Miltiadis H. Zgonis

Effect of interleukin-10 overexpression on the properties of healing tendon in a murine patellar tendon model.

PURPOSE Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine shown to inhibit scar formation in fetal wound healing. The role of IL-10 in adult tendon healing and scar formation, however, remains unknown. The objective of this study is to investigate the effect of IL-10 overexpression on the properties of adult healing tendon using a well-established murine model of tendon injury and a lentiviral-mediated method of IL-10 overexpression. METHODS A murine model of patellar tendon injury was used and animals divided into 3 groups. Mice received bilateral patellar tendon injections with a lentiviral vector containing an IL-10 transgene (n = 34) or no transgene (n = 34). Control mice (n = 34) received injections of sterile saline. All animals then were subjected to bilateral, central patellar tendon injuries 2 days after injection and were killed at 5, 10, 21, and 42 days after injury. IL-10 content was analyzed by immunohistochemistry (n = 4/group). Tendon healing was evaluated by histology (n = 4/group) and biomechanical analysis (n = 10/group). RESULTS Overexpression of IL-10 in patellar tendon was confirmed after injection of the lentiviral vector. IL-10 immunostaining was increased at day 10 in the IL-10 group relative to that in controls. Histologically, there was no significant difference in angular deviation between groups at day 21, but a trend toward decreased angular deviation in controls relative to that in empty vector group mice was seen at day 42. Biomechanically, the IL-10 group showed significantly increased maximum stress at day 42 relative to that in controls. Percent relaxation showed a trend toward an increase at day 10 and a significant increase at day 42 in the IL-10 group relative to that in controls. CONCLUSIONS This study demonstrates successful gene transfer of IL-10 into adult murine patellar tendon using a lentiviral vector. Although the effects of overexpression of IL-10 on adult tendon healing have not yet been fully elucidated, the current study may help to further clarify the mechanisms of tendon injury and repair.

Levels of Evidence Are Low for Clinical Management Questions on the Orthopaedic In-Training Examination

The Orthopaedic In-Training Examination (OITE) has been administered to orthopaedic surgery residents throughout the United States since 1963 and, over the past forty-five years, has become “an integral part of orthopaedic education.”1 The test was designed as an educational tool for residents, but it also is used to assess the performance of residents relative to that of others. Additionally, an implicit function of the test is to help to define the corpus of core knowledge for orthopaedic surgery residency. As noted by the participants of the Academic Orthopaedic Society symposium on musculoskeletal education2, “Testing organizations are influential in shaping the curriculum. Faculty are correct to teach what students need to know to pass their [tests].” The Evaluation Committee of the American Academy of Orthopaedic Surgeons (AAOS) writes the questions, amalgamates the examination, and scores the OITE. As such, test takers can reasonably infer that the material tested on the OITE reflects the Academy’s vision of the orthopaedic surgery core curriculum for residents in training, and, owing to the central position of the AAOS, the OITE is an important influence on the orthopaedic surgery curriculum for residents in the United States. For this reason, among others, it is worthwhile to examine the OITE for the purposes of studying what exactly residents are expected to master. One aspect of that analysis centers on the levels of evidence on which the OITE questions are based. Although the principles of evidence-based medicine3 do not limit the practitioner to the randomized trial (or any particular study type), there has been particular recent emphasis on the level of evidence associated with individual studies. In their editorial introducing the policy of explicitly listing this level, for example, the editors of this journal stated: “Orthopaedic surgeons have always based their clinical care on evidence… …

The correction of pelvic obliquity in patients with cerebral palsy and neuromuscular scoliosis: is there a benefit of anterior release prior to posterior spinal arthrodesis?

Study Design. A retrospective study of 61 patients with cerebral palsy (CP) and neuromuscular scoliosis treated by either a combined anterior-posterior spinal arthrodesis or a posterior-only arthrodesis with the unit rod. Objectives. Compare coronal and sagittal plane radiographic outcomes in patients undergoing either a combined anterior-posterior spinal fusion (PSF) or a posterior-only fusion with the unit rod for neuromuscular scoliosis in patients with CP. Summary of Background Data. Although an anterior release before posterior spinal arthrodesis is commonly done for larger and stiffer neuromuscular curves, it is unclear whether or not an all-posterior construct produces similar correction in pelvic obliquity as that seen with an anterior-posterior spinal fusion. Methods. Sixty-one consecutive children with CP and scoliosis were treated at a single institution between 1991 and 2003 with PSF using the unit rod with an anterior release (group A: 19 patients; average = 14.4 years) or without an anterior release (group B: 42 patients; average = 13.7 years). Side-bending, AP, and lateral radiographs were used to assess various sagittal and coronal plane parameters at baseline, after surgery, and at 2 years. Results. Before surgery, group A had larger major curves (91°A vs. 72°B; P = 0.001), less flexible major curves (21% A vs. 40% B; P = 0.01), with greater pelvic obliquity (26°A vs. 19°B, P = 0.02) than group B. In the subset of patients with a more severe preoperative pelvic obliquity (>20°), percent correction in pelvic obliquity was equivalent between groups A (71%) and B (74%, P = 0.91). With respect to coronal and sagittal plane radiographic outcomes, there were no significant group differences in major curve correction (58% A vs. 60% B), but group A trended toward greater % correction from preop bending films. At most recent follow-up, there were no differences with respect to loss of curve correction (7.6 A vs. 8.1°B, P = 0.80). The rate of major complications was 26% for both groups, but group A patients had significantly longer operative times. Conclusion. We demonstrate that excellent correction in severe pelvic obliquity can be achieved in smaller, more flexible curves using an all-posterior PSF, and in larger, less flexible curves using an anterior release with PSF.

Mechanical function near defects in an aligned nanofiber composite is preserved by inclusion of disorganized layers: Insight into meniscus structure and function

The meniscus is comprised of circumferentially aligned fibers that resist the tensile forces within the meniscus (i.e., hoop stress) that develop during loading of the knee. Although these circumferential fibers are severed by radial meniscal tears, tibial contact stresses do not increase until the tear reaches ∼90% of the meniscus width, suggesting that the severed circumferential fibers still bear load and maintain the mechanical functionality of the meniscus. Recent data demonstrates that the interfibrillar matrix can transfer strain energy to disconnected fibrils in tendon fascicles. In the meniscus, interdigitating radial tie fibers, which function to stabilize and bind the circumferential fibers together, are hypothesized to function in a similar manner by transmitting load to severed circumferential fibers near a radial tear. To test this hypothesis, we developed an engineered fibrous analog of the knee meniscus using poly(ε-caprolactone) to create aligned scaffolds with variable amounts of non-aligned elements embedded within the scaffold. We show that the tensile properties of these scaffolds are a function of the ratio of aligned to non-aligned elements, and change in a predictable fashion following a simple mixture model. When measuring the loss of mechanical function in scaffolds with a radial tear, compared to intact scaffolds, the decrease in apparent linear modulus was reduced in scaffolds containing non-aligned layers compared to purely aligned scaffolds. Increased strains in areas adjacent to the defect were also noted in composite scaffolds. These findings indicate that non-aligned (disorganized) elements interspersed within an aligned network can improve overall mechanical function by promoting strain transfer to nearby disconnected fibers. This finding supports the notion that radial tie fibers may similarly promote tear tolerance in the knee meniscus, and will direct changes in clinical practice and provide guidance for tissue engineering strategies. STATEMENT OF SIGNIFICANCE The meniscus is a complex fibrous tissue, whose architecture includes radial tie fibers that run perpendicular to and interdigitate with the predominant circumferential fibers. We hypothesized that these radial elements function to preserve mechanical function in the context of interruption of circumferential bundles, as would be the case in a meniscal tear. To test this hypothesis, we developed a biomaterial analog containing disorganized layers enmeshed regularly throughout an otherwise aligned network. Using this material formulation, we showed that strain transmission is improved in the vicinity of defects when disorganized fiber layers were present. This supports the idea that radial elements within the meniscus improve function near a tear, and will guide future clinical interventions and the development of engineered replacements.

Intra-articular changes precede extra-articular changes in the biceps tendon after rotator cuff tears in a rat model.

HYPOTHESIS Biceps tendon pathology is common with rotator cuff tears. The mechanisms for biceps changes, and therefore its optimal treatment, are unknown. Our objective was to determine the effect of rotator cuff tears on regional biceps tendon pathology. We hypothesized that histologic and compositional changes would appear before organizational changes, both would appear before mechanical changes, and changes would begin at the tendons insertion site. MATERIALS AND METHODS Detachment of supraspinatus and infraspinatus tendons or sham surgery was done in 65 Sprague-Dawley rats. Rats were euthanized at 1, 4, or 8 weeks for regional measurements of histologic, compositional, organizational (1, 4 and 8 weeks), or mechanical properties (4 and 8 weeks only). RESULTS One week after tendon detachments, decreased organization and more rounded cell shape were found in the intra-articular space of the biceps tendon. Aggrecan expression was increased along the entire length of the tendon, whereas all other compositional changes were only at the tendons proximal insertion into bone. With time, this disorganization and more rounded cell shape extended the length of the tendon. Organizational and cell shape changes also preceded detrimental mechanical changes: decreased modulus in the intra-articular space was found after 8 weeks. CONCLUSIONS Results support a degenerative component to pathology in the biceps tendon. In addition, changes resembling a tendon exposed to compressive loading occurring first in the intra-articular space indicate that the biceps tendon plays an increased role as a load-bearing structure against the humeral head in the presence of rotator cuff tears.

The effect of altered loading following rotator cuff tears in a rat model on the regional mechanical properties of the long head of the biceps tendon

Biceps tendon pathology is a common clinical problem often seen in conjunction with rotator cuff tears. A previous study found detrimental changes to biceps tendons in the presence of rotator cuff tears in a rat model. Therefore, the objective of this study was to utilize this model along with models of altered loading to investigate the effect of altered loading on the initiation of these detrimental changes. We created supraspinatus and infraspinatus rotator cuff tears in the rat and followed these tears with either increased or decreased loading. Mechanical properties were determined along the length of the biceps tendon 4 and 8 weeks following injury. At the insertion site, stiffness increased with decreased loading, while detrimental changes were seen with increased loading 4 weeks following detachments. Increased loading resulted in decreased mechanical properties along the entire tendon length at both time points. Decreased loading resulted in both increased and decreased tendon properties at different regions of the tendon at 4 weeks, but by 8 weeks, there were no differences between decreased loading and detachment alone. We could not conclude where changes begin in the tendon with altered loading, but did demonstrate that regional differences exist. These results support that there is an effect of altered loading, as decreased loading resulted in variable changes at 4 weeks that were no different from detachment alone by 8 weeks, and increased loading resulted in detrimental properties along the entire length at both 4 and 8 weeks.

Fibronectin splice variation in human knee cartilage, meniscus and synovial membrane: Observations in osteoarthritic knee

Fibronectin (FN) is a widely expressed molecule that can participate in development of osteoarthritis (OA) affecting cartilage, meniscus, and synovial membrane (SM). The alternatively spliced isoforms of FN in joint tissues other than cartilage have not been extensively studied previously. The present study compares FN splice variation in patients with varying degrees of osteoarthritic change. Joint tissues were collected from asymptomatic donors and patients undergoing arthroscopic procedures. Total RNA was amplified by PCR using primers flanking alternatively spliced Extra Domain A (EDA), Extra Domain B (EDB) and Variable (V) regions. EDB+, EDB− and EDA− and V+ variants were present in all joint tissues, while the EDA+ variant was rarely detected. Expression levels of EDB+ and EDV+ variants were similar in cartilage, synovium, and meniscal tissues. Synovial expression of V+ FN in arthroscopy patients varied with degree of cartilage degeneration. Two V− isoforms, previously identified in cartilage, were also present in SM and meniscus. Fibronectin splicing in meniscus and SM bears striking resemblance to that of cartilage. Expression levels of synovial V+ FN varied with degree of cartilage degeneration. V+ FN should be investigated as a potential biomarker of disease stage or progression in larger populations.

Decreased loading after rotator cuff tears leads to improved biceps tendon properties in a rat model

BACKGROUND The purpose of this study was to elucidate the mechanism of biceps tendon changes after rotator cuff tears. We hypothesized that increased loading on the biceps tendon after rotator cuff tears will result in further detrimental changes whereas decreased loading will result in increased organization and more normal tendon composition. In addition, we hypothesized that changes with altered loading will begin at the proximal insertion into bone and progress along the tendon length at later time points. MATERIALS AND METHODS Supraspinatus and infraspinatus tendon detachments in rats were followed by various loading protocols at various time points. Regional changes in cellularity, cell shape, collagen organization, and matrix proteins of the long head of the biceps tendon were determined by histologic measures and immunohistochemistry. RESULTS Increased loading after detachments resulted in more disorganized collagen after only 1 week and compositional changes by 4 weeks. By 8 weeks, decreased loading resulted in increased organization, decreased cellularity, a more elongated cell shape, and more normal tendon composition. Organizational changes with increased loading began in the intra-articular space and progressed along the tendon length with time. CONCLUSIONS Combined with previous findings of decreased mechanics with increased loading, these results show that increased compressive loading away from the proximal insertion into bone is a mechanism for biceps tendon pathology in the presence of rotator cuff tears. The striking improvements with decreased loading further support increased loading as a mechanism for biceps tendon pathology because removal of this load led to improvements in tendon histology, organization, and composition.

Biceps tendon properties worsen initially but improve over time following rotator cuff tears in a rat model

Damage to the biceps tendon is often seen in conjunction with rotator cuff tears. However, controversy exists regarding its role in the shoulder and its optimal treatment. A previous study determined that biceps tendons were detrimentally affected in the presence of rotator cuff tears in the rat model and this damage worsened over time. However, whether this damage progresses at later time points to provide a chronic model is unknown. The objective of this study was to determine the changes in the biceps tendon in the presence of a cuff tear over time. Our hypothesis was that histological, compositional, organizational, and mechanical properties would worsen with time. We detached the supraspinatus and infraspinatus tendons of 48 rats and evaluated these properties at 1, 4, 8, and 16 weeks postdetachment. Properties worsened through 8 weeks, but improved between 8 and 16 weeks. We therefore conclude that biceps tendon changes in this model are not truly chronic. Additionally, it has been shown that infraspinatus properties in this model return to normal by 16 weeks, when biceps properties improve, indicating that earlier repair of one or more of the rotator cuff tendons may lead to resolved pathology of the long head of the biceps tendon.

Maturation State and Matrix Microstructure Regulate Interstitial Cell Migration in Dense Connective Tissues

Few regenerative approaches exist for the treatment of injuries to adult dense connective tissues. Compared to fetal tissues, adult connective tissues are hypocellular and show limited healing after injury. We hypothesized that robust repair can occur in fetal tissues with an immature extracellular matrix (ECM) that is conducive to cell migration, and that this process fails in adults due to the biophysical barriers imposed by the mature ECM. Using the knee meniscus as a platform, we evaluated the evolving micromechanics and microstructure of fetal and adult tissues, and interrogated the interstitial migratory capacity of adult meniscal cells through fetal and adult tissue microenvironments with or without partial enzymatic digestion. To integrate our findings, a computational model was implemented to determine how changing biophysical parameters impact cell migration through these dense networks. Our results show that the micromechanics and microstructure of the adult meniscus ECM sterically hinder cell mobility, and that modulation of these ECM attributes via an exogenous matrix-degrading enzyme permits migration through this otherwise impenetrable network. By addressing the inherent limitations to repair imposed by the mature ECM, these studies may define new clinical strategies to promote repair of damaged dense connective tissues in adults.