Kevin A. Hildebrand

Tissue engineering of ligament and tendon healing.

Ligaments and tendons are bands of dense connective tissue that mediate normal joint movement and stability. Injury to these structures may result in significant joint dysfunction because they either heal by production of inferior matrix or do not heal at all. The process of ligament and tendon healing is complex and the roles of cellular and biochemical mediators continue to be elucidated. The expression of growth factors and growth factor receptors is modulated after injury, and cells from healing tissues are responsive to growth factors. Tissue engineering offers the potential to improve the quality of ligaments and tendons during the healing process. The concept is based on the manipulation of cellular and biochemical mediators to affect protein synthesis and improve tissue remodeling. Recently, novel techniques such as application of growth factors, gene transfer techniques, and cell therapy have shown promise and may become effective biologic therapies in the future. Many groups have been successful in introducing marker and therapeutic genes into ligaments and tendons. Cell therapy involves the introduction of mesenchymal progenitor cells as a pluripotent cell source into the healing environment. The combination of cell therapy with growth factor application via gene transfer offers new avenues to improve ligament and tendon healing.

The effects of platelet-derived growth factor-BB on healing of the rabbit medial collateral ligament : An in vivo study

We report a biologic approach to improve medial collateral ligament healing using growth factors normally expressed in healing tissue. Our previous in vitro work demonstrated that platelet-derived growth factor-BB and transforming growth factor- 1 promoted fibroblast proliferation and matrix synthesis, respectively. Therefore, these growth factors were used in vivo to determine whether they could improve medial collateral ligament healing, whether this effect was dose-dependent, and if combinations of growth factors could improve healing more than individual growth factors. Thirty-seven rabbits had various doses of growth factors applied to the ruptured right medial collateral ligaments using a fibrin sealant delivery vehicle. The five groups consisted of 1) two groups receiving two doses of platelet-derived growth factor-BB, 2) two groups receiving two doses of this growth factor plus transforming growth factor- 1, and 3) one group receiving fibrin sealant only. After sacrifice at 6 weeks, biomechanical and histologic evaluations of the healing ligament were performed. Femur-medial collateral ligament-tibia complexes of the knees given the higher dose of platelet-derived growth factor-BB had ultimate load, energy absorbed to failure, and ultimate elongation values that were 1.6, 2.4, and 1.6 times greater than the same complexes of the control group. Adding transforming growth factor- 1 did not lead to any further increases in the structural properties of the complex compared with treatment with platelet-derived growth factor-BB. These encouraging results suggest that use of platelet-derived growth factor-BB may improve the quality of the healing medial collateral ligament, and that it may also have a similar potential for promoting healing of other ligaments.

Engineering the healing of the rabbit medial collateral ligament

A biological approach to improve healing of the medical collateral ligament (MCL) was investigated by exploring the use of therapeutic growth factors based on in vitro and in vivo experiments. The in vitro cell culture studies involved screening a variety of growth factors to select those that exhibit the most positive effects on cell proliferation and extracellular matrix synthesis. The selected growth factors were applied in vivo to a rabbit model where the MCL was ruptured. Biomechanical and histological evaluations are performed to determine whether the selected growth factors can enhance the properties of the healed MCL, whether these improvements are dose dependent, and whether combinations of growth factors can enhance MCL healing to a greater extent than individual growth factors. In vitro studies showed that epidermal growth factor (EGF) and platelet derived growth factor-BB (PDGF-BB) have the greatest effect on ligament fibroblast proliferation, whereas transforming growth factor-β1 (TGF-β1) superiorly promotes extracellular matrix synthesis. These growth factors were then applied in vivo at different dosages, in isolation and in combination, and the ligaments were evaluated six weeks post-operatively. Tensile testing of the femur-MCL-tibia complexes (FMTCs) revealed that the specimens treated with a high dose of PDGF-BB have ultimate load, ultimate elongation and energy absorbed to failure values that are significantly greater than those from the other groups. The high dose of PDGF-BB was more effective than the low dose, indicating a dose dependency. The addition ofTGF-β1 to PDGF-BB did not lead to any further increases in the structural properties of the FMTC. These encouraging results suggest that PDGF-BB may be a potential growth factor to enhance the quality of the healing ligament.

High Rate of Joint Capsule Matrix Turnover in Chronic Human Elbow Contractures

The joint capsule is a key component in posttraumatic joint contractures. The capsule is described as thickened, but little data exist supporting the observation. Our hypotheses were that mRNA levels of (1) collagen; (2) decorin and biglycan; (3) matrix metalloproteinases; and (4) tissue inhibitors of matrix metalloproteinases were significantly elevated in anterior joint capsules obtained from 11 patients having surgery for posttraumatic contractures when compared with nine elbows, from organ donors, that were free of contractures. Reverse transcription-polymerase chain reaction was used to evaluate mRNA expression normalized to a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. In the joint capsules of the patients with elbow contractures, relative mRNA levels were increased for: collagen Types I, III, and V (1.5-2.5 times); biglycan (1.5 times); and matrix metalloproteinases-1, -2, -9, -13, and -15 (1.6-3.9 times). In contrast, expression of tissue inhibitors of matrix metalloproteinases-1, -2, and -4 were decreased (⅓-¾ times) in the capsules of patients with contractures. There was no difference between the groups in relative mRNA expression for decorin, matrix metalloproteinases-8, -14 and -16, and tissue inhibitor of matrix metalloproteinase-3. The results indicate that joint capsule matrix molecule mRNA levels are altered in the chronic stages of posttraumatic elbow contractures in humans, potentially creating an environment with high matrix turnover rates.

The effects of age on rabbit MCL fibroblast matrix synthesis in response to TGF-β1 or EGF

In this study, we examined the effects of age on collagen and total protein synthesis by ligament fibroblasts in response to growth factors. Three different doses of transforming growth factor-beta 1 (TGF-beta 1) or epidermal growth factor (EGF) were individually added to in vitro fibroblast cultures from the medial collateral ligament (MCL) of skeletally immature (age 3 months), mature (age 12 months) and senescent (age 48-51 months) rabbits. Analysis of the effects of age revealed that fibroblasts from senescent rabbits produced significantly less collagen in response to TGF-beta 1 or EGF stimulation when compared to fibroblasts from immature rabbits. Furthermore, increased age was found to result in significant reductions in the baseline levels of collagen synthesis but not total protein synthesis. Additionally, collagen and total protein synthesis by MCL fibroblasts were significantly affected by the TFG-beta 1 dose, but not by the EGF dose. When fibroblasts were normalized to their own controls, the increase in collagen and total protein synthesis due to TGF-beta 1 and EGF for the senescent group were found to be greater than those for the skeletally immature rabbits at all doses. This demonstrates that MCL fibroblasts from senescent rabbits are responsive to growth factors.

Myofibroblast upregulators are elevated in joint capsules in posttraumatic contractures.

We hypothesized specific growth factors are increased in the elbow capsules of patients with post traumatic elbow contractures. A model of surgically induced joint contracture in rabbit knees was developed to study the growth factor expression in joint contractures. This study demonstrates this model mimics the human condition and analyzes how the growth factor levels decrease with time in rabbit knees with contractures. Reverse transcription polymerase chain reaction was used to measure mRNA levels of transforming growth factor-β1, connective tissue growth factor, ED-A of fibronectin, and α-smooth muscle actin normalized to a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. In the joint capsules of patients with elbow contractures, mRNA levels were increased for transforming growth factor-β1, connective tissue growth factor, and α-smooth muscle actin. In the joint capsules of rabbit knees with contractures, mRNA levels were increased for transforming growth factor-β1, connective tissue growth factor, ED-A of fibronectin, and α-smooth muscle actin. The mRNA levels for transforming growth factor-β1, connective tissue growth factor, and α-smooth muscle actin decreased with time in rabbit knees. The elevated levels of these myofibroblast upregulators and fibrogenic growth factors could explain the previously reported increase in myofibroblasts and collagen mRNA levels. The rabbit knee model correlated well with the human post traumatic elbow contractures.Level of Evidence: Level II, prospective comparative study. See the Guidelines for Authors for a complete description of levels of evidence.

Regional variation is present in elbow capsules after injury.

Myofibroblast numbers and α-smooth muscle actin expression are increased in anterior joint capsules of patients with posttraumatic elbow contractures. The purpose of our study was to determine whether these changes occur regionally or throughout the entire joint capsule. We hypothesized that the α-smooth muscle actin mRNA expression and the myofibro- blast numbers in posterior joint capsules would be elevated in elbows obtained from patients with posttraumatic joint contractures compared with joint capsules obtained from organ donor elbows without contractures. Semiquantitative reverse transcription-polymerase chain reaction was used to evaluate relative mRNA levels of α-smooth muscle actin. Glyceraldehyde-3-phosphate dehydrogenase was used to normalize the levels. Immunohistochemical analysis was used to determine the myofibroblast cell numbers. Higher α-smooth muscle actin mRNA levels were observed in elbows of patients with contractures compared with organ donor elbows without contractures. Immunohistochemical studies determined that myofibroblast numbers and the percentage of total cells that were myofibroblasts were elevated (2-2.5- fold) in the joint capsules from patients with posttraumatic joint contractures compared with similar tissue obtained from organ donor elbows without contractures. These results suggest elevated myofibroblast numbers occur throughout the whole joint capsule in posttraumatic elbow contractures, although there is some regional variation.

The Basics of Soft Tissue Healing and General Factors that Influence Such Healing

Wound healing and repair of injured tissues follows several steps in the healthy individual. Following birth, the process is initiated by the inflammatory response and subsequent steps are based on this initial response. Whereas wound healing generally leads to a repair of the injured site, it does not lead to tissue regeneration. This difference between repair and regeneration has influence on tissues such as ligaments and tendons that function in a mechanically active environment. Thus, the dynamic interface between mechanics and biology influence the effectiveness of the healing response. The biology of the host is also influenced by a variety of factors including age, gender, genetics, and tissue history, factors that impact the outcome of the healing response. This review focuses on several of the issues surrounding the healing of tissues in general and ligaments and tendons more specifically. In addition, the use of interventions such as cell and gene therapy to improve healing is discussed.

Neuropeptide, mast cell and myofibroblast expression after rabbit deep flexor tendon repair

PURPOSEnIncreased numbers of myofibroblasts, mast cells, and neuropeptide-containing nerve fibers have been found in a number of fibrotic processes in connective tissues. The purpose of the present study was to investigate the occurrence of factors implicated in a hypothesized profibrotic neuropeptide-mast cell-myofibroblast pathway in deep flexor tendon healing.nnnMETHODSnIn a rabbit model of flexor tendon injury, with repair of the sharply transected deep flexor tendon using a modified Kessler and a running circumferential peripheral suture, segments of flexor tendons and sheaths were analyzed. The time points chosen-3, 6, 21, and 42 days after tendon repair-represent different stages in tendon healing. The messenger RNA levels of transforming growth factor-β1 and α-smooth muscle actin were measured with conventional reverse transcription-polymerase chain reaction, and the numbers of myofibroblasts, mast cells, and neuropeptide-containing nerve fibers were determined with immunohistochemistry.nnnRESULTSnThe messenger RNA levels for transforming growth factor-β1 and the myofibroblast marker α-smooth muscle actin were significantly increased in deep flexor tendons after injury and repair, at all studied time points, but remained unchanged or even down-regulated in the sheaths. Myofibroblasts, mast cells, and neuropeptide-containing nerve fibers all increased significantly in the healing tendons, exhibiting similar patterns of change in percentages of total cell number over time, reaching levels resembling that of the tendon sheaths with 33% to 50% of the total cell population.nnnCONCLUSIONSnAfter injury to the deep flexor tendon in a rabbit model, the proportion of myofibroblasts, mast cells, and neuropeptide-containing nerve fibers increases significantly. These findings support the hypothesis that the profibrotic neuropeptide-mast cell-myofibroblast pathway is activated in deep flexor tendon healing.

The contribution of the anterior cruciate ligament to knee joint kinematics: Evaluation of its in situ forces using a robot/universal force-moment sensor test system

Damage to one of the major soft tissue structures of the knee joint, namely, the anterior cruciate ligament (ACL), can lead to significant changes in joint kinematics, which the patient perceives as instability. In this manuscript, we illustrate the importance of the anatomical complexity and biomechanical function of the ACL in knee joint kinematics. Further, we introduce a new test system whereby the in situ forces of the ACL and multiple-degree of freedom (DOF) knee kinematics are determined. Using a robotic manipulator coupled with a universal force-moment sensor (UFS), these forces can be determined directly and without making mechanical contact with the tissue. We have found that the in situ force in the human ACL in response to 110N of anterior tibial load decreases significantly with knee flexion, changing from 110.6±14.8N at 15° of flexion to 71.1±29.5 N at 90° of flexion. Distribution of the in situ force within the human ACL was determined by consistently defining its distribution in anteromedial (AM) and posterolateral (PL) bundles of the ACL. In situ forces in the PL bundle in response to 110N of anterior tibial load showed a maximum of 75.2±18.3 N at 15°, and were significantly larger than those in the AM bundle. On the contrary, in situ forces in the AM bundle varied from a maximum of 47.4±34.2N at 60° to a minimum of 32.6±13.3N at 0°. In the porcine ACL, the magnitude of in situ force was not significantly affected as the constraints varied from 1-DOF to 5-DOF. However, the directions of the force were significantly different between 1-DOF and 5-DOF conditions. The ACL reconstruction study has shown that anatomical (proximal) ACL graft fixation in the tibial tunnel more closely reproduced the knee kinematics and in situ force of the native ACL than those in central and distal fixations. These findings have important implications in terms of the mechanisms of ACL injuries, complexity of the ACL function, and anatomy-and biomechanics-based approaches to ACL reconstruction.