Risk of intra-articular injection with longitudinal ultrasound-guided injection of collateral ligaments of the equine distal interphalangeal joint.

Desmopathy of the collateral ligaments of the distal interphalangeal joint is a common cause of equine foot lameness and carries a poor prognosis with conservative management. Intralesional injections may improve healing, although accuracy of radiographically guided injections is significantly less than when guided by MRI, which requires special needles. Longitudinal ultrasound-guided injection of the distal collateral ligament has not been evaluated objectively. In this prospective, anatomic study, seven equine cadaver limbs (14 collateral ligaments) were injected with methylene blue dye and radiographic contrast medium using ultrasound to guide the needle longitudinally into the collateral ligaments until contacting bone. The insertion site of the needle proximal to the coronary band was measured on the limb and the needles left in place for radiography and CT to evaluate the needle angulation, location of the contrast medium, and whether the contrast entered the distal interphalangeal joint. The limbs were frozen and sectioned with a band saw to identify the location of the dye. Fifty percentage of injections were in or around the collateral ligaments. However, the percentage of "successful" injections, defined as in the collateral ligament but not in the joint, was only 36%. All legs had dye and contrast in the joint after both ligaments had been injected. There were no significant differences between the needle angle and entry site for "successful" and "unsuccessful" injections. Findings from this study indicates that the success rate is low for injecting the distal portions of the distal interphalangeal joint collateral ligaments using ultrasound guidance alone.

Microparticles Locally Deliver Active Interleukin-1 Receptor Antagonist In Vivo.

Despite significant research in therapeutic protein delivery, localized and sustained delivery of active therapeutic proteins remains a challenge. Delivery is a particular challenge for therapeutic proteins with a short half-life. Herein, localized delivery of interleukin-1 receptor antagonist (IL-1Ra) by mineral coated microparticles (MPs) is assessed in a healing rat medial collateral ligament (MCL). The local tissue concentration and systemic serum concentration of IL-1Ra, the anti-inflammatory activity of IL-1Ra delivered with MPs, and whether IL-1Ra loaded MPs (IL-1Ra MPs) are immunogenic in a healing ligament are also examined. IL-1Ra MPs significantly increase the local concentration of IL-1Ra compared to soluble IL-1Ra at 7 and 14 days after treatment but do not elevate the systemic concentration of IL-1Ra at these time points, indicating localized delivery of IL-1Ra. IL-1Ra MPs significantly reduce inflammation caused by the MPs themselves, indicating the IL-1Ra is active. Finally, IL-1Ra MPs do not induce a foreign body response and decrease the immunogenicity of human IL-1Ra in a healing rat MCL. Overall, mineral coated microparticles have the ability to locally deliver active therapeutic proteins for an extended period of time.

Two Unique Cases of Ciprofloxacin-Associated Avulsion of Ligament and Tendon.

Ciprofloxacin is recognized to have a deleterious relationship with tendons, particularly Achilles tendinopathy, which makes up most case reports. Tendinopathy seems to occur because of induction of collagen-degrading enzymes causing damage and ischemia of the poorly vascularized regions preventing repair. The focus on the relationship of ciprofloxacin and the Achilles tendon leaves patients on fluoroquinolones with non-Achilles tendinopathy symptoms at risk of misdiagnosis. There have not been any documented instances of ligament damage with ciprofloxacin administration in the literature, although ligament and tendon compositions are similar and should have similar susceptibility. This report includes two cases, one presenting with right lateral thumb pain and a medical history of gastroenteritis treated with ciprofloxacin. Physical examination showed swelling of the right metacarpophalangeal joint and ultrasound confirmed disruption of the radial collateral ligament at insertion on first metacarpal; the second case is of a woman presenting with right hip pain in setting of chronic recurrent diverticulitis treated with ciprofloxacin. She received work-up for lumbar disc disease and spondylosis. After standard therapy with pharmacotherapy and physical therapy for radiculopathy failed, magnetic resonance imaging was performed showing near complete avulsion of the right hamstring tendons from the ischial tuberosity.

Development of Tissue-Engineered Ligaments: Elastin Promotes Regeneration of the Rabbit Medial Collateral Ligament.

When ligaments are injured, reconstructive surgery is sometimes required to restore function. Methods of reconstructive surgery include transplantation of an artificial ligament and autotransplantation of a tendon. However, these methods have limitations related to the strength of the bone-ligament insertion and biocompatibility of the transplanted tissue after surgery. Therefore, it is necessary to develop new reconstruction methods and pursue the development of artificial ligaments. Elastin is a major component of elastic fibers and ligaments. However, the role of elastin in ligament regeneration has not been described. Here, we developed a rabbit model of a medial collateral ligament (MCL) rupture and treated animal knees with exogenous elastin [100 µg/(0.5 mL·week)] for 6 or 12 weeks. Elastin treatment increased gene expression and protein content of collagen and elastin (gene expression, 6-fold and 42-fold, respectively; protein content, 1.6-fold and 1.9-fold, respectively), and also increased the elastic modulus of MCL increased with elastin treatment (2-fold) compared with the controls. Our data suggest that elastin is involved in the regeneration of damaged ligaments.

Interleukin expression after injury and the effects of interleukin-1 receptor antagonist.

Ligament healing follows a series of complex coordinated events involving various cell types, cytokines, as well as other factors, producing a mechanically inferior tissue more scar-like than native tissue. Macrophages provide an ongoing source of cytokines to modulate inflammatory cell adhesion and migration as well as fibroblast proliferation. Studying interleukins inherent to ligament healing during peak macrophage activation and angiogenesis may elucidate inflammatory mediators involved in subsequent scar formation. Herein, we used a rat healing model assayed after surgical transection of their medial collateral ligaments (MCLs). On days 3 and 7 post-injury, ligaments were collected and used for microarray analysis. Of the 12 significantly modified interleukins, components of the interleukin-1 family were significantly up-regulated. We therefore examined the influence of interleukin-1 receptor antagonist (IL-1Ra) on MCL healing. Transected rat MCLs received PBS or IL-1Ra at the time of surgery. Inhibition of IL-1 activation decreased pro-inflammatory cytokines (IL-1α, IL-1ß, IL-12, IL-2, and IFN-γ), myofibroblasts, and proliferating cells, as well as increased anti-inflammatory cytokines (IL-10), endothelial cells/blood vessel lumen, M2 macrophages, and granulation tissue size without compromising the mechanical properties. These results support the concept that IL-1Ra modulates MCL-localized granulation tissue components and cytokine production to create a transient environment that is less inflammatory. Overall, IL-1Ra may have therapeutic potential early in the healing cascade by stimulating the M2 macrophages and altering the granulation tissue components. However, the single dose of IL-1Ra used in this study was insufficient to maintain the more regenerative early response. Due to the transient influence on most of the healing components tested, IL-1Ra may have greater therapeutic potential with sustained delivery.

Lateral ankle ligament anesthesia significantly alters single limb postural control.

Lateral ankle anesthesia has been used as a model to explore effects of ligament deafferentation related to ankle sprain on single limb postural control with conflicting results. Time-to-boundary (TTB) is a postural control measurement technique found to be sensitive in detecting subtle deficits in postural control in those with chronic ankle instability. The objective of this study was to determine the effects of lateral ankle ligament anesthesia on TTB measures of single limb postural control in healthy adults. Twenty-two healthy adults with no history of lower extremity injury within the past 6 months or balance disorders participated in the study. All subjects received a lidocaine injection to the lateral ankle structures on one of two testing days. On both testing days, subjects performed 3 eyes open and 3 eyes closed, 10-s trials of barefoot single limb stance on a forceplate. The dependent variables were the mean of TTB minima(s) and standard deviation of TTB minima(s) in mediolateral (ML) and anteroposterior (AP) directions. Separate condition (anesthesia, control) by vision (eyes open, eyes closed) ANOVAs with repeated measures were used for each TTB variable to determine the effects of anesthesia on postural control. Alpha level was set a priori at p≤0.05. The anesthesia day TTBAP magnitude (p=0.008) and variability (p=0.044) measures were significantly lower than the control day, regardless of vision. Anesthesia of the lateral ankle ligamentous structures significantly reduced the magnitude and variability of TTBAP measures. These findings are similar to deficits found in those with chronic ankle instability.

Ultrasound phonophoresis of panax notoginseng improves the strength of repairing ligament: a rat model.

This study examined the phonophoretic effect of a therapeutic ultrasound coupled with a Panax notoginseng (PN) gel and compared it with a therapeutic ultrasound alone for medial collateral ligament repair in rats. Twenty mature male Sprague-Dawley rats receiving surgical transection to the left medial collateral ligament (MCL) were divided randomly into three groups: ultrasound (US, n = 7), ultrasound with PN coupling gel (PNUS, n = 7) and control (n = 6). The treatments started on day 3 after surgery for six days per week over a two-week period. The US group received 4 min of pulsed ultrasound (1 MHz) at the intensity of 0.5W/cm(2) with a normal ultrasonic coupling gel. The PNUS group received the same ultrasound treatment, but with a coupling gel that contained PN extract. The control group received a placebo ultrasound treatment similar to the other two groups. On day 17, the ligaments were mechanically tested for load-relaxation, stiffness and ultimate tensile strength (UTS). Values of the left side were normalized against that of the right side of each animal for analysis. Results revealed significantly higher normalized stiffness (p = 0.009) and UTS (p = 0.022) in the PNUS group than the other two groups, but insignificant difference in load-relaxation among all groups. This study reveals a positive ultrasonic phonophoretic effect of Panax notoginseng extract for improving the strength of ligament repair than ultrasound therapy alone.

Administrations of peripheral blood CD34-positive cells contribute to medial collateral ligament healing via vasculogenesis.

Neoangiogenesis is a key process in the initial phase of ligament healing. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, have been reported to contribute to neoangiogenesis; however, the therapeutic potential of CD34+ cells for ligament healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that ligament healing is supported by CD34+ cells via vasculogenesis. Granulocyte colony-stimulating factor-mobilized peripheral blood (GM-PB) CD34+ cells with atelocollagen (CD34+ group), GM-PB mononuclear cells (MNCs) with atelocollagen (MNC group), or atelocollagen alone (control group) was locally transplanted after the creation of medial collateral ligament injury in immunodeficient rats. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining at the injury site demonstrated that molecular and histological expression of human-specific markers for endothelial cells was higher in the CD34+ group compared with the other groups at week 1. Endogenous effect, assessed by capillary density and mRNA expression of vascular endothelial growth factor, was significantly higher in CD34+ cell group than the other groups. In addition to the observation that, as assessed by real-time RT-PCR, gene expression of ligament-specific marker was significantly higher in the CD34+ group than in the other groups, ligament healing assessed by macroscopic, histological, and biomechanical examination was significantly enhanced by CD34+ cell transplantation compared with the other groups. Our data strongly suggest that local transplantation of circulating human CD34+ cells may augment the ligament healing process by promoting a favorable environment through neovascularization.

Neuropeptides regulate expression of matrix molecule, growth factor and inflammatory mediator mRNA in explants of normal and healing medial collateral ligament.

Denervation degrades normal ligament properties and impairs ligament healing. This suggests that secreted neuromediators, such as neuropeptides, could be modulating cell metabolism in ligament and scar tissue. To test this hypothesis we investigated the effect of exogenous substance P (SP), neuropeptide Y (NPY) or calcitonin gene-related peptide (CGRP) on the mRNA levels for proteins associated with inflammation, angiogenesis, and matrix production in tissue-cultured specimens of normal and injured medial collateral ligament. SP and NPY induced increased mRNA levels for several inflammatory mediators in the 2-week post-injury specimens. All three neuropeptides induced decreases in mRNA levels for healing-associated growth factors and matrix molecules, including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and collagen types I and III. The results indicate that neuropeptides strongly influence the metabolic activity of cells in healing ligament, particularly at early time points after injury.

The effect of analgesic agents on the healing rat medial collateral ligament.

BACKGROUND: Studies have suggested that some nonselective nonsteroidal anti-inflammatory drugs, including piroxicam, may improve ligament healing, whereas other nonsteroidal anti-inflammatory drugs, including ibuprofen and the cyclooxygenase-2 inhibitor celecoxib, may have no effect on the mechanical properties or may even deter the healing process. These results might reflect variations in cyclooxygenase enzyme selectivity by different drugs or, alternatively, may be related to their analgesic properties because it is generally accepted that early activity improves ligament healing. HYPOTHESIS: Nonselective nonsteroidal anti-inflammatory drugs improve ligament healing, whereas other analgesics provide lesser degrees of improvement, and cyclooxygenase-2 inhibitors are detrimental. STUDY DESIGN: Controlled laboratory study. METHODS: One hundred fifty-five Sprague-Dawley rats were divided into 7 treatment groups (piroxicam, naproxen, rofecoxib, butorphanol, 2 doses of acetaminophen, and control). The right medial collateral ligament of each rat was transected, and the drugs were administered postoperatively on days 1 to 6. On day 14, the rats were sacrificed, and mechanical testing was performed on the medial collateral ligament. RESULTS: The piroxicam group demonstrated significantly greater load to failure (27%) compared with the control. No significant differences were observed between other groups. CONCLUSIONS: Piroxicam improves ligament healing, but this effect cannot be attributed to all nonselective nonsteroidal anti-inflammatory drugs. Opiate analgesics, acetaminophen, and cyclooxygenase-2 inhibitors do not appear to categorically affect ligament healing. CLINICAL RELEVANCE: In the treatment of ligament injury, piroxicam may be a drug of choice.

Effects of osteogenic protein-1 (OP-1, BMP-7) on gene expression in cultured medial collateral ligament cells.

Osteogenic protein-1 (OP-1, also called BMP-7), a member of the BMP family and the TGF-beta superfamily, induces formation of new bone and cartilage, but also regulates a wide array of processes. In the present study, the expression of several characteristic biochemical markers of ligaments, such as Six1, Scleraxis, aggrecan, and type I collagen in primary cultures of adult rat medial collateral ligament (MCL) cells was determined. The effects of OP-1 on cell proliferation and on gene expression were subsequently examined. OP-1 stimulated cell proliferation, alkaline phosphatase (AP) activity, and the steady-state mRNA levels of the transcription factor Runx2/Cbfa1 in a dose- and time-dependent manner. The mRNA levels of type I collagen only increased slightly, but the activity of the cloned collagen promoter increased by 2-fold in transiently transfected MCL cells. OP-1 also stimulated aggrecan mRNA expression. The mRNA levels of Six1 and Scleraxis were not detectably altered by OP-1. In control cultures, the steady-state mRNA levels of ActR-I, BMPR-IA, BMPR-IB, and BMPR-II increased as a function of time in culture. The mRNA levels of BMP-1 and -4 increased significantly after 12 days, but those of BMP-2 and -6 did not change. The GDF-1, -3, -5, -6, and -8 mRNA levels in the control cultures also increased as a function of time. OP-1 treatment stimulated mRNA expression of BMPR-IA and BMPR-II, but had little effect on ActR-I and BMPR-IB mRNA expression. OP-1 lowered the BMP-1, -2, and -6 mRNA levels without changing the BMP-4 mRNA level. OP-1 treatment also reduced the mRNA levels of GDFs detected. In summary, the present study demonstrated that OP-1 stimulated cell proliferation and mRNA expression of several biochemical markers in this ligament cell culture model and established the spatial and temporal appearance of several members of the TGF-beta superfamily.

Novel method for the quantitative assessment of cell migration: a study on the motility of rabbit anterior cruciate (ACL) and medial collateral ligament (MCL) cells.

A novel method of quantitating cell migration has been proposed for the potential utilization of tissue engineered scaffolds. Applying Alt's conservation law to describe the motion of first passage ACL and MCL cells, we have developed a quantitative method to assess innate differences in the motility of cells from these two ligamentous tissues. In this study, first passage ACL and MCL cells were cultured from four mature New Zealand white rabbits. One side of the cell monolayer was scraped completely away to create a wound model. The cell moved into the cell-free area, and cell density profiles were analyzed at 6 h and 12 h. Values of the random motility coefficient (mu) were then estimated by curve fitting the 6 h and 12 h data to a mathematical model, derived from the conservation law of cell flux. During 6 h of incubation in medium supplemented with 1% FBS, MCL cells (mu(MCL) = 4.63 +/- 0.65 X 10(-6) mm(2)/sec) were significantly (p < 0.05) more mobile than ACL cells (mu(ACL) = 2.51 +/- 0.31 X 10(-6) mm(2)/sec). At 12 h, the MCL cells also appeared to move faster (mu(ACL) = 4.39 +/- 0.63 X 10(-6) mm(2)/sec, mu(MCL) = 6.59 +/- 1.47 X 10(-6) mm(2)/sec), but the difference was not statistically significant (p = 0.18). Exposure of the cells to growth factors PDGF-BB or bFGF for 6 h had no significant effect on the migration of the ACL and MCL cells. However, exposure of the ACL cells (p < 0.05) and the MCL cells (p = 0.19) to 1 ng/mL of PDGFBB for 12 h enhanced their migration. Incubation with a high concentration (100 ng/mL) of PDGF-BB or bFGF at concentrations tested (1 or 100 ng/mL) for 12 h, produced little or no migratory stimulation on these ligament cells. Our findings support the previous qualitative observations made by numerous investigators. The novel methodology developed in this study may provide a basis for tissue engineering, and the results may be applied to tissue reconstruction techniques of the knee ligaments.

Collagenase degradation decreases collagen fibril diameters--an in vitro study of the rabbit medial collateral ligament.

Based on the similarity of fibril diameters in healing and grafted ligaments, it has been speculated that all small fibrils represent newly synthesized collagen. Alternatively, small fibrils in grafts could be due to enzymatic degradation of endogenous large fibrils. This study examined the effect of collagenase on collagen fibril diameters in normal NZW rabbit MCLs. Midsubstance MCL slivers were incubated in buffer for 72 or 144 h for comparison with slivers incubated in buffer containing 4 units/ml bacterial collagenase. The samples were examined under TEM for fibril diameter analysis. Mean fibril diameters of 3-day and 6-day collagenase-treated MCLs were significantly reduced, resembling 40-week scar values. These results suggest that collagenase treatment can alter collagen fibril diameter and shape in normal rabbit MCL, thus it is possible that despite their similarity to ligament scars, that at least some small fibrils in ligament grafts may be enzymatically reduced endogenous fibrils.

The effects of platelet-derived growth factor-BB on healing of the rabbit medial collateral ligament. An in vivo study.

We report a biologic approach to improve medial collateral ligament healing using growth factors normally expressed in healing tissue. Our previous in vitro work demonstrated that platelet-derived growth factor-BB and transforming growth factor-beta 1 promoted fibroblast proliferation and matrix synthesis, respectively. There-fore, these growth factors were used in vivo to determine whether they could improve medial collateral ligament healing, whether this effect was dose-dependent, and if combinations of growth factors could improve healing more than individual growth factors. Thirty-seven rabbits had various doses of growth factors applied to the ruptured right medial collateral ligaments using a fibrin sealant delivery vehicle. The five groups consisted of 1) two groups receiving two doses of platelet-derived growth factor-BB, 2) two groups receiving two doses of this growth factor plus transforming growth factor-beta 1, and 3) one group receiving fibrin sealant only. After sacrifice at 6 weeks, biomechanical and histologic evaluations of the healing ligament were performed. Femur-medial collateral ligament-tibia complexes of the knees given the higher dose of platelet-derived growth factor-BB had ultimate load, energy absorbed to failure, and ultimate elongation values that were 1.6, 2.4, and 1.6 times greater than the same complexes of the control group. Adding transforming growth factor-beta 1 did not lead to any further increase in the structural properties of the complex compared with treatment with platelet-derived growth factor-BB. These encouraging results suggest that use of platelet-derived growth factor-BB may improve the quality of the healing medial collateral ligament, and that it may also have a similar potential for promoting healing of other ligaments.