Marc T. Galloway

Mechanical Stimulation of Tendon Tissue Engineered Constructs: Effects on Construct Stiffness, Repair Biomechanics, and Their Correlation

The objective of this study was to determine how in vitro mechanical stimulation of tissue engineered constructs affects their stiffness and modulus in culture and tendon repair biomechanics 12 weeks after surgical implantation. Using six female adult New Zealand White rabbits, autogenous tissue engineered constructs were created by seeding mesenchymal stem cells (0.1 x 10(6) cells/ml) in collagen gel (2.6 mg/ml) and combining both with a collagen sponge. Employing a novel experimental design strategy, four constructs from each animal were mechanically stimulated (one 1 Hz cycle every 5 min to 2.4% peak strain for 8 h/day for 2 weeks) while the other four remained unstretched during the 2 week culture period. At the end of incubation, three of the mechanically stimulated (S) and three of the nonstimulated (NS) constructs from each animal were assigned for in vitro mechanical testing while the other two autogenous constructs were implanted into bilateral full-thickness, full-length defects created in the central third of rabbit patellar tendons (PTs). No significant differences were found in the in vitro linear stiffnesses between the S (0.15+/-0.1 N/mm) and NS constructs (0.08+/-0.02 N/mm; mean+/-SD). However, in vitro mechanical stimulation significantly increased the structural and material properties of the repair tissue, including a 14% increase in maximum force (p=0.01), a 50% increase in linear stiffness (p=0.001), and 23-41% increases in maximum stress and modulus (p=0.01). The S repairs achieved 65%, 80%, 60%, and 40% of normal central PT maximum force, linear stiffness, maximum stress, and linear modulus, respectively. The results for the S constructs exceed values obtained previously by our group using the same animal and defect model, and to our knowledge, this is the first study to show the benefits of in vitro mechanical stimulation on tendon repair biomechanics. In addition, the linear stiffnesses for the construct and repair were positively correlated (r=0.56) as were their linear moduli (r=0.68). Such in vitro predictors of in vivo outcome hold the potential to speed the development of tissue engineered products by reducing the time and costs of in vivo studies.

The Role of Mechanical Loading in Tendon Development, Maintenance, Injury, and Repair

Tendon injuries often result from excessive or insufficient mechanical loading, impairing the ability of the local tendon cell population to maintain normal tendon function. The resident cell population composing tendon tissue is mechanosensitive, given that the cells are able to alter the extracellular matrix in response to modifications of the local loading environment. Natural tendon healing is insufficient, characterized by improper collagen fibril diameter formation, collagen fibril distribution, and overall fibril misalignment. Current tendon repair rehabilitation protocols focus on implementing early, well-controlled eccentric loading exercises to improve repair outcome. Tissue engineers look toward incorporating mechanical loading regimens to precondition cell populations for the creation of improved biological augmentations for tendon repair.

Combined Effects of Scaffold Stiffening and Mechanical Preconditioning Cycles on Construct Biomechanics, Gene Expression, and Tendon Repair Biomechanics

Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases both construct stiffness and the biomechanical properties of the repair tissue after surgery. When optimized using response surface methodology, our results indicate that a mechanical stimulus with three components (2.4% strain, 3000 cycles/day, and one cycle repetition) produced the highest in vitro linear stiffness. Such positive correlations between construct and repair stiffness after surgery suggest that enhancing structural stiffness before surgery could not only accelerate repair stiffness but also prevent premature failures in culture due to poor mechanical integrity. In this study, we examined the combined effects of scaffold crosslinking and subsequent mechanical stimulation on construct mechanics and biology. Autologous tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 15 New Zealand white rabbits on type I collagen sponges that had undergone additional dehydrothermal crosslinking (termed ADHT in this manuscript). Both constructs from each rabbit were mechanically stimulated for 8h/day for 12 consecutive days with half receiving 100 cycles/day and the other half receiving 3000 cycles/day. These paired MSC-collagen autologous constructs were then implanted in bilateral full-thickness, full-length defects in the central third of rabbit patellar tendons. Increasing the number of in vitro cycles/day delivered to the ADHT constructs in culture produced no differences in stiffness or gene expression and no changes in biomechanical properties or histology 12 weeks after surgery. Compared to MSC-based repairs from a previous study that received no additional treatment in culture, ADHT crosslinking of the scaffolds actually lowered the 12-week repair stiffness. Thus, while ADHT crosslinking may initially stiffen a construct in culture, this specific treatment also appears to mask any benefits of stimulation among repairs postsurgery. Our findings emphasize the importance of properly preconditioning a scaffold to better control/modulate MSC differentiation in vitro and to further enhance repair outcome in vivo.

Effect of Implanting a Soft Tissue Autograft in a Central-Third Patellar Tendon Defect: Biomechanical and Histological Comparisons

Previous studies by our laboratory have demonstrated that implanting a stiffer tissue engineered construct at surgery is positively correlated with repair tissue stiffness at 12 weeks. The objective of this study was to test this correlation by implanting a construct that matches normal tissue biomechanical properties. To do this, we utilized a soft tissue patellar tendon autograft to repair a central-third patellar tendon defect. Patellar tendon autograft repairs were contrasted against an unfilled defect repaired by natural healing (NH). We hypothesized that after 12 weeks, patellar tendon autograft repairs would have biomechanical properties superior to NH. Bilateral defects were established in the central-third patellar tendon of skeletally mature (one year old), female New Zealand White rabbits (n = 10). In one limb, the excised tissue, the patellar tendon autograft, was sutured into the defect site. In the contralateral limb, the defect was left empty (natural healing). After 12 weeks of recovery, the animals were euthanized and their limbs were dedicated to biomechanical (n = 7) or histological (n = 3) evaluations. Only stiffness was improved by treatment with patellar tendon autograft relative to natural healing (p = 0.009). Additionally, neither the patellar tendon autograft nor natural healing repairs regenerated a normal zonal insertion site between the tendon and bone. Immunohistochemical staining for collagen type II demonstrated that fibrocartilage-like tissue was regenerated at the tendon-bone interface for both repairs. However, the tissue was disorganized. Insufficient tissue integration at the tendon-to-bone junction led to repair tissue failure at the insertion site during testing. It is important to re-establish the tendon-to-bone insertion site because it provides joint stability and enables force transmission from muscle to tendon and subsequent loading of the tendon. Without loading, tendon mechanical properties deteriorate. Future studies by our laboratory will investigate potential strategies to improve patellar tendon autograft integration into bone using this model.

The native cell population does not contribute to central-third graft healing at 6, 12, or 26 weeks in the rabbit patellar tendon

Investigators do not yet understand the role of intrinsic tendon cells in healing at the tendon‐to‐bone enthesis. Therefore, our first objective was to understand how the native cell population influences tendon autograft incorporation in the central‐third patellar tendon (PT) defect site. To do this, we contrasted the histochemical and biomechanical properties of de‐cellularized patellar tendon autograft (dcPTA) and patellar tendon autograft (PTA) repairs in the skeletally mature New Zealand white rabbit. Recognizing that soft tissues in many animal models require up to 26 weeks to incorporate into bone, our second objective was to investigate how recovery time affects enthesis formation and graft tissue biomechanical properties. Thus, we examined graft structure and mechanics at 6, 12, and 26 weeks post‐surgery. Our results showed that maintaining the native cell population produced no histochemical or biomechanical benefit at 6, 12, or 26 weeks. These findings suggest that PTA healing is mediated more by extrinsic rather than intrinsic cellular mechanisms. Moreover, while repair tissue biomechanical properties generally increased from 6 to 12 weeks after surgery, no further improvements were noted up to 26 weeks.

Effect of Surgery to Implant Motion and Force Sensors on Vertical Ground Reaction Forces in the Ovine Model

Activities of daily living (ADLs) generate complex, multidirectional forces in the anterior cruciate ligament (ACL). While calibration problems preclude direct measurement in patients, ACL forces can conceivably be measured in animals after technical challenges are overcome. For example, motion and force sensors can be implanted in the animal but investigators must determine the extent to which these sensors and surgery affect normal gait. Our objectives in this study were to determine (1) if surgically implanting knee motion sensors and an ACL force sensor significantly alter normal ovine gait and (2) how increasing gait speed and grade on a treadmill affect ovine gait before and after surgery. Ten skeletally mature, female sheep were used to test four hypotheses: (1) surgical implantation of sensors would significantly decrease average and peak vertical ground reaction forces (VGRFs) in the operated limb, (2) surgical implantation would significantly decrease single limb stance duration for the operated limb, (3) increasing treadmill speed would increase VGRFs pre- and post operatively, and (4) increasing treadmill grade would increase the hind limb VGRFs pre- and post operatively. An instrumented treadmill with two force plates was used to record fore and hind limb VGRFs during four combinations of two speeds (1.0 m/s and 1.3 m/s) and two grades (0 deg and 6 deg). Sensor implantation decreased average and peak VGRFs less than 10% and 20%, respectively, across all combinations of speed and grade. Sensor implantation significantly decreased the single limb stance duration in the operated hind limb during inclined walking at 1.3 m/s but had no effect on single limb stance duration in the operated limb during other activities. Increasing treadmill speed increased hind limb peak (but not average) VGRFs before surgery and peak VGRF only in the unoperated hind limb during level walking after surgery. Increasing treadmill grade (at 1 m/s) significantly increased hind limb average and peak VGRFs before surgery but increasing treadmill grade post op did not significantly affect any response measure. Since VGRF values exceeded 80% of presurgery levels, we conclude that animal gait post op is near normal. Thus, we can assume normal gait when conducting experiments following sensor implantation. Ultimately, we seek to measure ACL forces for ADLs to provide design criteria and evaluation benchmarks for traditional and tissue engineered ACL repairs and reconstructions.

MPC-Collagen Gel Biologic Augmentations do not Promote Patellar Tendon Integration Into Bone

The high failure rate of rotator cuff repair is often attributed to the fact that a normal zonal insertion site is not regenerated between tendon and bone. [1,2] Interestingly, a previous study in our laboratory demonstrated that a patellar tendon autograft (PTA) used to repair a central-third patellar tendon (PT) defect also does not regenerate a normal zonal insertion site at 12 weeks following surgery. Therefore, using our model of tendon healing, our objective was to design a biologic augmentation (BA) that could be implemented at the insertion site between tendon and bone to help promote integration. The results of this study have potential application in developing new techniques for rotator cuff repair.Copyright

Comparative Histological and Biomechanical Effects of Prostaglandin-E2 and Bacterial Collagenase on the Rabbit Patellar Tendon

Ninety-seven percent of tendon ruptures are found in areas of chronic tendon degeneration, which is thought to be a cell-mediated process involving increased extracellular matrix turnover and remodeling [1,2]. The degenerative aspects seen within regions of tendon degeneration include collagen matrix disorganization, collagen fibril thinning, cellular hyperplasia, and neovascularization [1–3]. The etiology of tendon degeneration is unclear at this point. One theory involves the introduction of multiple mechanical insults (mechanical overuse) that act to trigger a degenerative pathway of increased matrix degradation by matrix metalloproteinases (MMP). Inflammation is not seen within regions of degenerative tendon. However, inflammatory mediators such as prostaglandin-E2 (PGE2) may have a role as they have been shown to be upregulated by fibroblasts as a result of mechanical over-stimulation in culture [4]. Multiple injections of PGE2 within the midsubstance of the rabbit patellar tendon (PT) also produced collagen fibril disorganization and thinning [5].Copyright

Effect of Mechanical Stimulation on the Biomechanics of Stem Cell: Collagen Sponge Constructs for Patellar Tendon Repair

Tendons (rotator cuff, Achilles and patellar tendons) are among the most commonly injured soft tissues [1]. Many techniques for repair/reconstruction have been attempted (e.g. sutures, resorbable biomaterials, autografts, and allografts) with varying success. A tissue engineered repair using mesenchymal stem cells (MSCs) is and attractive option [2–4] but the stiffness and strength of currently available constructs are insufficient for clinical use [6].Copyright