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.

Programmed biomolecule delivery to enable and direct cell migration for connective tissue repair.

Dense connective tissue injuries have limited repair, due to the paucity of cells at the wound site. We hypothesize that decreasing the density of the local extracellular matrix (ECM) in conjunction with releasing chemoattractive signals increases cellularity and tissue formation after injury. Using the knee meniscus as a model system, we query interstitial cell migration in the context of migratory barriers using a novel tissue Boyden chamber and show that a gradient of platelet-derived growth factor-AB (PDGF-AB) expedites migration through native tissue. To implement these signals in situ, we develop nanofibrous scaffolds with distinct fiber fractions that sequentially release active collagenase (to increase ECM porosity) and PDGF-AB (to attract endogenous cells) in a localized and coordinated manner. We show that, when placed into a meniscal defect, the controlled release of collagenase and PDGF-AB increases cellularity at the interface and within the scaffold, as well as integration with the surrounding tissue.

Engineering meniscus structure and function via multi-layered mesenchymal stem cell-seeded nanofibrous scaffolds.

Despite advances in tissue engineering for the knee meniscus, it remains a challenge to match the complex macroscopic and microscopic structural features of native tissue, including the circumferentially and radially aligned collagen bundles essential for mechanical function. To mimic this structural hierarchy, this study developed multi-lamellar mesenchymal stem cell (MSC)-seeded nanofibrous constructs. Bovine MSCs were seeded onto nanofibrous scaffolds comprised of poly(ε-caprolactone) with fibers aligned in a single direction (0° or 90° to the scaffold long axis) or circumferentially aligned (C). Multi-layer groups (0°/0°/0°, 90°/90°/90°, 0°/90°/0°, 90°/0°/90°, and C/C/C) were created and cultured for a total of 6 weeks under conditions favoring fibrocartilaginous tissue formation. Tensile testing showed that 0° and C single layer constructs had stiffness values several fold higher than 90° constructs. For multi-layer groups, the stiffness of 0°/0°/0° constructs was higher than all other groups, while 90°/90°/90° constructs had the lowest values. Data for collagen content showed a general positive interactive effect for multi-layers relative to single layer constructs, while a positive interaction for stiffness was found only for the C/C/C group. Collagen content and cell infiltration occurred independent of scaffold alignment, and newly formed collagenous matrix followed the scaffold fiber direction. Structural hierarchies within multi-lamellar constructs dictated biomechanical properties, and only the C/C/C constructs with non-orthogonal alignment within layers featured positive mechanical reinforcement as a consequence of the layered construction. These multi-layer constructs may serve as functional substitutes for the meniscus as well as test beds to understand the complex mechanical principles that enable meniscus function.

Prospective intraoperative syndesmotic evaluation during ankle fracture fixation: stress external rotation versus lateral fibular stress.

OBJECTIVES: We hypothesized that the method of stress external rotation more accurately reproduces the mechanism of injury, and therefore this diagnostic method more likely detects ankle instability than the fibular stress examination. DESIGN: Prospective cohort comparison study. SETTING: Level 1 trauma center. PATIENTS: Twenty-eight consecutive patients with unstable ankle fractures presenting within 7 days from the time of injury. Previous ankle surgical history or age younger than 18 years was excluded. INTERVENTION: Stress external rotation and lateral fibular stress examination was performed intraoperatively. MAIN OUTCOME MEASURE: Radiographic measurement of the tibiofibular clear space, tibiofibular overlap, and medial clear space were recorded. RESULTS: After normalization of the fluoroscopic measurements, there was no difference in detecting changes in tibiofibular clear space or tibiofibular overlap. However, there was a significant difference in detecting medial clear space widening with stress external rotation. Compared with lateral fibular stress, stress external rotation demonstrated a 35% increase (P < 0.05) in medial clear space widening. This difference correlates with the 1-2-mm difference of additional widening with stress external rotation. CONCLUSIONS: Untreated instability impacts patient outcomes. The difference in widening with stress external rotation was significantly greater than lateral fibular stress and appreciable on standard fluoroscopic views. Stress external rotation radiographs are a more reliable indicator of mortise instability than traditional lateral fibular stress. LEVEL OF EVIDENCE: Diagnostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Repair of dense connective tissues via biomaterial-mediated matrix reprogramming of the wound interface.

Repair of dense connective tissues in adults is limited by their intrinsic hypocellularity and is exacerbated by a dense extracellular matrix (ECM) that impedes cellular migration to and local proliferation at the wound site. Conversely, healing in fetal tissues occurs due in part to an environment conducive to cell mobility and division. Here, we investigated whether the application of a degradative enzyme, collagenase, could reprogram the adult wound margin to a more fetal-like state, and thus abrogate the biophysical impediments that hinder migration and proliferation. We tested this concept using the knee meniscus, a commonly injured structure for which few regenerative approaches exist. To focus delivery and degradation to the wound interface, we developed a system in which collagenase was stored inside poly(ethylene oxide) (PEO) electrospun nanofibers and released upon hydration. Through a series of in vitro and in vivo studies, our findings show that partial digestion of the wound interface improves repair by creating a more compliant and porous microenvironment that expedites cell migration to and/or proliferation at the wound margin. This innovative approach of targeted manipulation of the wound interface, focused on removing the naturally occurring barriers to adult tissue repair, may find widespread application in the treatment of injuries to a variety of dense connective tissues.

Biomaterial-mediated delivery of degradative enzymes to improve meniscus integration and repair.

Endogenous repair of fibrous connective tissues is limited, and there exist few successful strategies to improve healing after injury. As such, new methods that advance repair by promoting cell growth, extracellular matrix (ECM) production, and tissue integration would represent a marked clinical advance. Using the meniscus as a test platform, we sought to develop an enzyme-releasing scaffold that enhances integrative repair. We hypothesized that the high ECM density and low cellularity of native tissue present physical and biological barriers to endogenous healing, and that localized collagenase treatment might expedite cell migration to the wound edge and tissue remodeling. To test this hypothesis, we fabricated a delivery system in which collagenase was stored inside electrospun poly(ethylene oxide) (PEO) nanofibers and released upon hydration. In vitro results showed that partial digestion of the wound interface improved repair by creating a microenvironment that facilitated cell migration, proliferation and matrix deposition. Specifically, treatment with high-dose collagenase led to a 2-fold increase in cell density at the wound margin and a 2-fold increase in integrative tissue compared to untreated controls at 4 weeks (P≤0.05). Furthermore, when composite scaffolds containing both collagenase-releasing and structural fiber fractions were placed inside meniscal tears in vitro, enzyme release acted locally and resulted in a positive cellular response similar to that of global treatment with aqueous collagenase. This innovative approach to targeted enzyme delivery may aid the many patients that exhibit meniscal tears by promoting integration of the defect, thereby circumventing the pathologic consequences of partial meniscus removal, and may find widespread application in the treatment of injuries to a variety of dense connective tissues.

Organized nanofibrous scaffolds that mimic the macroscopic and microscopic architecture of the knee meniscus.

The menisci are crescent-shaped fibrocartilaginous tissues whose structural organization consists of dense collagen bundles that are locally aligned but show a continuous change in macroscopic directionality. This circumferential patterning is necessary for load transmission across the knee joint and is a key design parameter for tissue engineered constructs. To address this issue we developed a novel electrospinning method to produce scaffolds composed of circumferentially aligned (CircAl) nanofibers, quantified their structure and mechanics, and compared them with traditional linearly aligned (LinAl) scaffolds. Fibers were locally oriented in CircAl scaffolds, but their orientation varied considerably as a function of position (P<0.05). LinAl fibers did not change in orientation over a similar length scale (P>0.05). Cell seeding of CircAl scaffolds resulted in a similar cellular directionality. Mechanical analysis of CircAl scaffolds revealed significant interactions between scaffold length and region (P<0.05), with the tensile modulus near the edge of the scaffolds decreasing with increasing scaffold length. No such differences were detected in LinAl specimens (P>0.05). Simulation of the fiber deposition process produced "theoretical" fiber populations that matched the fiber organization and mechanical properties observed experimentally. These novel scaffolds, with spatially varying local orientations and mechanics, will enable the formation of functional anatomic meniscus constructs.

Sacrificial nanofibrous composites provide instruction without impediment and enable functional tissue formation.

The fibrous tissues prevalent throughout the body possess an ordered structure that underlies their refined and robust mechanical properties. Engineered replacements will require recapitulation of this exquisite architecture in three dimensions. Aligned nanofibrous scaffolds can dictate cell and matrix organization; however, their widespread application has been hindered by poor cell infiltration due to the tight packing of fibers during fabrication. Here, we develop and validate an enabling technology in which tunable composite nanofibrous scaffolds are produced to provide instruction without impediment. Composites were formed containing two distinct fiber fractions: slow-degrading poly(ε-caprolactone) and water-soluble, sacrificial poly(ethylene oxide), which can be selectively removed to increase pore size. Increasing the initial fraction of sacrificial poly(ethylene oxide) fibers enhanced cell infiltration and improved matrix distribution. Despite the removal of >50% of the initial fibers, the remaining scaffold provided sufficient instruction to align cells and direct the formation of a highly organized ECM across multiple length scales, which in turn led to pronounced increases in the tensile properties of the engineered constructs (nearly matching native tissue). This approach transforms what is an interesting surface phenomenon (cells on top of nanofibrous mats) into a method by which functional, 3D tissues (>1 mm thick) can be formed, both in vitro and in vivo. As such, this work represents a marked advance in the engineering of load-bearing fibrous tissues, and will find widespread applications in regenerative medicine.

Fabrication and evaluation of biomimetic-synthetic nanofibrous composites for soft tissue regeneration.

Electrospun scaffolds hold promise for the regeneration of dense connective tissues, given their nanoscale topographies, provision of directional cues for infiltrating cells and versatile composition. Synthetic slow-degrading scaffolds provide long-term mechanical support and nanoscale instructional cues; however, these scaffolds suffer from a poor infiltration rate. Alternatively, nanofibrous constructs formed from natural biomimetic materials (such as collagen) rapidly infiltrate but provide little mechanical support. To take advantage of the positive features of these constructs, we have developed a composite scaffold consisting in both a biomimetic fiber fraction (i.e., Type I collagen nanofibers) together with a traditional synthetic (i.e., poly-[ε-caprolactone], PCL) fiber fraction. We hypothesize that inclusion of biomimetic elements will improve initial cell adhesion and eventual scaffold infiltration, whereas the synthetic elements will provide controlled and long-term mechanical support. We have developed a method of forming and crosslinking collagen nanofibers by using the natural crosslinking agent genipin (GP). Further, we have formed composites from collagen and PCL and evaluated the long-term performance of these scaffolds when seeded with mesenchymal stem cells. Our results demonstrate that GP crosslinking is cytocompatible and generates stable nanofibrous type I collagen constructs. Composites with varying fractions of the biomimetic and synthetic fiber families are formed and retain their collagen fiber fractions during in vitro culture. However, at the maximum collagen fiber fractions (20%), cell ingress is limited compared with pure PCL scaffolds. These results provide a new foundation for the development and optimization of biomimetic/synthetic nanofibrous composites for in vivo tissue engineering.

Venous thromboembolism in patients with blunt trauma: are comprehensive guidelines the answer?

OBJECTIVES: This study was designed to determine the outcome of implemented guidelines for venous thromboembolism (VTE) prophylaxis. METHODS: This study was a retrospective review of a series of consecutive blunt orthopaedic trauma patients with thromboembolic complications. The patients were compared to control subjects over the same 10-year period. Univariate and multivariate statistical methods were used to determine the odds of VTE in the setting of this management guideline and risk factors for thromboembolic complications that may be refractory to this strategy. RESULTS: In the 10 years following institution of clinical management guidelines at our institution, the rate of VTE events was 3.2%, and the rate of pulmonary embolus was 0.3%. Risk factors for VTE that were refractory to our clinical management guidelines were pelvic fractures, major lower extremity injury, greater than 3 days of mechanical ventilation, increasing injury severity, and spinal cord injury. CONCLUSIONS: The implementation of a clinical management strategy for decreasing the incidence of VTE in blunt trauma patients and other potentially preventable complications is essential. Our data suggest that patients with certain injuries are particularly at risk for VTE and warrant special attention in clinical management and risk stratification, despite effective clinical management guidelines.

Delay in surgical débridement of open tibia fractures: an analysis of national practice trends.

BACKGROUND: Débridement and irrigation (D&I) of open tibia fractures less than 6 hours from the time of injury has been promoted as orthopaedic dogma despite limited evidence. The goal of this study was to determine the duration between emergency room presentation and D&I in open tibia fractures and to examine factors associated with delay in treatment. METHODS: The National Trauma Data Bank Version 3.0 identified 6099 blunt trauma patients with open tibia fractures. Time was calculated from emergency room arrival to first D&I. Risk factors associated with delay in treatment greater than 6 hours and greater than 24 hours were then calculated using univariate and multivariate statistical methods. RESULTS: Median time to D&I was 4.9 hours. Forty-two percent of patients with open tibia fractures experienced a delay in treatment of greater than 6 hours and 24% of patients experienced a delay to treatment of greater than 24 hours. Risk factors associated with greater than 6- and 24-hour delay on univariate and multivariate logistic regression were age, head or thoracic injury with Abbreviated Injury Score greater than 2, and presentation between 6 pm and 2 am. Level I and university hospitals carry a greater risk of delay that was independent of injury severity in multivariate analysis. CONCLUSIONS: A significant percentage of patients with open tibia fractures undergo their first surgical intervention of D&I greater than 6 hours after presentation to the emergency room. Patients with delayed D&I have more severe injuries, are treated at university or Level I centers, and present later in the day.

Does morbid obesity negatively affect the hospital course of patients undergoing treatment of closed, lower-extremity diaphyseal long-bone fractures?

Obesity is prevalent in the developed world and is associated with significant costs to the health care system. The effect of morbid obesity in patients operatively treated for long-bone fractures of the lower extremity is largely unknown. The National Trauma Data Bank was accessed to determine if morbidly obese patients (body mass index >40) with lower extremity fractures have longer length of hospital stay, higher cost, greater rehabilitation admission rates, and more complications than nonobese patients. We identified patients with operatively treated diaphyseal femur (6920) and tibia (5190) fractures. Polytrauma patients and patients younger than 16 years were excluded. Morbidly obese patients were identified by ICD-9 and database comorbidity designation (femur, 131 morbidly obese; tibia, 75 morbidly obese). Patients meeting these criteria who were not morbidly obese were used as controls. Sensitivity analyses were performed to analyze patients with isolated trauma to the tibia or femur. Morbidly obese patients were more likely to be admitted to a subacute facility. Length of stay trended higher in morbidly obese patients. There was no significant relationship between obesity and inpatient mortality or inpatient complications. These trends held true when considering patients with multiple injuries and patients who had isolated long-bone injuries. Our study showed that morbidly obese patients may have greater rehabilitation needs following long-bone fractures in the lower extremity. Our study showed no difference in mortality or complications, although further studies are needed to confirm these findings.

Is upper extremity trauma an independent risk factor for lower extremity venous thromboembolism? An 11-year experience at a Level I trauma center.

INTRODUCTION: Venous thromboembolism (VTE) is an important problem in orthopedic trauma patients. An association between VTE and upper extremity injury has not been reported. The purpose of this investigation was to determine whether upper extremity trauma is an independent risk factor for lower extremity VTE. This study also attempted to identify associations between VTEs and demographic and injury variables in patients that sustained upper extremity trauma. METHODS: Eleven years of data from the trauma registry at our Level I trauma center was retrospectively reviewed in an injury-matched cohort study. From an initial pool of 646 patients who sustained upper extremity trauma, 32 subjects (4.95%) were identified as having major upper extremity injuries as well as thromboembolic complications. Thirty-two injury-matched controls were randomly selected from the 646 patients with major upper extremity injuries. Regression analysis was performed to determine variables that were significantly associated with lower extremity thromboembolic complications. RESULTS: Overall incidence of VTE in patients sustaining upper extremity injury was 4.95% (deep vein thrombus 4.64%, pulmonary embolism 0.31%) and was similar to the 4.95% VTE rate in patients without upper extremity injury. Major head injury (p = 0.022) occurred at increased frequency in the VTE group. Patients with increased length of hospital stay (p < 0.001) and length of time on a ventilator (p = 0.002) were at significantly higher risk for thromboembolic complications. No patient with isolated upper extremity trauma had complications from VTE. CONCLUSION: Lower extremity VTE occurs at similar rates in patients sustaining upper extremity injury compared to those patients that do not. Major upper extremity orthopedic trauma is not an independent risk factor for lower extremity VTE, and current clinical management guidelines for VTE prophylaxis are adequate for patients sustaining major upper extremity trauma.

Use of medical comorbidities to predict complications after hip fracture surgery in the elderly.

BACKGROUND: Comorbidities before and complications following hip fracture surgery can impact the return of function. We hypothesized that the American Society of Anesthesiologists (ASA) classification of medical comorbidities is a useful surrogate variable for the patient's general medical condition and would be a strong predictor of perioperative medical complications following hip fracture surgery. METHODS: A retrospective review of the cases of 197 elderly patients who had undergone operative management of a hip fracture was performed. The ASA class, data regarding perioperative medical and surgical complications, and demographic data were obtained. Medical complications were defined as those requiring intervention by an internist or medical specialist. Differences in complication rates among the ASA classes were determined. RESULTS: Medical complications were more common in patients in ASA class 3 (p < 0.001) and those in class 4 (p = 0.001) than in those in class 2. Patients in ASA class 3 had a 3.78 times greater chance of having a medical complication than did those in class 2 (p < 0.001). Patients in ASA class 4 had a 7.39 times greater chance of having medical complications than did those in class 2 (p = 0.001). No significant relationship was identified between the ASA class and surgical complications. CONCLUSIONS: The ASA class is strongly associated with medical problems in the perioperative period following hip fracture surgery in the elderly. Patients identified as being at higher risk (in ASA class 3 or 4) preoperatively should be closely managed medically so that perioperative medical complications can be managed and evolving medical issues can be addressed in a timely fashion.

Open tibial shaft fractures: II. Definitive management and limb salvage.

Definitive treatment of open fractures of the tibial diaphysis is challenging. The high-energy nature of these fractures, as well as the contamination of the fracture site and devitalization of the soft-tissue envelope, greatly increases the risk of infection, nonunion, and wound complications. The goals of definitive treatment include wound coverage or closure; prevention of infection; restoration of length, alignment, rotation, and stability; fracture healing; and return of function. Advances in orthobiologics, modern plastic surgical techniques, and fracture stabilization methods, most notably locked intramedullary nailing, have led to improved prognosis for functional recovery and limb salvage. Despite improved union and limb salvage rates, the prognosis for severe type III open fracture of the tibial shaft remains guarded, and outcomes are often determined by patient psychosocial variables.

Open tibial shaft fractures: I. Evaluation and initial wound management.

Open fractures of the tibial diaphysis are often associated with severe bone and soft-tissue injury. Contamination of the fracture site and devitalization of the soft-tissue envelope greatly increase the risk of infection, nonunion, and wound complications. Management of open tibial shaft fractures begins with a thorough patient evaluation, including assessment of the bone and soft tissue surrounding the tibial injury. Classification of these injuries according to the system of Gustilo and Anderson at the time of surgical débridement is useful in guiding treatment and predicting outcomes. Administration of antibiotic prophylaxis as soon as possible after injury as well as urgent and thorough débridement, irrigation, and bony stabilization are done to minimize the risk of infection and improve outcomes. The use of antibiotic bead pouches and negative-pressure wound therapy has proved to be efficacious for the acute, temporary management of severe bone and soft-tissue defects.

Compartment syndrome in tibial fractures.

OBJECTIVES: Compartment syndrome is a devastating complication of tibial fractures. The purpose of this study was to investigate the rate of clinically determined compartment syndrome requiring surgical intervention in tibial fractures by anatomical region and to identify the associated patient and injury factors. DESIGN: Retrospective cohort. SETTING: University level I trauma center. PATIENTS/PARTICIPANTS: Acute tibial fractures in 414 patients from January 1, 2004 through October 31, 2006. METHODS: Tibial fractures in 414 patients met the inclusion and exclusion criteria. The fractures were classified into 3 groups (proximal, diaphyseal, and distal) based on the anatomic location of the fractures (AO/OTA fractures 41, 42, and 43, respectively). To determine the patient and injury factors associated with the development of compartment syndrome in tibial fractures, the following data were obtained: patient age and sex, mechanism of injury, presence of associated fractures, presence of concomitant head/chest/abdominal/pelvic injury, blood pressure upon admission, open versus closed fracture (Gustilo-Anderson classification if open), status of the fibula, and AO/OTA classification of the tibial fracture. MAIN OUTCOME MEASURES: Rate of clinically determined compartment syndrome requiring fasciotomy by anatomical region of the tibia. RESULTS: The rate of compartment syndrome was highest in the diaphyseal group (8.1%, P < 0.05) followed by proximal (1.6%) and distal (1.4%) groups. The diaphyseal group was further analyzed according to patient and injury factors. Patients who developed compartment syndrome were significantly younger (27.5 years +/- 11.7 SD versus 39.0 years +/- 16.7 SD, P = 0.003, Student t test) than those who did not develop compartment syndrome. The mean arterial pressures upon admission of the patients who developed compartment syndrome were also found to be slightly higher (107 versus 98.5 mm Hg, P = 0.039, Student t test) but not significantly so after Bonferroni adjustment. In multivariate regression analysis, decreasing age remained the only statistically significant independent predictor for the development of compartment syndrome (P = 0.006, regression coefficient = -0.0589) in diaphyseal tibial fractures. CONCLUSIONS: Tibial fractures of the diaphysis are more frequently associated with development of compartment syndrome than proximal or distal tibial fractures. More specifically, young patients with diaphyseal fractures are at risk for developing this complication and warrant increased vigilance and suspicion for compartment syndrome. A prospective study with sufficient power is needed to further identify risk factors associated with compartment syndrome in tibial fractures.