Growth factor free, peptide-functionalized gelatin hydrogel promotes arteriogenesis and attenuates tissue damage in a murine model of critical limb ischemia.

Critical limb ischemia (CLI) occurs when blood flow is restricted through the arteries, resulting in ulcers, necrosis, and chronic wounds in the downstream extremities. The development of collateral arterioles (i.e. arteriogenesis), either by remodeling of pre-existing vascular networks or de novo growth of new vessels, can prevent or reverse ischemic damage, but it remains challenging to stimulate collateral arteriole development in a therapeutic context. Here, we show that a gelatin-based hydrogel, devoid of growth factors or encapsulated cells, promotes arteriogenesis and attenuates tissue damage in a murine CLI model. The gelatin hydrogel is functionalized with a peptide derived from the extracellular epitope of Type 1 cadherins. Mechanistically, these "GelCad" hydrogels promote arteriogenesis by recruiting smooth muscle cells to vessel structures in both ex vivo and in vivo assays. In a murine femoral artery ligation model of CLI, delivery of in situ crosslinking GelCad hydrogels was sufficient to restore limb perfusion and maintain tissue health for 14 days, whereas mice treated with gelatin hydrogels had extensive necrosis and autoamputated within 7 days. A small cohort of mice receiving the GelCad hydrogels were aged out to 5 months and exhibited no decline in tissue quality, indicating durability of the collateral arteriole networks. Overall, given the simplicity and off-the-shelf format of the GelCad hydrogel platform, we suggest it could have utility for CLI treatment and potentially other indications that would benefit from arteriole development.

Maintaining the balance: the critical role of plasmin activity in orthopedic surgery injury response.

The musculoskeletal system plays vital roles in the body, facilitating movement, protecting vital structures, and regulating hematopoiesis and mineral metabolism. Injuries to this system are common and can cause chronic pain, loss of range of motion, and disability. The acute phase response (APR) is a complex process necessary for surviving and repairing injured musculoskeletal tissue. To conceptualize the APR, it is useful to divide it into 2 distinct phases, survival and repair. During the survival-APR, a "damage matrix" primarily composed of fibrin, via thrombin activity, is produced to contain the zone of injury. Once containment is achieved, the APR transitions to the repair phase, where reparative inflammatory cells use plasmin to systematically remove the damage matrix and replace it with new permanent matrices produced by differentiated mesenchymal stem cells. The timing of thrombin and plasmin activation during their respective APR phases is crucial for appropriate regulation of the damage matrix. This review focuses on evidence indicating that inappropriate exuberant activation of plasmin during the survival-APR can result in an overactive APR, leading to an "immunocoagulopathy" that may cause "immunothrombosis" and death. Conversely, preclinical data suggest that too little plasmin activity during the repair-APR may contribute to failed tissue repair, such as a fracture nonunion, and chronic inflammatory degenerative diseases like osteoporosis. Future clinical studies are required to affirm these findings. Therefore, the temporal-spatial functions of plasmin in response to musculoskeletal injury and its pharmacologic manipulation are intriguing new targets for improving orthopedic care.

Sterility of Miniature C-arm Fluoroscopy in Hand and Upper Extremity Surgery.

Previous studies have demonstrated that sterile equipment is frequently contaminated intraoperatively, yet the incidence of miniature c-arm (MCA) contamination in hand and upper extremity surgery is unclear. To examine this incidence, a prospective study of MCA sterility in hand and upper extremity cases was performed in a hospital main operating room (MOR) ( n = 13) or an ambulatory surgery center operating room (AOR) ( n = 16) at a single tertiary care center. Case length, MCA usage parameters, and sterility of the MCA through the case were examined. We found that MOR surgical times trended toward significance ( p = 0.055) and that MOR MCAs had significantly more contamination prior to draping than AOR MCAs ( p < 0.001). In MORs and AORs, 46.2 and 37.5% of MCAs respectively were contaminated intraoperatively. In MORs and AORs, 85.7 and 80% of noncontaminated cases, respectively, used the above hand- table technique, while 50 and 83.3% of contaminated MOR and AOR cases, respectively, used a below hand-table technique. Similar CPT codes were noted in both settings. Thus, a high-rate of MCA intraoperative contamination occurs in both settings. MCA placement below the hand-table may impact intraoperative contamination, even to distant MCA areas. Regular sterilization of equipment and awareness of these possible risk factors could lower bacterial burden.

Variable Response to Antifibrinolytics Correlates with Blood-loss and Transfusion in Posterior Spinal Fusion.

PURPOSE: Posterior spinal fusion (PSF) activates the fibrinolytic protease plasmin, which is implicated in blood loss and transfusion. While antifibrinolytic drugs have improved blood loss and reduced transfusion, variable blood loss has been observed in similar PSF procedures treated with the same dose of antifibrinolytics. However, both the cause of this and the appropriate measures to determine antifibrinolytic efficacy during high-blood-loss spine surgery are unknown, making clinical trials to optimize antifibrinolytic dosing in PSF difficult. We hypothesized that patients undergoing PSF respond differently to antifibrinolytic dosing, resulting in variable blood loss, and that specific diagnostic markers of plasmin activity will accurately measure the efficacy of antifibrinolytics in PSF. METHODS: A prospective study of 17 patients undergoing elective PSF with the same dosing regimen of TXA was conducted. Surgery-induced plasmin activity was exhaustively analyzed in perioperative blood samples and correlated to measures of inflammation, bleeding, and transfusion. RESULTS: While markers of in vivo plasmin activation (PAP and D-dimer) suggested significant breakthrough plasmin activation and fibrinolysis (P < 0.01), in vitro plasmin assays, including TEG, did not detect plasmin activation. In vivo measures of breakthrough plasmin activation correlated with blood loss (R2 = 0.400, 0.264; P < 0.01), transfusions (R2 = 0.388; P < 0.01), and complement activation (R2 = 0.346, P < 0.05). CONCLUSIONS: Despite all patients receiving a high dose of TXA, its efficacy among patients was variable, indicated by notable intra-operative plasmin activity. Markers of in vivo plasmin activation best correlated with clinical outcomes. These findings suggest that the efficacy of antifibrinolytic therapy to inhibit plasmin in PSF surgery should be determined by markers of in vivo plasmin activation in future studies. LEVEL OF EVIDENCE: Level II-diagnostic.

Quantitative Analysis of Growth Factors From Cancellous Bone Graft Collected With a Reamer-Irrigator-Aspirator System From Native Long Bones Versus Previously Reamed Long Bones.

OBJECTIVE: Collection of bone graft with the Reamer-Irrigator-Aspirator (RIA) system has become common practice across the field of orthopaedic surgery. While RIA bone graft is typically obtained from native long bones, grafting material can likewise be harvested from long bones that have previously undergone the placement and removal of an intramedullary nail, a process termed re-reamed RIA (RRR). The purpose of this study was to evaluate the total protein and growth factor concentrations present in native-RIA (NR) compared with RRR samples. METHODS: NR and RRR bone grafts were collected intraoperatively with the RIA system and processed to evaluate both the aqueous and the hard tissue components. Total protein concentration and specific growth factors were analyzed using standard bicinchoninic acid and multiplex assays, respectively. Analyte levels were then normalized to the total amount of protein detected. RESULTS: Total protein levels were comparable between NR and RRR samples for both the aqueous filtrate and the hard tissue samples. When normalized, while levels of bone morphogenic protein-2 and vascular endothelial growth factor were comparable in the hard tissue component, the aqueous filtrate from the RRR sample was found to have elevated levels of growth factors, with bone morphogenic protein-2 reaching statistical significance. CONCLUSIONS: This study demonstrates that ample protein is found within both NR and RRR samples, with comparable or elevated levels of osteogenic growth factors found within RRR samples. Future, larger, prospective studies will be required to evaluate the osteogenic potential and clinical efficacy of NR and RRR cancellous bone grafts to validate their equivalency.

A Mini-Open Approach to Medial Pinning in Pediatric Supracondylar Humeral Fractures May Be Safer Than Previously Thought.

BACKGROUND: Displaced pediatric supracondylar humeral fractures (SCHFs) are stabilized after reduction by smooth pins. Although some SCHFs are biomechanically stable after lateral-only entry pinning (lateral pinning), an additional medial entry pin (cross-pinning) confers superior stabilization in some SCHFs. There is a recognized risk of iatrogenic ulnar nerve injury with medial entry pinning. The best existing evidence has estimated an iatrogenic ulnar nerve injury rate of approximately 3.4% in cross-pinning. In similar studies, the rate of iatrogenic nerve injury (all nerves) in lateral pinning is estimated at 1.9%. This study aimed to use a large, single-center, single-technique (mini-open) retrospective case series to determine the rate of iatrogenic ulnar nerve injury in cross-pinning. METHODS: Patients undergoing percutaneous cross-pinning via the mini-open technique for SCHFs from 2007 to 2017 were retrospectively reviewed. Injury characteristics, operative variables, fixation technique, and complications, such as iatrogenic nerve injury, were recorded. Patients who underwent operative treatment at another hospital, had no postoperative follow-up, or died due to polytrauma were excluded. RESULTS: In this study, 698 patients undergoing cross-pinning during the study period were identified. Patients treated with cross-pinning had severe fractures, including a total of 198 preoperative neurovascular injuries (28.4%), 32 patients (4.6%) with skin tenting, and 19 patients (2.7%) with open fractures. Iatrogenic nerve injury was reported in 3 cases (0.43%), all of which affected the ulnar nerve. In 2 of 3 cases of iatrogenic nerve injury, the ulnar nerve symptoms resolved at a mean follow-up of 15 weeks. CONCLUSIONS: The mini-open approach for medial pin insertion is safer than previous estimates. Here, in the largest single-center study of cross-pinning for SCHFs, the iatrogenic ulnar nerve injury rate of 0.43% was nearly 10 times lower than estimated rates from recent meta-analyses. Considering all nerves, the iatrogenic injury rate for this cross-pinning cohort was also lower than the estimated iatrogenic nerve injury rate for lateral pinning. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

The Deleterious Effects of Impaired Fibrinolysis on Skeletal Development Are Dependent on Fibrin(ogen), but Independent of Interlukin-6.

Chronic diseases in growing children, such as autoimmune disorders, obesity, and cancer, are hallmarked by musculoskeletal growth disturbances and osteoporosis. Many of the skeletal changes in these children are thought to be secondary to chronic inflammation. Recent studies have likewise suggested that changes in coagulation and fibrinolysis may contribute to musculoskeletal growth disturbances. In prior work, we demonstrated that mice deficient in plasminogen, the principal protease of degrading and clearing fibrin matrices, suffer from inflammation-driven systemic osteoporosis and that elimination of fibrinogen resulted in normalization of IL-6 levels and complete rescue of the skeletal phenotype. Given the intimate link between coagulation, fibrinolysis, and inflammation, here we determined if persistent fibrin deposition, elevated IL-6, or both contribute to early skeletal aging and physeal disruption in chronic inflammatory conditions. Skeletal growth as well as bone quality, physeal development, and vascularity were analyzed in C57BL6/J mice with plasminogen deficiency with and without deficiencies of either fibrinogen or IL-6. Elimination of fibrinogen, but not IL-6, rescued the skeletal phenotype and growth disturbances in this model of chronic disease. Furthermore, the skeletal phenotypes directly correlated with both systemic and local vascular changes in the skeletal environment. In conclusion, these results suggest that fibrinolysis through plasmin is essential for skeletal growth and maintenance, and is multifactorial by limiting inflammation and preserving vasculature.

Surgical Technique: Closed Reduction and Percutaneous Pinning of Posterolaterally Displaced Supracondylar Humerus Fractures.

SUMMARY: Gartland type III posterolateral (IIIB) supracondylar humerus fractures are common among the pediatric population and can lead to concomitant injury, including compromise of the brachial artery and median nerve and long-term deformity, such as cubitus varus. These fractures can be difficult to reduce, and there is little consensus regarding the optimal technique for closed reduction and percutaneous pinning. Here, we discuss the management of Gartland III posterolateral supracondylar humerus fractures, including an in-depth technical description of the methods of operative fixation. We describe a lateral pin-only fixation technique for Gartland III posterolateral supracondylar humerus fractures that uses the intact periosteum during reduction of the distal fragment to assist in realigning the medial and lateral columns anatomically. We also discuss a safe method for placing a medial-based pin if there is persistent rotational instability at the fracture site after placement of the laterally based pins.

The Heterogeneity of Pediatric Knee Infections: A Retrospective Analysis.

BACKGROUND: Musculoskeletal infection is a major cause of morbidity in the pediatric population. Despite the canonical teaching that an irritable joint and signs of infection likely represent an infected joint space, recent evidence in the pediatric hip has demonstrated that alternative diagnoses are equally or more likely and that combinations of pathologies are common. The knee is the second most commonly infected joint in children, yet there remains a paucity of available data regarding the epidemiology and workup of the infected pediatric knee. The authors hypothesize that there is heterogeneity of pathologies, including combinations of pathologies, that presents as a potentially infected knee in a child. The authors aim to show the utility of magnetic resonance imaging and epidemiologic and laboratory markers in the workup of these patients. METHODS: A retrospective review of all consults made to the pediatric orthopaedic surgery team at a single tertiary care center from September 2009 through December 2015 regarding a concern for potential knee infection was performed. Excluded from the study were patients with penetrating trauma, postoperative infection, open fracture, no C-reactive protein (CRP) within 24 hours of admission, sickle cell disease, an immunocompromised state, or chronic osteomyelitis. RESULTS: A total of 120 patients were analyzed in this study. There was marked variability in pathologies. Patients with isolated osteomyelitis or osteomyelitis+septic arthritis were older, had an increased admission CRP, were more likely to be infected with Staphylococcus aureus, required an increased duration of antibiotics, and had an increased incidence of musculoskeletal complications than patients with isolated septic arthritis. CONCLUSIONS: When considering a child with an irritable knee, a heterogeneity of potential underlying pathologies and combinations of pathologies are possible. Importantly, the age of the patient and CRP can guide a clinician when considering further workup. Older patients with a higher admission CRP value warrant an immediate magnetic resonance imaging, as they are likely to have osteomyelitis, which was associated with worse outcomes when compared with patients with isolated septic arthritis. LEVEL OF EVIDENCE: Level III-retrospective research study.

The Size of Intramedullary Fixation Affects Endochondral-Mediated Angiogenesis During Fracture Repair.

OBJECTIVES: To explore the effect of intramedullary pin size on the biology of a healing fracture, specifically endochondral angiogenesis. We hypothesized that fracture fixation with a smaller pin would permit greater interfragmentary strain resulting in increased total amount of vascular endothelial growth factor within the callus and greater angiogenesis compared to fixation with a larger pin. METHODS: Transverse mid-shaft femur fractures in 8-week-old mice were fixed with either a 23-gauge (G) or 30-G pin. Differences in interfragmentary strain at the fracture site were estimated between cohorts. A combination of histology, gene expression, serial radiography, and microcomputed tomography with and without vascular contrast agent were used to assess fracture healing and vascularity for each cohort. RESULTS: Larger soft-tissue callus formation increased vascular endothelial growth factor-A expression, and a corresponding increase in vascular volume was observed in the higher strain, 30-G cohort. Radiographic analysis demonstrated earlier hard callus formation with greater initial interfragmentary strain, similar rates of union between pin size cohorts, yet delayed callus remodeling in mice with the larger pin size. CONCLUSIONS: These findings suggest that the stability conferred by an intramedullary nail influences endochondral angiogenesis at the fracture.

Autologous chondrocyte grafting promotes bone formation in the posterolateral spine.

BACKGROUND CONTEXT: Pseudarthrosis following spinal fusion remains problematic despite modern surgical and grafting techniques. In surgical spinal fusion, new bone forms via intramembranous and endochondral ossification, with endochondral ossification occurring in the hypoxic zones of the fusion bed. During bone development and fracture healing, the key cellular mediator of endochondral ossification is the hypertrophic chondrocyte given its ability to function in hypoxia and induce neovascularization and ossification. We therefore hypothesize that hypertrophic chondrocytes may be an effective bone graft alternative. PURPOSE: Spinal fusion procedures have increased substantially; yet 5% to 35% of all spinal fusions may result in pseudoarthrosis. Pseudoarthrosis may occur because of implant failure, infection, or biological failure, among other reasons. Advances in surgical techniques and bone grafting have improved fusion; however pseudarthrosis rates remain unacceptably high. Thus, the goal of this study is to investigate hypertrophic chondrocytes as a potential biological graft alternative. METHODS: Using a validated murine fracture model, hypertrophic chondrocytes were harvested from fracture calluses and transplanted into the posterolateral spines of identical mice. New bone formation was assessed by X-ray, microcomputed tomography (µCT), and in vivo fluorescent imaging. Results were compared against a standard iliac crest bone graft and a sham surgery control group. Funding for this work was provided by the Department of Orthopaedics and Rehabilitation, the OREF (Grant #16-150), and The Caitlin Lovejoy Fund. RESULTS: Radiography, µCT, and in vivo fluorescent imaging demonstrated that hypertrophic chondrocytes promoted bone formation at rates equivalent to iliac crest autograft. Additionally, µCT analysis demonstrated similar fusion rates in a subset of mice from the iliac crest and hypertrophic chondrocyte groups. CONCLUSIONS: This proof-of-concept study indicates that hypertrophic chondrocytes can promote bone formation comparable to iliac crest bone graft. These findings provide the foundation for future studies to investigate the potential therapeutic use of hypertrophic chondrocytes in spinal fusion.