Justin A. Jacobson

Remodeling of Murine Intrasynovial Tendon Adhesions following Injury: MMP and Neotendon Gene Expression

Tendon injury frequently results in the formation of adhesions that reduce joint range of motion. To study the cellular, molecular, and biomechanical events involved in intrasynovial tendon healing and adhesion formation, we developed a murine flexor tendon healing model in which the flexor digitorum longus (FDL) tendon of C57BL/6 mice was transected and repaired using suture. This model was used to test the hypothesis that murine flexor tendons heal with differential expression of matrix metalloproteases (MMPs), resulting in the formation of scar tissue as well as the subsequent remodeling of scar and adhesions. Healing tendons were evaluated by histology, gene expression via real‐time RT‐PCR, and in situ hybridization, as well as biomechanical testing to assess the metatarsophalangeal (MTP) joint flexion range of motion (ROM) and the tensile failure properties. Tendons healed with a highly disorganized fibroblastic tissue response that was progressively remodeled through day 35 resulting in a more organized pattern of collagen fibers. Initial repair involved elevated levels of Mmp‐9 at day 7, which is associated with catabolism of damaged collagen fibers. High levels of Col3 are consistent with scar tissue, and gradually transition to the expression of Col1. Scleraxis expression peaked at day 7, but the expression was limited to the original tendon adjacent to the injury site, and no expression was present in granulation tissue involved in the repair response. The MTP joint ROM with standardized force on the tendon was decreased on days 14 and 21 compared to day 0, indicating the presence of adhesions. Peak expressions of Mmp‐2 and Mmp‐14 were observed at day 21, associated with tendon remodeling. At day 28, two genes associated with neotendon formation, Smad8 and Gdf‐5, were elevated and an improvement in MTP ROM occurred. Tensile strength of the tendon progressively increased, but by 63 days the repaired tendons had not reached the tensile strength of normal tendon. The murine model of primary tendon repair, described here, provides a novel mechanism to study the tendon healing process, and further enhances the understanding of this process at the molecular, cellular, and biomechanical level.

Adhesions in a Murine Flexor Tendon Graft Model: Autograft versus Allograft Reconstruction

Reconstruction of flexor tendons often results in adhesions that compromise joint flexion. Little is known about the factors involved in the formation of flexor tendon graft adhesions. In this study, we developed and characterized a novel mouse model of flexor digitorum longus (FDL) tendon reconstruction with live autografts or reconstituted freeze‐dried allografts. Grafted tendons were evaluated at multiple time points up to 84 days post‐reconstruction. To assess the flexion range of the metatarsophalangeal joint, we developed a quantitative outcome measure proportional to the resistance to tendon gliding due to adhesions, which we termed the Gliding Coefficient. At 14 days post‐grafting, the Gliding Coefficient was 29‐ and 26‐fold greater than normal FDL tendon for both autografts and allografts, respectively (p < 0.001), and subsequently doubled for 28‐day autografts. Interestingly, there were no significant differences in maximum tensile force or stiffness between live autograft and freeze‐dried allograft repairs over time. Histologically, autograft healing was characterized by extensive remodeling and exuberant scarring around both the ends and the body of the graft, whereas allograft scarring was abundant only near the graft–host junctions. Gene expression of GDF‐5 and VEGF were significantly increased in 28‐day autografts compared to allografts and to normal tendons. These results suggest that the biomechanical advantages for tendon reconstruction using live autografts over devitalized allografts are minimal. This mouse model can be useful in elucidating the molecular mechanisms in tendon repair and can aid in preliminary screening of molecular treatments of flexor tendon adhesions.

Erythropoietin accelerates functional recovery after peripheral nerve injury.

BACKGROUND Erythropoietin is a naturally occurring hormone with multiple effects on a number of different cell types. Recent data have suggested neuroprotective and perhaps even neurotrophic roles for erythropoietin. We hypothesized that these functional effects could be demonstrable in standard models of peripheral nerve injury. METHODS Experiments were undertaken to evaluate the effect of erythropoietin on the previously reported standard course of healing of sciatic injuries in mice. The injury groups included mice that were subjected to (1) sham surgery, (2) a calibrated sciatic crush injury, (3) transection of the sciatic nerve followed by epineural repair, or (4) a transection followed by burial of the proximal stump in the adjacent muscle tissue (neurectomy). Either erythropoietin or saline solution was administered to the mice in each of these experimental groups twenty-four hours preinjury, immediately after surgical creation of the injury, twenty-four hours postinjury, or one week postinjury. All mice were evaluated on the basis of the published model for recovery of sciatic nerve motor function by measuring footprint parameters at specific times after the injury. Immunohistochemistry was also performed to assess the erythropoietin-receptor expression profile at the site of injury. RESULTS In general, the mice treated with erythropoietin recovered sciatic nerve motor function significantly faster than did the untreated controls. This conclusion was based on a sciatic function index that was 60% better in the erythropoietin-treated mice at seven days postinjury (p < 0.05). Although the group that had been given the erythropoietin immediately postinjury showed the best enhancement of recovery, the timing of the administration of the drug was not critical. Histological analysis demonstrated enhanced erythropoietin-receptor positivity in the nerves that recovered fastest, suggesting that accelerated healing correlates with expression of the receptor in nerve tissue. CONCLUSIONS Erythropoietin treatment of an acute sciatic nerve crush injury leads to an effect consistent with functional neuroprotection. This protective effect may have clinical relevance, especially since it was detectable even when erythropoietin had been administered up to one week after injury.

Evaluation of dense polylactic acid/beta-tricalcium phosphate scaffolds for bone tissue engineering.

Advances in biomaterial fabrication have introduced numerous innovations in designing scaffolds for bone tissue engineering. Often, the focus has been on fabricating scaffolds with high and interconnected porosity that would allow for cellular seeding and tissue ingrowth. However, such scaffolds typically lack the mechanical strength to sustain in vivo ambulatory stresses in models of load bearing cortical bone reconstruction. In this study, we investigated the microstructural and mechanical properties of dense PLA and PLA/beta-TCP (85:15) scaffolds fabricated using a rapid volume expansion phase separation technique, which embeds uncoated beta-TCP particles within the porous polymer. PLA scaffolds had a volumetric porosity in the range of 30 to 40%. With the embedding of beta-TCP mineral particles, the porosity of the scaffolds was reduced in half, whereas the ultimate compressive and torsional strength were significantly increased. We also investigated the properties of the scaffolds as delivery vehicles for growth factors in vitro and in vivo. The low-surface porosity resulted in sub optimal retention efficiency of the growth factors, and burst release kinetics reflecting surface coating rather than volumetric entrapment, regardless of the scaffold used. When loaded with BMP2 and VEGF and implanted in the quadriceps muscle, PLA/beta-TCP scaffolds did not induce ectopic mineralization but induced a significant 1.8-fold increase in neo vessel formation. In conclusion, dense PLA/beta-TCP scaffolds can be engineered with enhanced mechanical properties and potentially be exploited for localized therapeutic factor delivery.

Bone Marrow-Derived Matrix Metalloproteinase-9 Is Associated with Fibrous Adhesion Formation after Murine Flexor Tendon Injury

The pathogenesis of adhesions following primary tendon repair is poorly understood, but is thought to involve dysregulation of matrix metalloproteinases (Mmps). We have previously demonstrated that Mmp9 gene expression is increased during the inflammatory phase following murine flexor digitorum (FDL) tendon repair in association with increased adhesions. To further investigate the role of Mmp9, the cellular, molecular, and biomechanical features of healing were examined in WT and Mmp9−/− mice using the FDL tendon repair model. Adhesions persisted in WT, but were reduced in Mmp9−/− mice by 21 days without any decrease in strength. Deletion of Mmp9 resulted in accelerated expression of neo-tendon associated genes, Gdf5 and Smad8, and delayed expression of collagen I and collagen III. Furthermore, WT bone marrow cells (GFP+) migrated specifically to the tendon repair site. Transplanting myeloablated Mmp9−/− mice with WT marrow cells resulted in greater adhesions than observed in Mmp9−/− mice and similar to those seen in WT mice. These studies show that Mmp9 is primarily derived from bone marrow cells that migrate to the repair site, and mediates adhesion formation in injured tendons. Mmp9 is a potential target to limit adhesion formation in tendon healing.

Screw pull-out force is dependent on screw orientation in an anterior cervical plate construct.

Two common justifications for orienting cervical screws in an angled direction is to increase pull-out strength and to allow use of longer screws. This concept is widely taught and has guided implant design. Fixed versus variable angle systems may offer strength advantages. The purpose of our study is to test the influence of screw orientation and plate design on the maximum screw pull-out load. Variable and fixed angle 4.0×15 mm and 4.0×13 mm self-tapping screws were used to affix a Medtronic Atlantis cervical plate to polyurethane foam bone samples (density 0.160/cm3). This synthetic product is a model of osteoporotic cancellous bone. The fixed angle screws can only be placed at 12 degrees convergent to the midline and 12 degrees in the cephalad/caudal (“12 degrees up and in”) direction. Three groups were tested: (1) all fixed angle screws, (2) variable angle, all screws 12 degrees up and in, (3) variable angle, all screws 90 degrees to the plate. Plate constructs were pulled off with an Instron DynaMight 8841 servohydrolic machine measuring for maximum screw pull-out force. There was no difference between group 1, fixed angle (288.4±37.7 N) (mean±SD) and 2, variable angle group (297.7±41.31 N P≤0.73). There was a significant increase in maximum pull-out force to failure for the construct with all screws at 90 degrees (415.2±17.4 N) compared with all screws 12 degrees “up and in” (297.4±41.3 N, P≤0.0016). Group 3 done with 13 mm screws, showed a trend toward better pull-out strength, compared to group 2 w/15 mm screws (345.2±20.5 vs. 297.4±41.3, P≤0.06). In this plate pull-out model, screw orientation influences maximum force to failure. When all 4 screws are 90 degrees to the plate the construct has the greatest ability to resist pullout. Fixed angle designs show no advantage over variable angle. These findings are contrary to current teaching.

Evaluation of Polylactic Acid/Beta-Tricalcium Phosphate Scaffolds as Segmental Bone Graft Substitutes

The use of processed structural allografts for treatment of massive segmental defects in long bones can be complicated by poor incorporation and remodeling of the devitalized graft, foreign-body reaction and micro-damage accumulation which often leads to catastrophic graft failure [1]. It is therefore useful to develop a bioengineered, biodegradable scaffold that is able to stimulate healing of the defect region. The use of bioengineered scaffolds has been limited due to their poor mechanical strength that does not permit withstanding large in vivo loads and due to their poor osteoinductive properties. We therefore investigated the use of rigid polylactic acid/beta-tricalcium phosphate (PLA/βTCP) composites used in conjunction with osteoinductive factors such as growth hormones (parathyroid hormone (PTH)) and growth factors (bone morphogenic protein-2 (BMP-2) & vascular endothelial growth factor (VEGF)) to stimulate bone formation and vessel ingrowth in the segmental defect region. We examined the physical characteristics of the scaffolds, and evaluated their osteoinductive potential in a clinically-relevant mouse model of a femoral segmental defect with or without PTH treatment. Finally, we used an ectopic bone formation model to assess the efficacy of the scaffold in site-specific delivery of bone anabolic factors.Copyright