposome vector complexes led to the formation of larger diameter collagen fibrils and an increase in tensile strength at six weeks.²⁸ In our research centre, studies have shown that in a healing MCL, Type III collagen is elevated early and requires one year to return to normal after an MCL injury while Type V collagen remains elevated even after one year.^20,22 Therefore, it was hypothesised that lowering the ratio of Type III/I or V/I collagen using antisense gene therapy may minimise the formation of poor tissue. Specifically, the increases in collagen fibril diameters in the healing tissue should enhance the biomechanical properties of the healing MCL. AS-collagen Types III and V-ODNs were transfected into human patellar tendon fibroblasts (HPTFs) in vitro using lipofectamine, a non-viral lipid vector. The results showed that AS-ODNs were effective in inhibiting collagen Type III and Type V synthesis and mRNA levels with respect to sense and missense controls, suggesting that antisense gene therapy may be a promising approach.^26,71

Mesenchymal Stem Cells
Mesenchymal stem cells (MSC) offer another potential to improve ligament and tendon healing. Preliminary studies have shown that MSCs were present up to 28 days after transplantation.⁷² In addition, when mediated with MSCs, repaired tendons demonstrated significant increases in ultimate stress, modulus and strain energy density when transplanted in a collagen matrix.⁷³

Biological Scaffolds: Small Intestinal Submucosa (SIS)
Biological scaffolds such as small intestinal submucosa (SIS) are being explored to determine their efficacy in enhancing the tensile properties of the healing MCL. Small intestinal submucosa is harvested from the porcine intestine. It consists of an organised Type I collagen matrix with preferred alignment and contains active growth factors which both contribute to its ability to promote tissue healing.⁷⁴ In a study using a rabbit MCL model, a 6 mm wide MCL gap injury was created after which SIS was secured onto the gap with nonresorbable sutures. It was found that the modulus and tensile strength of the healing MCL with SIS were nearly double that of those without the use of SIS after 12 weeks.²⁷ Ongoing studies include examining the fibre organisation and crimp pattern as well as the fibril diameters and distribution of the healing ligament. Longer term experiments will examine whether or not the mechanical properties will continue to be superior to the non-SIS controls over time.

Mechanical Factors
Finally, mechanical factors such as scaffolding and microgrooved surfaces to guide the fibroblasts to form collagen fibrils that are more aligned to those for normal ligaments can be used.⁷⁶ Fibroblasts, when cyclically stretched in culture on smooth silicone surfaces, became largely perpendicular in orientation to the direction of the applied load. Whereas, cells cultured on microgrooved silicone surfaces were elongated and oriented themselves in alignment with the direction of stretch via contact guidance.⁷⁶ Static culture of cells on microgrooved surfaces also produced an organised collagen matrix with fibres that were aligned parallel to the microgrooves.⁷⁵ This suggests that orienting cells along the longitudinal direction of the ligament midsubstance may lead to production of an aligned matrix that more closely resembles the intact state of ligaments. Therefore, to improve the quality of the healing ligament substance, it is important to take into consideration both cell orientation and mechanical loading.

ROBOTIC TECHNOLOGY: IMPROVING THE UNDERSTANDING OF ACL RECONSTRUCTION

The Robotic/UFS Testing System
The knee moves in six degrees of freedom (DOF) - three translations and three rotations. This motion can be conveniently described with respect to three anatomical axes: the axis of the tibial shaft, the axis defined by the femoral insertion sites of the collateral ligaments, and the floating axis perpendicular to these two axes. Thus, the translations are compression-distraction, medial-lateral, and anterior-posterior, while the rotations are internal-external tibial rotation, flexionextension, and varus-valgus rotation.^77,78

Using this motion description, our research centre has developed a novel robotic/universal force-moment sensor (UFS) testing system to study the multiple DOF knee kinematics as well as to directly measure the in situ forces in the knee ligaments without mechanically contacting these tissues.^79,80

The robotic/UFS testing system can operate in position control, force control, and hybrid control modes. In position control, the tibia is moved to a specified location and the resulting forces are recorded by the UFS.⁸¹ In force control mode, a targetted external load can be applied to the tibia as the resulting knee kinematics are recorded.^79,80,82 Hybrid control can be achieved by using position control to move the joint along a pre-selected path of motion while the forces and moments in specified degrees of freedom are controlled.

Since the path of motion is precisely repeated, the in situ force in a ligament can be calculated by the changes in forces after cutting a ligament, based on the principle of superposition (Figure 5). For example, when an external loading condition (F₁) is applied to the intact knee, the kinematics in the five unconstrained DOF (P0 to P1) are recorded. Next, the ligament of interest is cut, the previously recorded kinematics in the five DOF (P0 to P1) are repeated using the position control mode, and a set of new forces (F₂) are measured. Therefore, the vector difference between F1 and F2 is equal to the in situ force in the cut ligament. The determination of the magnitude, direction, and point of application of forces using the robotic/UFS testing system was first validated by comparing the results from simple tensile tests using an Instron testing machine.⁸²

Figure 5: The in situ force in a ligament can be calculated by the changes in forces after cutting a ligament, based on the principle of superposition. Adapted from Woo, S.L-Y., Debski, R.E., Wong, E., Yagi, M., and Tarinelli, D.: Use of Robotic Technology for Diarthroidial Joint Research. J. of Science & Medicine in Sport. 2 (4):283-297, 1999. Reprinted by permission.

The robotic/UFS testing system has many advantages in gaining knee kinematic data and in situ forces in knee ligaments, as compared with previous methodologies such as buckle transducers, implantable transducers, transducers at ligament insertion sites, linkage systems and cutting studies.^83-91 This testing system can not only repeat both the initial knee position and the path of motion, but it also can obtain the forces and force distribution within the ACL without making physical contact with the ligament. Most importantly, this method of approach allows data to be obtained from the same knee, which eliminates interspecimen variation and increases the statistical power.

The Intact Human ACL
The robotic/UFS testing system has been used in numerous studies to elucidate important roles of the ACL.^{79,81,82,92-98} For example, the distribution of the in situ force in the two bundles of the ACL under both anterior tibial and rotatory loading conditions was determined. The posterolateral (PL) bundle of the ACL carried more of the in situ force under anterior tibial loads near full extension while the anteromedial (AM) bundle carried more force at higher flexion angles (Figure 6).⁹⁷

Figure 6: Distribution of forces in ACL in response to anterior tibial load

Figure 7: Response of ACL to rotatory load

Additionally, under a combined load of both internal and valgus torque, the AM bundle carried more of the in situ force at both 15 and 30 degrees of knee flexion; but, the PL experienced a significant level of in situ force.⁹² These studies demonstrate the complexity of the ACL and its important contribution to knee function.

Studies using the 6-DOF testing system have shown the importance of coupled motions in the knee.^93,95 The ACL was found to limit anterior tibial translation even under combined valgus and internal rotatory loads.⁹⁴ After transection of the ACL, anterior tibial translation increased by as much as 4mm under these combined rotatory loads (Figure 7). There were also small increases in internal rotation for combined internal rotatory and valgus loads versus isolated internal rotatory loads, indicating coupling of internal rotation with valgus rotation. These results confirm the importance of coupled motions previously elucidated in an earlier study using a canine model.⁵¹ Additionally, the results of this study suggest that, after sectioning of the MCL, the ACL is the primary ligament that could restrain varus-valgus rotations. This is true because of the coupling of internal and external rotations with valgus and varus rotations, respectively.

Clinically, it is important to remember that the geometry of the ACL is complex and the PL bundle of the ACL carries greater force near extension, while the AM bundle carries greater force at higher flexion angles. In addition, the ACL plays a significant contribution in resisting rotatory loads.

ACL Grafts
Bone-Patellar Tendon-Bone (BPTB) versus Quadriceps/Hamstring Grafts (QSTG)
Currently, there is no consensus view regarding the best of the two most popular autografts, i.e. bone-patellar tendon-bone (BPTB) and quadriceps/hamstrings (QSTG). Clinical studies have yielded no difference between them as they have resulted in similar outcomes. However, randomised studies of both short- and long-term follow-ups revealed 20-25% of patients have reported less than satisfactory results following ACL reconstruction.^{38-41, 44-45}

Thus, in our research centre, the robotic/UFS testing system has been used to assess the ability of the BPTB graft versus the QSTG graft in resisting anterior tibial loads and rotatory loads that simulated the pivot shift test.⁹⁹ Results indicate that under anterior tibial loads, both grafts were successful in restraining anterior tibial translation when compared with that of the ACL-deficient knee. However, under rotatory loads, neither replacement graft was able to reduce the anterior tibial translation, when compared with that of the ACL-deficient knee (Figure 8). Although both grafts were able to restore the in situ forces in the intact ACL under anterior tibial loads, neither were successful in restoring the in situ forces to those experienced by the knee with an intact ACL under rotatory loads. These results suggest that serious problems may exist in the area of reconstruction procedures and not the grafts.

Specifically, these include such issues as femoral tunnel placement and graft tunnel motion.

Figure 8: Response of QSTG and BPTB grafts at 30º knee flexion. Adapted from Woo, S.L-Y., Kanamori, A., Zeminski, J., Yagi, M., Papageorgiou, C., and Fu, F.H.: The Effectiveness of Anterior Cruciate Ligament Reconstruction by Hamstrings and Patellar Tendon: A Cadaveric Study Comparing Anterior Tibial Load vs. Rotational Loads. J. of Bone and Joint Surgery, 84A(6):907-914, 2002. Reprinted by permission.

Femoral tunnel placement
Placement of the femoral tunnel in ACL reconstructions was studied by looking at the effectiveness of moving the tunnel more laterally and away from the rotational centre of the knee at improving the function of the ACL graft.⁹⁶ Using a BPTB graft for the graft, the femoral tunnel was placed at the 10 o’clock and 11 o’clock positions (for a left knee). The 10 o’clock position approximates the femoral insertion site of the PL bundle of the ACL, while the 11 o’clock position is close to the insertion site of the AM bundle. In response to an anterior tibial load, no significant differences in anterior tibial translation (ATT) or in situ forces between the intact and reconstructed knee for both the 10 and 11 o’clock positions were found. However, under a combined rotatory load, the coupled ATT for the 10 o’clock position was smaller than that for the 11 o’clock position. The in situ force in the replacement graft fixed at the 10 o’clock position was also significantly higher than those for the 11 o’clock position. These results indicate that, under rotatory loads, the 10 o’clock position restores function more effectively than the 11 o’clock position, but neither position can restore the knee to its intact state (Figure 9). Clinically, these results suggest that, in isolation, replacing the AM or PL bundle of the ACL alone does not restore the complex function of the intact ACL and, therefore, demonstrates the importance of reconstructing both the AM and PL bundles to restore the function of the ACL.

Figure 9: Comparison of the anterior tibial load and in situ force between the 10 o’clock and 11 o’clock graft positions under a combined rotatory load. Adapted from Loh, J., Fukuda, Y., Tsuda, E., Steadman, R., Fu, F., and Woo, S.L-Y.: Knee Stability and Graft Function Following Anterior Cruciate Ligament Reconstruction: Comparison between 11 o’clock and 10 o’clock Femoral Tunnel Placement. J. of Arthroscopic & Related Research, 19(3):297-304, 2003 with permission from The Arthroscopy Association of North America.

Anatomical Reconstruction
Using a QSTG graft, the anatomical reconstruction in which both the AM and PL bundles are replaced was studied.⁹⁸ A single bundle reconstruction at the 11 o’clock position was also done for comparative purposes. In the anatomical reconstructions, one tibial tunnel and two femoral tunnels (one at the insertion site of the PL bundle and the other at the insertion site of the AM bundle) were used. In response to rotatory loads, it was found that the anatomically reconstructed knee had significantly lower ATT than both the single bundle reconstruction and ACL deficient knees. However, both the single bundle and anatomically reconstructed knees experienced significantly higher ATT than the intact knee. Although not perfect, the anatomically reconstructed knee could better restore the in situ force of the intact ligament, suggesting that closer restoration of the anatomy of the ACL may produce an improved biomechanical outcome of ACL reconstruction (Figure 10).

Figure 10: Comparison of the anterior tibial load and in situ force between the single (11 o’clock) and anatomical graft positions under a combined rotatory load. Adapted from Yagi, M., Wong, E.K., Kanamori, A., Debski, R.E., Fu, F.H., and Woo, S.L-Y.: The Biomechanical Analysis of an Anatomical ACL Reconstruction. Am. J. of Sports Medicine, 30(5):660-666, 2002. Permission Requested. Reprinted by permission of Sage Publications Inc.

In summary, it is clear that there are extremely complex issues involved with ACL reconstructions. As the loading conditions applied in these laboratory studies were relatively simple and were only designed to mimic clinical examinations, further studies that include more realistic in vivo loading conditions must be implemented in order to truly understand both the function of the intact ACL and to evaluate the effectiveness of ACL reconstruction procedures.

FUTURE DIRECTIONS IN LIGAMENT RESEARCH
Through biomechanics research, major advances have been made to increase our understanding of ligament function, healing, and reconstruction and, more importantly, have led to better clinical management of ligament injuries. Laboratory studies have helped to establish conservative management with early controlled mobilisation for healing of an isolated MCL rupture. In effect, the clinical paradigm of surgicalrepair followed by immobilisation for these injuries has been changed for the better.^19,24 In the case of combined ACL/MCL injuries, the ACL is recommended to be reconstructed using an autograft while the MCL can be treated conservatively.^50,54

Knowing that a healed ligament, such as the MCL, has  inferior quality after injury, the frontier of research has been at the cellular and tissue level by looking at the possibilities of applying growth factors, gene transfer/gene therapy, and biological scaffolds. For example, the efficacy of SIS and the roles of AS-ODNs of Types III and V collagen in enhancing MCL healing in vivo are being pursued.^{26-28,71,100-103} We also believe that seeding SIS with fibroblasts and then applying mechanical loading in vitro may further enhance the formation and alignment of the collagen fibres within the scaffold. Eventually, a combination of approaches such as seeding cells on a scaffold that is conditioned with the ideal combination of mechanical stimuli and gene therapy could be developed to treat ligament injuries. These functional tissue engineering approaches have the potential to enhance the properties of the healing tissues. While early successes have been achieved at the molecular or cellular levels, major research efforts are still needed to translate these findings to in vivo situations and, eventually, clinical applications. To do this, one must integrate biomechanics with other biological sciences to improve the outcomes in ligament healing. Once successful, these newly developed technologies may be extended to other ligaments and tendons that do not have the healing capability of the MCL.

Development of the novel 6-DOF robotic/UFS testing system has enabled us to evaluate the function of the knee ligaments and its replacement grafts on a qualitative basis. This unique technology has offered the potential to pursue new approaches for ACL reconstructions, especially with regards to the importance of graft function under complex loading conditions. For example, important surgical parameters which have been identified include the proximal fixation of the graft at the tibial tunnel, more lateral placement of the femoral tunnel, and the need for a more anatomical reconstruction. To do this, it will be necessary to incorporate computer simulation of joint motion and mathematical modelling of the ligament. At our research centre, we have been able to develop experimentally validated models to predict the in situ forces in the ACL. This combined approach offers potential for the study of mechanisms of ACL injury as well as customised surgical planning and optimised rehabilitation of patients.

In the future, in vivo kinematic data will be collected from human subjects and reproduced on the robotic/UFS testing system on a human cadaveric specimen. This specimen will be matched according to such parameters as size, age, etc. to the in vivo patient. The kinematic data obtained from the robotic/UFS testing system can then be used as inputs into a computational model to experimentally measure and computationally determine the in situ forces in ligaments during in vivo activities. By comparing the force data obtained from the computational model to that obtained from the robotic/UFS testing system, the model can be validated. Once validated, these computational models can then be used to develop a database containing the in situ forces in ligaments, as well as the stress and strain data for patients of different ages, genders, and sizes. This database will assist in surgical planning and the optimisation of rehabilitation protocols and has the potential to improve patient outcomes following ACL injuries.

Ligament research has indeed reached an exciting time as improved methods of treating ligament injuries are becoming possible. Obviously, a multidisciplinary collaboration where biologists, biochemists, clinicians, and bioengineers can work together in a seamless transition with no walls between the disciplines will greatly help to achieve these goals. With such an approach, it is to be hoped that patients can recover completely from their ligament injuries and resume normal activities including participation in sports.

ACKNOWLEDGEMENTS
Some of the cited work was done in collaboration with the colleagues of the senior author while he was at the University of California, San Diego. The financial support of NIH grants #AR-39683 and #AR-41820 is gratefully appreciated. The authors also thank many of the students, residents, and research fellows of the MSRC for their contributions. Figures 2 and 3 the authors gratefully acknowledge the support of the San Diego Veterans Administration Medical Centre, NIE Grants AM00304, AM14918 and GM24900, Japan Society for the Promotion of Science and Kobe University to conduct this work.

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Copyright: 23 April 2004