ACL graft-suture fixation can be constructed with needle or needleless techniques. Needleless techniques have advantages of decreased injury, preparation time and cost. The Nice Knot (NK) is common among upper extremity procedures; however, its efficacy in ACL reconstruction relative to other needleless methods is not well known. The purpose of this study was to biomechanically compare quadriceps tendon (QT) grafts prepared with the NK versus the modified Prusik Knot (PK). Twenty QT grafts were harvested from 10 embalmed human cadaver specimens. 10 were prepared with the PK and 10 with the NK using a No.2 FiberWire (Arthrex, Naples, FL). The prepared grafts were then mounted in a materials testing machine (ElectroPuls E10000, Instron, Norwood, MA) and subjected to tensile loading based on an established protocol. Each tendon-suture specimen was preconditioned with 3 cycles of 0–100N at 1Hz followed by a constant load of 50N for 1 minute and cyclic loading of 200 cycles from 50–200N at 1Hz and then loaded to failure at a displacement rate of 20mm/min. Load and displacement data for each tendon-suture construct was recorded by the testing machine.Abstract
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Methods
To evaluate mechanical properties of three suture-tendon constructs, the Krackow stitch (KS), the modified Prusik knot (PK) and the Locking SpeedWhip (LSW), using human cadaveric quadriceps grafts (QT). Thirty QT grafts were obtained from human cadaver specimens and an equal number of tendon-suture constructs were prepared for three stitches: KS, PK and LSW. The constructs were mounted in a materials testing machine (ElectroPuls E10000, Instron, Norwood, MA) and subject to tensile loading based on an established protocol. Load and displacement data for each tendon-suture construct were recorded.Abstract
Objectives
Methods
Initial performance of sutured quadriceps tendon (QT) ACL graft constructs is not well studied in human tissue and the results of animal tissue testing may not extend to the human model. Two common methods of preserving human tissue are to freeze the specimens immediately after death or embalm with formalin solution. The purpose of this study is to compare elongations and loads in biomechanical testing of fresh-frozen to that of embalmed quadriceps tendon-suture constructs. Twenty QT grafts were harvested from human cadaver specimens, 10 fresh-frozen and 10 embalmed. The grafts were prepared with the modified Prusik knot using a No.2 FiberWire (Arthrex, Naples, FL), mounted in a materials testing machine (ElectroPuls E10000, Instron, Norwood, MA) and subjected to tensile loading based on an established protocol. Each tendon was preconditioned with 3 cycles of 0–100N at 1Hz followed by a constant load of 50N for 1 minute and 200 cycles from 50–200N at 1Hz and then loaded to failure at a displacement rate of 20mm/min. Load and displacement data for each tendon-suture construct was recorded.Abstract
Objectives
Methods
Lubricin is a proteoglycan that is a boundary lubricant in synovial joints and both a surface and collagen inter-fascicular lubricant in ligaments. The purpose of this study was to characterise the mRNA levels for lubricin in the anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), and lateral collateral ligament (LCL) in aging and surgically-induced menopausal rabbits. We hypothesised that lubricin mRNA levels would be increased in ligaments from aging and menopausal rabbits compared with ligaments from normal rabbits. All four knee ligaments (ACL, PCL, MCL, LCL) were isolated from normal (1-year-old rabbits, n=8), aging (3-year-old rabbits, n=6), and menopausal (1-year-old rabbits fourteen weeks after surgical ovariohysterectomy, n=8) female New Zealand White rabbits. RT-qPCR was used to evaluate the mRNA levels for lubricin normalised to the housekeeping gene 18S. After removing outliers, data for normal, aging, and menopausal rabbits for each knee ligament (ACL, PCL, MCL, LCL) were compared using ANOVA with linear contrasts or Kruskal-Wallis test with Conover post-hoc analysis. For ACLs, the mRNA levels for lubricin were increased in menopausal and aging rabbits compared with normal rabbits (p<0.056). For PCLs, trends for increased lubricin mRNA levels were found when comparing menopausal and aging rabbits with normal rabbits (p<0.092). For MCLs, the mRNA levels for lubricin were increased in menopausal and aging rabbits compared with normal rabbits (p<0.050). For LCLs, no differences in lubricin mRNA levels were detected comparing the three groups. For all four knee ligaments (ACL, PCL, MCL, LCL), no differences in lubricin mRNA levels were detected comparing the ligaments from menopausal rabbits with those from aging rabbits. Lubricin plays a role in collagen fascicle lubrication in ligaments (1,2). Increased lubricin gene expression was associated with mechanical changes (including decreased modulus and increased failure strain) in the aging rabbit MCL (3). Detection of similar molecular changes in the ACL, and possibly the PCL, may indicate that their mechanical properties may also change as a result of increased lubricin gene expression, thereby potentially pre-disposing these ligaments to damage accumulation. Compared to aging ligaments, aging tendons exhibited decreased lubricin gene and protein expression, and increased stiffness (4). Although opposite changes than aging ligaments, these findings support the relationship between lubricin and modulus/stiffness. The similarities between ligaments in the aging and menopausal groups may suggest that surgically-induced menopause results in a form of accelerated aging in the rabbit ACL, MCL and possibly PCL.
We observed that severe muscle weakness leads to OA, whereas a transient inflammatory stimulus did not have a significant effect on cartilage degradation. This arises the thought that a severe but transient inflammation may not be an independent risk factor for OA. Biomechanical disturbances and joint inflammation are known risk factors, which may provoke or advance osteoarthritis (OA). However, the effect of interactions of such risk factors on the onset and progression of OA are still poorly understood. Therefore, the goal of this study was to investigate the in vivo effects of muscle weakness, joint inflammation, and the combination of these two risk factors, on the onset and progression of OA in the rabbit knee.Summary Statement
Introduction
Previous work in a rabbit model of post-traumatic joint contractures shows that the mast cell stabilizer ketotifen decreases contracture severity. We show here that ketotifen decreases collagen gel contraction mediated by rabbit joint capsule fibroblasts when mast cells are present. Ketotifen was shown to decrease contracture severity and associated joint capsule fibrosis in an animal model of post-traumatic joint contractures. Ketotifen prevents the release of profibrotic growth factors from mast cells (MC). An Summary
Introduction
Subtle deformities of the acetabulum and proximal femur are recognised as biomechanical risk factors for the development of hip osteoarthritis (OA) as well as a cause of hip and groin pain. We undertook this study to examine relationships between a number of morphological measurements of the acetabulum and proximal femur and the hip pain in a 20-year longitudinal study. In 1989 women of 45–64 years of age were recruited. Each had an AP-Pelvis radiograph at Year-2. These radiographs were analysed using a validated programme for measuring morphology. All morphological measurements were read blinded to outcome. At year 3 all participants were asked whether they experienced hip pain (side specific). This was repeated at visits up to and including 20-years. Logistic regression analysis (with robust standard errors and clustering by subject identifier) was performed using hip pain as a binary outcome. The model adjusted for baseline age, BMI and joint space and included only participants who were pain free on initial questioning.Introduction
Methods
Recent work has shown that joint contracture severity can be decreased with the mast cell stabilizer ketotifen in association with decreased numbers of myofibroblasts and mast cells in the joint capsule of a rabbit model of post-traumatic contractures. Neuropeptides such as Substance P (SP) can induce mast cells to release growth factors. Using a gel contraction assay, we test the hypothesis that joint capsule cell-mediated contraction of a collagen gel can be enhanced with SP, but the effect is magnified in the presence of mast cells. Anterior elbow joint capsules were obtained at the time of surgical release from 2 men (age 34 and 54) and 1 woman (age 40) with chronic (> 1 year) post-traumatic joint contractures. The human mast cell line HMC-1 (Mayo Clinic, Rochester), SP and the NK1 receptor antagonist RP67580 (Sigma, Oakville, ON) were used. NK1 is the SP receptor. Neutralized Collagen solution composed with 58% Vitrogen 100 purified collagen mixed with HMC-1 cells only (7.5 105), human capsule cells (2.5 105), or human capsule cells (2.5 105) and 7.5 105 mast cells (1:3) were cast into 24- well tissue culture plates. In some experiments, SP (1 × 10−5 M) +/− RP67580 (0.5 mM) were added. The gels were maintained with 0.5 ml DMEM composed with 2% BSA and incubated at 37C for 12 h for gelation to occur. The gels were then detached from the wall and the bottom of culture plate wells, and photographed at regular intervals up to 72 hours. Gel contraction studies were carried out on passage 4 and done in triplicate for each patient. The average value of each patients triplicate was combined to give a mean contraction at each time point. Statistical analysis involved an ANOVA with posthoc Bonferroni correction. P < 0.001 was significant.Purpose
Method
The hypothesis is that cells isolated from capsules of joints with contractures will contract collagen gels at a faster rate when compared to cells obtained from capsules of joints free of contractures. Post-traumatic joint contractures were produced by removing cortical bone windows from the femoral condyles of three skeletally mature rabbits and immobilizing the knees for four weeks with a K-wire. The contralateral knees served as an unoperated control. At sacrifice, the posterior capsules were immediately placed in medium and the tissue was minced. Upon confluence, cells were trypsinised and gel contraction studies were carried out on passage four cells. Five x 105 cells/ml were mixed with 58% neutralised bovine collagen solution and five hundred microlitres of collagen gel/cells solution were then cast into wells of a tissue culture plate. Gelation occurred overnight at 37C in a humidified incubator containing 5% CO2. At cultured day zero, day one, day three, the gels were released from the well walls. The areas of the gel were measured using an image analyzer immediately after release (zero hour), and one hour, two hour, three hour and four hour post-release. The amount the collagen gels were contracted depended on the time of preincubation of cells and collagen before release and the source of the joint capsule cells. In general, increasing the time of preincubation heightened the contractile response of the cells. The collagen gel contraction was small for the day zero groups over the first four hours, but for the day three groups the rate of contraction was markedly increased. In all cases the collagen gel contraction was larger for the contracture capsule cells when compared to the control capsule cells. The patterns of the contraction over the four hours post release were similar for contracture and control groups. Cells from capsules of joints with post-traumatic contractures have intrinsically heightened in vitro contractile properties when compared to normal cells. Future work will determine whether the response is exaggerated to fibrotic stimuli such as TGF-beta1 in these capsule cells from post-traumatic joint contractures.
The hypothesis is that mast cell numbers and neuropeptide containing nerve fibres are increased in the elbow joint anterior capsule of patients with post-traumatic contractures when compared to normal capsules. Capsules were obtained from two patients with chronic contractures following radial head fractures and two organ donor elbows free of contractures. Four sections from each capsule were double-labelled with specific antibodies to the mast cell marker chymase and the neuropeptide calcitonin gene-related peptide (CGRP). Species specific secondary fluorescent antibodies were used to detect the marker antibodies and cells were identified with a fluorescent nuclear marker (DAPI). Images were captured using a microscope (200x magnification) and five randomly selected areas were sampled for each section obtained from all joint capsules. Chymase positive cell numbers and numbers of nerve fibers (minimum length fifty micrometres) were gathered. The number of chymase positive mast cells was 6x greater in the contracture capsules when compared to normal capsules. In the contracture capsule, chymase positive mast cells represented 39% of total cells while in control capsules they represented 7% of total cells. Total cell numbers were similar in the capsules of both groups. The number of CGRP positive nerve fibres was increased 3x in the contracture capsule when compared to normal capsule. Mast cell numbers and neuropeptide positive fibre numbers are increased in the elbow joint anterior capsule of patients with post-traumatic contractures when compared to normal tissues. Neuropeptides such as CGRP can induce mast cell degranulation. Mast cells release profibrotic molecules such as transforming growth factor beta1 (TGF-b1), a myofibroblast upregulator. It has been described that TGF-b1 and myofibroblast numbers are elevated in human elbow joint capsules in post-traumatic contractures. While these trends are encouraging, more subjects are needed to determine whether the mast cell and neuropeptide nerve fibre findings can be generalised to larger numbers. If future work supports a myofibroblast - mast cell - neuropeptide - fibrosis axis in the joint capsule in post-traumatic contractures, then methods to modulate this axis, such as mast cell stabilisers, may be evaluated in animal models.
Our aim was to determine the effect of denervation on repair-associated mRNA levels in the MCL after partial tear. Cohorts of rabbits underwent partial MCL tear with or without concomitant femoral nerve transection. Ligaments were harvested, RNA extracted and RT-PCR was performed using rabbit-specific primers for repair-associated molecules at three days, two wks, six wks and sixteen wks post-injury. Angiogenesis genes MMP3, MMP13, matrix components Collagen I and III and growth factors TGF-ß and NGF mRNA levels were increased in the denervated group at two-weeks post-injury (p<
0.05). Denervation significantly alters mRNA levels during the early stages of rabbit MCL healing. To determine the effect of denervation on repair-associated mRNA levels in the injured medial collateral ligament (MCL). Previous experiments revealed that denervation impairs healing of the MCL. We hypothesized that denervation would decrease repair-associated mRNA levels in the injured MCL when compared with normally innervated injured MCL. Adult, skeletally mature female rabbits were assigned to one of four groups: unoperated control, femoral nerve transection alone (denervated controls), MCL partial tear and denervated MCL partial tear. At three days, two weeks, six weeks or sixteen weeks post-surgery, cohorts of six rabbits from each experimental group were killed. Control rabbits were assessed at two weeks. Ligaments were harvested, RNA extracted and RT-PCR was performed using rabbit-specific primers. In the denervated injury group, mRNA levels of angiogenesis genes MMP-3 and MMP-13, matrix components Collagen I and III and growth factors TGF-ß and NGF had all increased at two-weeks post-injury, in comparison to non-denervated (p<
0.05). We also found increased levels of MMP-3 and NGF mRNA in the denervated group at sixteen weeks post injury (p<
0.05). The mRNA levels of the housekeeping gene GAPDH were increased in the denervated group only at three days post injury (p<
0.05). Of note, TGF-ß mRNA levels were significantly decreased in the denervated group at three days post injury (p<
0.05). Contrary to our initial hypothesis, denervation increases mRNA levels for many important molecules during the early stages of MCL healing. Additional research will be required to explain how and why denervation impairs ligament healing. No previous study has shown that innervation regulates mRNA levels in healing ligament.
The objective of this report was to evaluate myofibroblast numbers in human elbow anterior joint capsules. Joint capsules were obtained from six patients with post-traumatic contractures and from six elbow joints of age-matched organ donors. Frozen sections were labeled with α-smooth muscle actin (α-SMA), a marker of myofibroblasts. Myofibroblasts were identified in both experimental and control tissues. Myofibroblast numbers and percentage of total cells were significantly elevated in the capsules of patients (919 ± 187; 36 ± 0.04%) when compared to organ donor control tissue (485 ± 335; 9 ± 0.04%). Future work will look at the expression of myofibroblast modulators in human elbow joint contractures. The purpose of this study was to determine whether myofibroblasts are associated with human elbow joint contractures. Myofibroblast numbers and percentage of myofibroblasts to total cells were significantly increased in anterior elbow joint capsules of patients with post-traumatic contractures. Methods to alter myofibroblast expression may be strategies to prevent or treat post-traumatic elbow joint contractures. Joint capsules were obtained from six patients (age 33±13 yrs, preoperative flexion-extension arc range of motion 58°±15°) and from six elbow joints of organ donors free of contractures (age 26±15 yrs). Frozen sections were double labeled using monoclonal antibodies to α-smooth muscle actin (α-SMA) with peroxidase conjugated secondary antibodies, and affinity purified antibodies to laminin with Elexa Fluor 488 conjugated secondary antibodies. The laminin antibodies label components of blood vessels, to differentiate between α-SMA expression associated with blood vessels or myofibroblasts. Endogenous peroxidases were quenched and 10% normal goat serum was used as a blocking agent. DAB/peroxide substrate was added for thirteen minutes. DAPI was applied to label nuclei. Cell nuclei associated with α-SMA and not with laminin were counted as myofibroblasts. Myofibroblast numbers and percentage of total cells were significantly increased (t-test, p <
0.05) in the joint capsules of the patients when compared to organ donor control tissue. Total cell numbers were not significantly different in the patient and control tissue. Modulators of α-SMA expression and myofibroblast formation include growth factors and matrix molecule components. Future work will look at the expression of these modulators in human elbow joint contractures.
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