Temporal and spatial expression of TGF-beta1 in an Achilles tendon section model after application of platelet-rich plasma.

BACKGROUND: To investigate the effect of platelet-rich plasma (PRP) on TGF-beta1 expression during tendon healing. METHODS: We used 48 skeletally mature New Zealand White rabbits. 24 rabbits received the PRP, and 24 rabbits served as an untreated control group. Equal numbers of animals were sacrificed at 1st, 2nd, 3rd, and 4th week. The surgical procedure involved a transverse incision to transect the Achilles tendon. A volume of 1ml of PRP was then injected into the tendon mass in the PRP group. Histological and immunohistochemical evaluations with an anti-TGF-beta primary antibody were performed. RESULTS: The pattern of expression of TGF-beta1 in the PRP group was characterized by a significant upregulation during the first 2 weeks and subsequently significant downregulation in the 3rd and 4th week in comparison with the controls. CONCLUSIONS: Our results suggest that PRP may affect the tendon healing process by altering the expression of TGF-beta1.

Effects of long-term immobilization and recovery on human triceps surae and collagen turnover in the Achilles tendon in patients with healing ankle fracture.

The aim of the present study was to analyze how human tendon connective tissue responds to an approximately 7-wk period of immobilization and a remobilization period of a similar length, in patients with unilateral ankle fracture, which is currently unknown. Calf muscle cross-sectional area (CSA) decreased by 15% (5,316 to 4,517 mm2) and strength by 54% (239 to 110 N.m) in the immobilized leg after 7 wk. During the 7-wk remobilization, the CSA increased by 9% (to 4,943 mm2) and strength by 37% (to 176 Nm). Achilles tendon CSA did not change significantly during either immobilization or remobilization. Local collagen turnover was measured as the peritendinous concentrations of NH2-terminal propeptide of type I collagen (PINP) and COOH-terminal telopeptide region of type I collagen (ICTP), markers thought to be indexes of type I collagen synthesis and degradation, respectively. Both markers were increased (PINP: 257 vs. 56 ng/ml; ICTP: 9.8 vs. 2.1 microg/l) in the immobilized leg compared with the control leg after the 7 wk of immobilization, and levels decreased again in the immobilized leg during the recovery period (PINP: 103 vs. 44 ng/ml; ICTP: 4.2 vs. 1.9 microg/l). A significant reduction in calf muscle CSA and strength was found in relation to 7 wk of immobilization. Immobilization increased both collagen synthesis and degradation in tendon near tissue. However, it cannot be excluded that the facture of the ankle in close proximity could have affected these data. Remobilization increased muscle size and strength and tendon synthesis and degradation decreased to baseline levels. These dynamic changes in tendon connective tissue turnover were not associated with macroscopic changes in tendon size.

Implications of trauma and subsequent articulation on the release of Proteoglycan-4 and tissue response in adult human ankle cartilage.

The purpose of this study was to investigate the effects of trauma and subsequent articulation on adult human ankle cartilage subjected to an injurious impact. Trauma was initiated through impaction on talar cartilage explants. Articulation and loading were applied in a joint bioreactor over 5 consecutive days. The early (24 h) effects of impaction included a reduced chondrocytes viability (51% vs. 81% for non-impacted; p = 0.03), increased levels of apoptosis (43% vs. 27%; p = 0.03), and an increase in the histopathology score (4.4 vs. 1.7; p = 0.02) as compared to non-impacted cartilage explants. One of the key findings was that damage also stimulated the PRG4 release (2.2 vs. 1.5 µg/ml). Subsequent articulation for 5 days did not lead to further changes in tissue histopathology and cell viability, neither for injured nor non-injured samples. However, articulation led to an increased apoptosis in the injured samples (p = 0.03 for the interaction term). Articulation also caused a significant increase of PG/GAG release into the culture medium (p = 0.04) for both injured and non-injured samples; however, the synthesis of PG was not affected by articulation (p = 0.45) though the PG synthesis was higher in injured samples (p < 0.01). With regard to the PRG4 release, impacted samples continued to show higher amounts (p = 0.01), adding articulation led to a reduction (p = 0.02). The current study demonstrated that adult human talar cartilage increases both the PRG4 release and biosynthetic activity as an immediate cellular response to injury. Articulation played a less contributing role to biosynthesis and remodeling, behaving mostly neutral, in that no further damage emerged. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:667-676, 2017.

Inflammatory Cytokines and Matrix Metalloproteinases in the Synovial Fluid After Intra-articular Ankle Fracture.

BACKGROUND: Posttraumatic osteoarthritis (PTOA) can occur after intra-articular fracture despite anatomic fracture reduction. It has been hypothesized that an early inflammatory response after intra-articular injury could lead to irreversible cartilage damage that progresses to PTOA. Therefore, in addition to meticulous fracture reduction, it would be ideal to prevent this initial inflammatory response but little is known about the composition of the synovial environment after intra-articular fracture. The purpose of this work was to characterize the inflammatory cytokine and matrix metalloproteinase (MMP) composition in the synovial fluid (SF) of patients with acute intra-articular ankle fractures. METHODS: Twenty-one patients with an intra-articular ankle fracture were included in this study. All patients had a contralateral ankle joint that was pain free, had no radiographic evidence of arthritis, and no history of trauma. The uninjured ankle served as a matched control. SF was obtained from bilateral ankles at the time of surgery which occurred at a mean of 17 days post-fracture (range 8-40). The SF was analyzed for granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, IL-2, IL-6, IL-8, IL-10, IL-12p70, MMP-1, MMP-2, MMP-3, MMP-9, MMP-10, CTXII, sGAG, and bilirubin/biliverdin (markers of hemearthrosis) using either multiplex assay or ELISA using commercially available kits. Mean concentrations of each factor were compared between SF from fractured and control ankles, and correlation analysis was done to determine potential relationships between levels of cytokines and time from fracture and age at fracture. RESULTS: Twelve of 18 measured factors including GM-CSF, IL-10, IL-1ß, IL-6, IL-8, TNF-α, MMP-1, MMP-2, MMP-3, MMP-9, MMP-10, and bilirubin/biliverdin were found to be significantly higher in the fractured ankles. Mean concentrations of ECM degradation markers (sGAG and CTXII) were not found to be significatnly different between groups. CONCLUSION: These data indicate that after intra-articular ankle fracture the SF exhibits a largely pro-inflammatory and extra-cellular matrix degrading environment similar to that described in idiopathic osteoarthritis. IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, and MMP-10 were significantly elevated and may play a role in the development of PTOA. CLINICAL RELEVANCE: In addition to anatomic fracture reduction, these data lend credence to reducing acute intra-articular inflammation through the development of antagonists to these pro-inflammatory and degrading mediators. Likewise, intra-articular lavage might reduce this inflammatory burden.

Defect type, localization and marker gene expression determines early adverse events of matrix-associated autologous chondrocyte implantation.

INTRODUCTION: Since the first description of autologous chondrocyte implantation (ACI) in 1994 different methods and improvements were established for this regenerative treatment option of large chondral defects. This study analyzes safety and short-term clinical results from characterized ACI using a collagen based biphasic scaffold and evaluates prognostic factors. METHODS: 433 patients with a mean age of 33.4 years and localized grade III to IV cartilage defects (ICRS classification) in the knee or ankle were included. Mean defect size was 5.9 cm(2). Prior seeding of the scaffold, expanded chondrocytes were characterized by RT-PCR on 6 different marker genes (type I and II collagen, aggrecan, interleukin-1 ß (IL-1ß), vascular endothelial growth factor receptor 1 (FLT-1) and bone sialoprotein-2 (BSP-2)). Clinical outcome was evaluated using a questionnaire for defect history, basic demographics, time elapsed from surgery, 10-point outcome assessments of pain, function and swelling. Moreover, adverse events (AEs) or subsequent treatments were recorded and analysed. RESULTS: Patients improved significantly over baseline (p < 0.0001) in pain, function and swelling. Subjects with later than 12 months follow-up reported nominally greater mean changes. Graft failure incidence was 6% for patients with greater than one year follow-up. Graft-related complications were significantly higher for patellar (p < 0.0001) and degenerative defects (p = 0.005). Elevated expression of FLT-1 (p = 0.02) or IL-1 ß mRNA (p = 0.03) was associated with graft-related AEs. A borderline association was found for low collagen type II expression (p = 0.08). CONCLUSION: Early graft-related AEs after ACI with a biphasic collagen scaffold are related to defect type, location and marker gene expression. The levels of significance observed for gene expression with respect to graft-related AEs were subordinate to those identified in the analysis of lesion history and location.

A critical appraisal of pinhole scintigraphy of the ankle and foot.

BACKGROUND: Scintigraphy is an established imaging technique for injuries of the ankle and foot that are not apparent on plain radiographs. The scintigraphic technique has varied, with planar and pinhole images being used. MATERIALS AND METHODS: The incremental value of pinhole scintigraphy over planar imaging was studied in 16 patients with established diagnoses. Inter-reporter reproducibility was also measured. RESULTS: Pinhole scintigraphy improved the diagnostic specificity in nearly one half of the patients (48%). It did not contribute substantial information in 46% and led to confusion in the diagnosis of one patient. Inter-reporter agreement was good, with a kappa value of 0.78. Diagnoses varied from fractures of the talar dome to avulsion fractures of the malleoli and impingement syndromes. CONCLUSIONS: Pinhole images add a significant incremental value to planar scintigraphy of the foot and ankle. Although this had been perceived intuitively in the past, it has not been critically evaluated. The technique has good inter-reporter agreement.

Protective effect of P188 in the model of acute trauma to human ankle cartilage: the mechanism of action.

OBJECTIVE: Because P188 poloxamer is effective in promoting cell survival in models of acute trauma, the objectives were to understand the mechanism of its action focusing on glycogen synthase kinase-3 (GSK3) activation, interleukin-6 (IL-6), and p38 signaling. DESIGN: Sixteen normal human tali were impacted using a 4-mm diameter indenter with an impulse of 1 Ns. Eight-millimeter cartilage plugs containing the 4-mm impacted core and 4-mm adjacent nonimpacted ring were removed and cultured with or without P188. Cell lysates were analyzed using Western blots with antibodies against total and phosphorylated extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38, ATF-2, GSK3, Stat1, and Stat3. Additional tests were performed with the p38 inhibitor (p38i) SB203580. RESULTS: Studied pathways were activated after impaction with the peak of activity at 1 hour. P188 completely attenuated phosphorylation of Stat1 and ATF-2 and inhibited p38, Stat3, JNK, ERK, and GSK3. The p38i partially offset phosphorylation of Stat3, GSK3, and ERK suggesting a role of p38 in these three pathways. Additionally, the p38i improved cell survival (P = 0.053) and reduced apoptosis (by approximately 20%, P = 0.046, versus almost 40% by P188), thus confirming that P188 acts (at least in part) through the p38 pathway. CONCLUSION: Our results report a novel mechanism through which P188 exerts its protective effects on cartilage in the model of acute injury. In addition to its effect on cellular membrane, P188 affects stress-related p38 signaling, apoptosis-related GSK3, and inflammation-related IL-6 signaling. Taken together, these findings suggest that P188 alone or in combination with proanabolic agents may have a therapeutic potential in preventing progressive cartilage degeneration and the development of posttraumatic osteoarthritis.

A review of the differences between normal and osteoarthritis articular cartilage in human knee and ankle joints.

BACKGROUND: Osteoarthritis (OA) is the most common joint disease yet its pathophysiology is still poorly understood. It is more prevalent in some lower limb joints than others; in particular the knee is more commonly affected than the ankle. Research into articular cartilage and OA has primarily focussed on using animal models. However, it is apparent that articular cartilage differs between species, so more research is concentrating on human cartilage. OBJECTIVE: This paper reviews recent studies that have been undertaken to elucidate the reasons for this, and to discover if the findings would alter the conception that articular cartilage is not capable of repair. METHOD: Primary research papers into human knee and ankle cartilage published since 1997 have been reviewed. RESULTS: Differences in the structure, metabolism, physical properties and response to trauma have been found, implying that ankle cartilage may be more resistant to damage. CONCLUSIONS: More research is needed before definitive conclusions can be reached, but the findings so far suggest that OA should not be accepted as the inevitable outcome of joint injury and individuals and practitioners, such as podiatrists, may be able to use simple measures to prevent or delay its onset.

Mechanical injury potentiates proteoglycan catabolism induced by interleukin-6 with soluble interleukin-6 receptor and tumor necrosis factor alpha in immature bovine and adult human articular cartilage.

OBJECTIVE: Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNFalpha. METHODS: Injured or uninjured cartilage disks were incubated with or without TNFalpha and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6-blocking antibody Fab fragment and subjected to injury and TNFalpha treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6. RESULTS: In bovine cartilage samples, injury combined with TNFalpha and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNFalpha exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6-blocking Fab significantly reduced proteoglycan degradation. CONCLUSION: Our findings indicate that mechanical injury potentiates the catabolic effects of TNFalpha and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFalpha appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6-blocking Fab.

Collagen type I metabolism after bone surgery.

This study follows the postoperative course of serum collagen type I metabolites in patients after uncomplicated implantation of a cemented total hip endoprothesis (TEP; n = 12, mean age: 69.3 years), a cemented hemiendoprothesis (HEP; n = 13, mean age 79.7 years), a dynamic condylar or hip screw (DCS/DHS; n = 12, mean age 75.1 years) and osteosynthetic treatment of a Weber B or C fracture (OS; n = 17, mean age 54.3 years). The course of the propeptide of human type I procollagen (PICP) as an anabolic marker as well as of I-carboxyterminal telopeptide (ICTP) as a catabolic marker of bone metabolism was characterized. Measurements were done preoperatively and weekly for 3 weeks after surgery. The concentrations of both markers increased and reached a maximum in the 2nd or 3rd week after surgery. However, the PICP values differed, depending on the kind of surgical intervention and the type of bone healing. Secondary fracture healing with formation of callus occurred in the DCS/DHS group, which developed the highest median PICP concentrations (initial 83 microg/l, second week 337 microg/l; P < 0.001). In contrast, the primary bone healing in the OS group showed increasing ICTP but unchanged PICP concentrations. Patients in the cemented TEP and HEP groups as a kind of artificial bone healing had comparable concentrations. To consider the effective metabolism of collagen type I, the PICP/ICTP ratio was calculated. Although the median PICP and ICTP concentrations of the studied groups differed, the PICP/ICTP ratios were similar. In comparison to 54 young and healthy volunteers (median PICP/ICTP ratio: 37), the ratios of the studied groups were still normal but low (median ratios: < 20). This could be an effect of decreasing collagen type I metabolism with age. Although the results are in agreement with animal studies and histomorphometric investigations, the clinical use of PICP and ICTP determination as a tool for the detection of complicated bone healing is limited by the marked interindividual variability and the uncertain bone specificity.

Biochemical changes after trauma and skeletal surgery of the lower extremity: quantification of the operative burden.

OBJECTIVE: To quantify changes in variables of inflammation, coagulation, and fibrinolysis in blunt trauma patients with lower extremity fractures who underwent different types of surgical procedures. DESIGN: Prospective, cohort study. SETTING: Level I university trauma center. PATIENTS: We allocated 83 blunt trauma patients in stable condition and 22 patients eligible for elective hip replacement to four treatment groups. INTERVENTIONS: In 34 multiply traumatized patients with femoral fracture (group PTFF) and in 28 patients with an isolated femoral fracture (group IFF), primary unreamed intramedullary nailing for stabilization of the femoral shaft fracture was performed. In 22 patients, an elective uncemented total hip arthroplasty (group THA) was inserted for osteoarthritis, and in 21 control patients, an isolated ankle fracture (group AF) was acutely stabilized. MEASUREMENTS AND MAIN RESULTS: From serially sampled central venous blood, the perioperative concentrations of interleukin (IL)-6, of tumor necrosis factor-alpha, of prothrombin fragments 1 + 2, and of D-dimer cross-linked fibrin degradation products were evaluated. Intramedullary instrumentation for an isolated femur fracture caused a significant perioperative increase in the concentrations of IL-6 (preoperative IL-6, 52 +/- 12 pg/mL; IL-6 30 mins postinsertion, 78 +/- 14 pg/mL; p = .02). This increase was comparable with group THA (preoperative IL-6, 46 +/- 16 pg/mL; IL-6 30 mins postinsertion, 67 +/- 11 pg/mL; p = .03). A positive correlation occurred between both groups (r = .83, p < .0004). Multiple trauma patients demonstrated significantly (p = .0002) higher IL-6 concentrations than all other groups throughout the study period and showed a significant increase after femoral nailing (preoperative IL-6, 570 +/- 21 pg/mL; IL-6 30 mins postinsertion, 690 +/- 24 pg/mL; p = .003), whereas no perioperative change was seen in group AF. The highest IL-6 increases were associated with a longer ventilation time (group PTFF) and a longer period of positive fluid balances (groups PTFF, IFF, THA). The coagulatory variables demonstrated similar perioperative increases in groups IFF and THA, but not in groups PTFF and AF. The IL-6 concentrations and the prothrombin fragments 1 + 2 concentrations correlated between groups THA and IFF at 30 mins and at 1 hr after surgery (r2 = .64, p < .02). In all patients the clinical variables were stable perioperatively. CONCLUSIONS: Major surgery of the lower extremity causes changes to the inflammatory, fibrinolytic, and coagulatory cascades in patients with stable cardiopulmonary function. The inflammatory response induced by femoral nailing is biochemically comparable to that induced by uncemented total hip arthroplasty. In multiple trauma patients, increases, which occurred in addition to those induced by the initial trauma, were measured. Definitive primary femoral stabilization by intramedullary nailing imposes an additional burden to the patient with blunt trauma. A careful preoperative investigation is required to evaluate whether primary definitive stabilization can be performed safely.

Función dinámica de los músculos peroneus en apoyo unipodal / Dynamic function of fibular muscles during the unipodal support stage

Los autores hacen una descripción, adentrándose en la fisiología articular, del movimiento de las articulaciones del pie en general y del tobillo en particular. Analizan la acción de los dos músculos retromaleolares, peroneo lateral largo (PLL) y peroneo lateral corto (PLC) cuando el pie está apoyado en el suelo durante el movimiento lineal, marcha y carrera. Describen las ventajas mecánicas que propicia la ubicación, trayecto e inserción de ambos músculos para facilitar el movimiento, tanto en la fase de apoyo unipodal, como en la fase de impulso. Para concluir afirman que es la acción en carga del pie la que ha solicitado de todas las modificaciones mecánicas necesarias, en el complejo articular del tobillo, para economizar al máximo el gasto energético, utilizando como ejemplo la acción de los músculos PLL y PLC (AU)

The authors carry out a description of foot joints movement and more particularly of the ankle joint one, taking into account the joint physiology. They analyse the action of retromalleolar muscles, peroneus longus (PLL) and peroneus brevis (PLC), during the unipodal phase of linear movement (running and walking). The mechanical advantage in the movement as result of location, trajectory and insertion of muscles are described for both the unipodal support and impulse phases. In order to conclude, the writers argue that mechanical modifications of ankle joint during the human evolution process have been conditioned by the foot load force and energy consumption reduction principle. They use as example PLL and PLC muscles (AU)