Vascular physiology and long-term healing of partial ligament tears.

Functional outcomes of anterior cruciate ligament (ACL) injury are generally poorer than those of medial collateral ligament (MCL) tears. Following ligament damage, all phases of ligament healing require an adequate blood supply. We hypothesized that the differences in healing properties of the ACL and MCL would reflect their vascular responses to joint injury. This paper examines the long-term changes in blood flow and vascular volume of rabbit knee ligaments after direct injury, and under conditions of chronic joint instability induced by section of the posterior cruciate ligament (PCL). Standardized injuries were surgically induced in adult rabbit knee ligaments: partial MCL transection, partial ACL transection, or complete PCL transection (joint instability). Sixteen weeks later the blood flow and vascular volume of the ACL and MCL were measured and compared to control and sham-operated animals. Direct ligament injury induced significant increases in standardized blood flow and vascular volume of both ACL and MCL after 16 weeks; however, the vascular volume of the ACL was not higher than the control levels in the MCL. We conclude that direct injury to both the anterior cruciate and MCLs induces long-term physiological responses. Joint laxity is a common sequel to PCL injury. Chronic joint laxity failed to induce adaptive vascular responses in the ACL, while the MCL shows significant amplification of blood supply. Although both MCL and ACL showed increased weight after PCL transection, the lack of a long-term vascular response in the ACL may be a major factor in its the diminished healing potential.     

Immediate ACL reconstruction prevents microvascular pathophysiology in the Uninjured MCL that is not fully reversed by delayed ACL reconstruction

Anterior cruciate ligament (ACL) injury induces maladaptive vascular responses that degrade medial collateral ligament (MCL) function. The purpose of this study was to determine if early or delayed ACL reconstruction can prevent or reverse the abnormal changes in vascular function that occur in the uninjured MCL after ACL injury. Twenty‐four rabbits were divided into four groups (n = 6); control, ACL‐deficient (ACL‐X), immediate ACL reconstructed (ACL‐IR) and delayed ACL reconstructed (ACL‐DR). After 8 weeks, MCLs were assessed for blood flow, responses to acetylcholine (ACh) and phenylephrine (Phe) and autoregulatory responses, using laser speckle perfusion imaging. In ACL‐X knees, blood flow in the MCL increased by 2.5‐fold compared to control. MCL hyperemia was diminished in ACL‐DR knees and was unaltered in ACL‐IR knees. MCL vasculature was unresponsive to ACh and Phe in ACL‐X. These responses were partially restored by ACL reconstruction. Autoregulatory responses were not significantly different between groups. ACL‐DR decreased hyperemia in the MCL and partially attenuated abnormal MCL vascular responses. ACL‐IR was more effective at preventing MCL hyperemia and preserving vascular responsiveness to ACh and Phe. This suggests that the vascular alterations in the uninjured rabbit MCL are largely caused by abnormal mechanical loading resulting from ACL deficiency and can be prevented through early reconstruction. Early ACL reconstruction could limit the progression of microvascular dysfunction of the MCL, and preserve physiological joint homeostasis. This might prevent joint degeneration and delay the progression of osteoarthritis. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1390–1396, 2011     

Vascular adaptation of intact joint stabilizing structures in the posterior cruciate ligament deficient rabbit knee.

Loss of the posterior cruciate ligament (PCL) of the knee has a significant impact on joint stability and biomechanical function. Changes in joint biomechanics may result in mal-adaptive tissue degeneration and functional alteration of supporting ligaments. This study examines the effects of joint laxity on the vascular physiology of the intact anterior cruciate (ACL) and medial collateral (MCL) ligaments after PCL transection in rabbits.One-year-old female New Zealand white rabbits were assigned to control (n=12), sham-operated (n=12) or PCL transected (2, 6 or 16 weeks, n=12 per time point) groups. Half of the animals (n=6 per group) were used for ACL and MCL blood flow determination using coloured microsphere infusion (ml/min/100 g), and half were used for vascular volume determination (given as vascular index, micro l/g).In the MCL, PCL transection induced large, significant (4-5-fold) increases in blood flow (peak at 2 weeks) and vascular index (peak at 6 weeks) compared to sham-operated animals that returned towards control values by 16 weeks. In contrast, the ACL showed no increase in blood flow in lax joints, and a relatively small (2-fold) increase in vascular index at 6 weeks only. The wet weight and water content of both the MCL and ACL were significantly increased in PCL-deficient joints.We conclude that joint laxity (instability) subsequent to loss of the PCL in rabbits impacts the vascular physiology of intact supporting ligaments, inducing both vasomotor and angiogenic responses in the MCL. Changes in wet weight and water content of both the MCL and ACL demonstrate prolonged physiological adaptation of intact structures in lax joints.     

Bilogic characteristics of fibroblast cells cultured from the knee ligaments

OBJECTIVE: To culture fibroblast cells from the knee ligaments and to study the biological characteristics of these cells. METHODS: Cells of the anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) from New Zealand white rabbit were cultured in vitro. Cellular growth and expression of the collagen were analyzed. Moreover, an in vitro wound closure model was established and the healing of the ACL and the MCL cells was compared. RESULTS: Maximal growth for all these cells were obtained with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, but RPMI 1640 and Ham's F12 media were not suitable to maintain these cells. Morphology of both ACL and MCL cells from New Zealand white rabbit was alike in vitro, but the MCL cells grew faster than the ACL cells. Both cell types produced similar amount of collagen in culture, but the ratio of collage type I to type III produced by ACL cells was higher than that produced by MCL cells. Wound closure assay showed that at 36 hours after injury, cell-free zones created in the ACL cultures were occupied partially by the ACL cells; in contrast, the wounded zone in the MCL cultures was almost completely covered by the cells. CONCLUSIONS: Although the ACL cells and the MCL cells from New Zealand white rabbit show similar appearance in morphology in culture, the cellular growth and the biochemical synthesis of collagen as well as the healing in vitro were significantly different. These differences in intrinsic properties of the two types of cells in vitro might contribute to the differential healing potentials of these ligaments in vivo.     

Healing of the medial collateral ligament following a triad injury: a biomechanical and histological study of the knee in rabbits.

The effect of a partial medial meniscectomy and anterior cruciate ligament (ACL) transection on medial collateral ligament (MCL) healing was studied in skeletally mature rabbits. Two groups of animals, group I (isolated MCL rupture) and group II (MCL rupture with ACL transection and partial medial meniscectomy), were examined. At 6 and 12 weeks postoperatively, histological examination of the healing MCL and biomechanical evaluation of the varus-valgus (V-V) knee rotation and tensile properties of the femur-MCL-tibia complex (FMTC) were performed. Group II animals experienced substantial joint degeneration by 6 weeks. Progressive osteophyte formation was observed adjacent to the MCL insertions along with proximal migration of the MCL tibial insertion between 6 and 12 weeks. Histologic examination of the healing MCL substance from both groups showed disorganized collagen, inflammation, and fibroblast proliferation that decreased over time. For group II knees, the V-V knee rotation was found to be significantly elevated (4.7 to 5.2 times the contralateral control), and did not decrease with time. In contrast, the V-V knee rotations of the group I specimens were 1.8 times greater than control immediately following injury, and approached control values by 12 weeks. Tensile testing of the FMTCs revealed that the ultimate load increased with time for both groups, but group I had significantly higher values than group II. The linear stiffness in group I was not different than that group II and did not increase with time. For the mechanical (material) properties of the healed MCL substance, the modulus of the healing tissue for group II was only 40% that of group I. The structural properties of the FMTC and the mechanical properties of the MCL substance from both groups at 6 and 12 weeks were significantly different from the contralateral controls. We further demonstrated that immediately after ACL reconstruction, the V-V rotation of group II knees could be restored to group I levels. Recent clinical studies of MCL healing following isolated complete ligament tears have suggested that nonoperative management without immobilization leads to excellent treatment outcome. However, in more severe injuries involving additional tissues, poor quality of the healed ligament tissue and articular degeneration are observed. Our results demonstrate the deleterious effects of an untreated triad injury on the healing of the MCL substance and its insertions. Examination of the MCL substance suggests that a much larger healing mass is formed following a triad injury, which partially compensates for inferior ligament mechanical properties.(ABSTRACT TRUNCATED AT 400 WORDS)     

Type I procollagen gene expression in normal and early healing of the medial collateral and anterior cruciate ligaments in rabbits: an in situ hybridization study

The purpose of this study was to compare the levels of procollagen type I messenger RNA (mRNA) in normal and healing medial collateral ligament (MCL) and anterior cruciate ligament (ACL) in a rabbit model. Our method of injury involved a surgical model with identical partial lacerations in the midsubstance of the MCL and ACL. Paraffin sections of normal ligaments, and ligaments 3, 7, 14, and 28 days postlaceration were studied by in situ hybridization to compare and follow the level of type I procollagen mRNA in the two ligaments. A complementary DNA (cDNA) probe corresponding to alpha 1(I) procollagen mRNA was labeled with [32P]d-CTP. After hybridization, autoradiography, and staining of the sections, the level of procollagen mRNA was assessed by microscopic examination. A higher level of procollagen mRNA was consistently detected in normal MCL than in normal ACL, suggesting higher collagen synthetic activity in the MCL. At the injury sites of the MCL and ACL, the levels of type I procollagen mRNA increased at all post-laceration periods, reaching its highest level at 14 days postsurgery. The MCL healing site had a considerably higher level of procollagen mRNA than the ACL healing site (i.e., injury site) at all postoperative intervals. The results demonstrate that procollagen mRNA levels in MCL tissue are higher than those in ACL tissue under normal conditions, as well as in response to injury. The differences in the procollagen mRNA levels of MCL and ACL may reflect the synthesis of collagen in these tissues, and may contribute to the differences in their healing capacities.     

Vascular structure and function in the medial collateral ligament of anterior cruciate ligament transected rabbit knees

To determine if decreased vascular responsiveness in the medial collateral ligament (MCL) of anterior cruciate ligament transected (ACL‐t) rabbit knees is due to pericyte deficiency associated with angiogenesis. Vascular responses to potassium chloride (KCl), phenylephrine, acetylcholine, and sodium nitroprusside (SNP) were evaluated in ACL‐t rabbit knees (n = 6) and control knees (n = 5) using laser speckle perfusion imaging. Ligament degeneration was determined by ultrasound imaging. Vascular and pericyte volume were measured using quantitative immunohistochemical volumetric analysis using CD31 and α‐smooth muscle actin antibodies with co‐localization analysis. Perfusion was increased in the ACL‐t rabbits 2.5‐fold. Responsiveness to phenylephrine, SNP, and acetylcholine was significantly decreased in the ACL knee while no change in KCl responses was seen. MCL ultrasound imaging revealed decreased collagen organization, increased ligament thickness, and increased water content in the ACL‐t MCL. Vascular Volume was increased fourfold in ACL deficient knees, while pericyte volume to endothelial volume was not changed. No difference in CD31 and α‐SMA co‐localization was found. Blood vessels in the MCL of ACL‐t knees do not lack smooth muscle. The MCL vasculature can undergo constrictive response to KCl, but have impaired receptor mediated responses and impaired nitric oxide signaling. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1104–1110, 2014.     

Ultrastructural differences between the cells of the medical collateral and the anterior cruciate ligaments

The anterior cruciate ligament (ACL) does not heal after an interstitial tear, in contrast to the medial collateral ligament (MCL), whose interstitial tears heal readily. The light microscopic and ultrastructural differences between the cells of the two ligaments were studied in rabbit knees to observe the healing characteristics of the two ligaments. A rabbit knee was chosen because the rabbit ACL, like that of humans, does not heal following interstitial injury. The cells populating the MCL have the characteristics of fibroblasts. The cells of the ACL resemble fibrocartilage cells. The phenotypic differences in the cells of the two ligaments may be important determinants of the differences in healing.     

The healing medial collateral ligament following a combined anterior cruciate and medial collateral ligament injury--a biomechanical study in a goat model.

The ideal treatment of a combined anterior cruciate ligament (ACL) and medial collateral ligament (MCL) injury to the knee is still debated. In particular, the question of whether reconstruction of the ACL can provide the knee with sufficient multidirectional stability to allow for effective MCL healing needs to be better elucidated. Therefore, the first objective of this study was to quantify the changes in the function of goat knees between time-zero and 6 weeks following a combined ACL/MCL injury treated with ACL reconstruction. Using a robotic/universal force-moment sensor testing system, the kinematics of the knee and in situ forces in the ACL/ACL graft as well as in the sham-operated and healing MCL were evaluated in response to (1) a 67 N anterior-posterior (A-P) tibial load and (2) a 5 Nm varus-valgus (V-V) moment. The second objective was to evaluate the structural properties of the healing femur-MCL-tibia complex (FMTC) and the mechanical properties of the healing MCL at 6 weeks under uniaxial tension.In response to the 67 N A-P tibial load, the A-P translations for the experimental knee increased by as much as 4.5 times from time-zero to 6 weeks (p<0.05). Correspondingly, the in situ forces in the ACL graft decreased by as much as 45% (p<0.05). There was no measurable changes of the in situ force in the healing MCL. In response to a 5 Nm V-V moment, V-V rotations were twice as much as controls, but similar for both time periods. From time-zero to 6 weeks, the in situ forces in the ACL graft dropped by over 71% (p<0.05), while the in situ force in the healing MCL was as much as 35+/-19 N.In terms of the structural properties of the healing FMTC, the stiffness and ultimate load values at 6 weeks reached 53% and 29% of sham-operated contralateral controls, respectively (p<0.05). For the mechanical properties of the healing MCL substance, the values for tangent modulus and tensile strength were only 13% and 10% of sham-operated controls, respectively (p<0.05). These results suggest that the ACL graft stabilized the knee initially, but became loose over time. As a result, the healing MCL may have been required to take on excessive loads and was unable to heal sufficiently as compared to an isolated MCL injury.     

膝内侧半月板“湾征”对内侧副韧带断裂的诊断意义

目的内侧副韧带损伤后,关节镜下可见内侧半月板上滑膜缘完全显示,类似海湾形状,称为"海湾全景征"(简称"湾征"),判断其作为诊断膝内侧副韧带断裂标志体征的可靠性及意义。方法 2007年3月-2011年3月,纳入59例MRI检查提示内侧副韧带断裂患者作为观察组,其中男38例,女21例;年龄16～39岁,平均23.2岁;单纯内侧副韧带断裂12例,合并外侧半月板损伤16例,前交叉韧带损伤27例,前、后交叉韧带损伤3例,髌骨脱位1例。68例MRI检查提示无内侧副韧带断裂患者作为对照组,其中男45例,女23例;年龄25～49岁,平均31.8岁;前交叉韧带损伤38例,前、后交叉韧带损伤4例,前交叉韧带合并外侧半月板损伤26例。两组治疗前后行关节镜探查比较"湾征"出现情况。结果观察组膝内侧副韧带修复重建前关节镜探查均见"湾征",明确内侧副韧带断裂;修复重建后"湾征"消失。对照组交叉韧带重建前后均未见"湾征"。结论 "湾征"可作为关节镜下膝内侧副韧带断裂的诊断指征,以及术中韧带修复重建成功与否的判断依据。     

The early effects of joint immobilization on medial collateral ligament healing in an ACL-deficient knee: a gross anatomic and biomechanical investigation in the adult rabbit model.

In this study, the short-term effects of immobilization on joint damage and medial collateral ligament (MCL) healing were investigated in unstable, anterior cruciate ligament (ACL)-deficient knees in rabbits. Forty-six 12-month-old female New Zealand white rabbits were separated into three groups. Animals from each group had surgery on their right knees: group I, sham controls (n = 9); group II, complete transection of the ACL and removal of a 4 mm segment (gap injury) of MCL midsubstance with no immobilization of the limb (n = 19); and group III, same injuries to the ACL and MCL (as group II) but with immobilization of the limb (n = 18). No surgical repair of disrupted ligaments was performed. Left knees served as unoperated contralateral controls. All animals were allowed unrestricted cage activity until sacrifice in subgroups at 3, 6, and 14 weeks of healing when biomechanical properties of all MCLs were measured. All knee joints were systematically examined for gross evidence of damage to articular cartilage, menisci, and periarticular soft tissues. To monitor relative in vivo loads on injured limbs during healing, hindlimb weight bearing was assessed at biweekly intervals. Results indicated that animals in both groups II and III bore relatively lower loads (compared to preinjury values) on their injured hindlimbs. Mechanical testing of MCLs showed only minor changes in sham controls, while group II and III healing MCLs demonstrated significantly lower force and stress at MCL complex failure compared to contralateral controls. In specific comparisons of group III to group II animals, we noted that immobilization prevented joint damage over the early intervals studied. In addition, immobilization resulted in MCL laxity similar to contralateral control values but inhibited development of structural strength and stiffness in healing MCLs. These results suggest that in the rabbit, short-term immobilization of an ACL-deficient knee offers some advantages to the joint and to certain low load behaviors of the healing MCL, but it also results in a smaller quantity of scar tissue that is less able to resist higher loads. Longer-term studies involving remobilization are necessary before the effects of brief immobilization on joint damage and MCL healing in this ACL-deficient model can be fully defined.     

Differences in cellular properties and responses to growth factors between human ACL and MCL cells

The anterior cruciate ligament (ACL) has poor healing responses compared with those of the medial collateral ligament (MCL). It has been implied that this is partially due to the poor reparative capacity of ACL cells for ligament injury. The present study was designed to elucidate the reparative capacities of human ACL and MCL cells by investigating their cellular properties and their responses to growth factors. Human ACL and MCL were obtained from seven fresh human cadavers. The cells were isolated from each tissue, and primary cultures were used for the examination. The growth rates of all the human ACL cells were lower than those of the human MCL cells; consistent with this, the doubling time of the ACL cells was 30 ± 7.4% longer than that of the MCL cells. The chemotactic migration of human ACL cells was 33 ± 8.1% slower and the synthesis of DNA and collagen in human ACL cells was 29 ± 6.3% and 31 ± 9.7% lower, respectively, in comparison with those of MCL cells. Cellular responses, in terms of DNA synthesis, in human ACL cells to either basic-fibroblast growth factor (1.0 and 10.0 ng/ml) or transforming growth factor-β (1.0 ng/ml) were lower than those of human MCL cells. However, no differences in the cellular responses in terms of collagen synthesis were found. Composite data show that human ACL cells have poorer cellular properties and lower responses to growth factors compared with those of human MCL cells, which suggests that the reparative capacity of human ACL cells may be poorer than that of human MCL cells. Received for publication on Sept. 2, 1998; accepted on Jan. 29, 1999     

Differential sensitivity to NO-induced apoptosis between anterior cruciate and medial collateral ligament cells

It is well known that the anterior cruciate ligament (ACL) of the knee joint has poorer healing responses than the medial collateral ligament (MCL). Nitric oxide (NO) induces free radicals and plays a key role in the induction of apoptosis in various wound-healing models. We hypothesized that the poor healing response of the ACL may be ascribed to high susceptibility to apoptosis, and we investigated the difference in susceptibility to apoptosis between ACL and MCL cells after treatment with sodium nitroprusside, a NO donor. Apoptosis was evaluated by phase contrast microscopy, electron microscopy, DNA gel electrophoresis, and flow cytometric analysis. Although morphological changes and DNA ladders were observed in both ACL and MCL cells after 2mM sodium nitroprusside treatment, ACL cells were more prone to apoptosis at 1mM. Based on flow cytometric analysis, DNA fragmentation at 1mM sodium nitroprusside was significantly greater in ACL cells than in MCL cells (58.6% ± 1.6% vs. 11.9% ± 2.2%). Caspase-3 inhibitor (Ac-Asp-Glu-Val-Asp-CHO) and caspase-9 inhibitor (Ac-Leu-Glu-His-Asp-CHO) completely inhibited this DNA fragmentation. In conclusion, the ACL and MCL cells exhibit essential differences, and the differential sensitivity to NO-induced apoptosis between the ACL and MCL cells may be a reflection of these differences.     

Posttraumatic incarceration of medial collateral ligament into knee joint with anterior cruciate ligament injury

Medial collateral ligament of the knee is an important coronal stabiliser and often injured in isolation or as combination of injuries. The article reports a case of incarcerated medial collateral ligament (MCL) injury in combination with anterior cruciate ligament (ACL) injury in 20 year old male who presented to us 4 weeks after injury. Clinical examination and MRI was correlated to complete ACL tear with torn distal MCL and incarceration into the joint. Patient was taken up for ACL hamstring graft reconstruction with mini-arthrotomy and repair of the torn MCL. Patient was followed up with dedicated rehabilitation protocol with good functional results. At one year follow-up, patient exhibited full range of motion with negative Lachman, Pivot shift and valgus stress tests. This article highlights the rare pattern of MCL tear and also reviews the literature on this pattern of injury.     

Characterization of the intrinsic properties of the anterior cruciate and medial collateral ligament cells: an in vitro cell culture study.

The poor healing abilities of the anterior cruciate ligament (ACL) in contrast to those of the medial collateral ligament (MCL) are well known. Different intrinsic properties of the constituent cells of these ligaments have been proposed to be one of the factors in the differential repair mechanisms. To examine this hypothesis, we have established primary cell lines of ACL and MCL from the tissue explants of approximately similar dimensions and have studied their behavior in vitro. The outgrowth of cells from ACL explants was slower than from MCL explants, as shown by the size of the surrounding clusters of cells. Both ACL and MCL cultures exhibited typical fibroblastic morphology. No significant differences were observed in either attachment or growth of cells from the attached explants derived from various segments of ACL and MCL. Growth curves of ACL and MCL cultures at both passage numbers 2 and 6 showed a slower rate of proliferation of ACL cells than MCL cells (p less than 0.005). DNA synthesis measured in terms of [3H]thymidine incorporation (CPM/10(3) cells) of both log phase (ACL = 607.5 +/- 5.4 vs. MCL = 1356.4 +/- 11.3) and confluent (ACL = 83.0 +/- 3.6 vs. MCL = 189.8 +/- 5.4) cultures, supports the conclusion that differential proliferation rates of these cells exist in culture. FITC-phalloidin staining (for actin) of later passage cultures (P3-P5) showed a spread-out appearance of ACL cells and an elongated appearance of MCL cells. Relatively more stress fibers were seen within ACL cells. SDS-PAGE and Western blot analysis of cellular proteins revealed higher actin (43 kDa) content in ACL cells than in MCL cells. In vitro wound closure assay was performed by creating a uniform wound of 0.6 mm width in the confluent layer of ACL and MCL cultures. By 48 h postwounding, cell-free zones created in ACL cultures were occupied partially by single cells in a nonconfluent fashion. In contrast, the wounded zone in the MCL cultures was almost completely covered by the cells. Results presented in this report demonstrate a lower proliferation and migration potential of ACL cells in comparison with MCL cells. These differences in intrinsic properties of ACL and MCL cells that were observed in vitro might contribute to the differential healing potentials of these ligaments in vivo.     

Medial collateral knee ligament healing: Combined medial collateral and anterior cruciate ligament injuries studied in rabbits

We examined the histological appearance and biochemical properties of the healing medial collateral ligament (MCL) of a rabbit knee after combined MCL and anterior cruciate ligament (ACL) injury treated with ACL reconstruction and with or without MCL repair. By so doing, we hoped to understand better our previous biomechanical observations (Ohno et al. 1995) and possibly learn where to focus future investigation into improving the quality of the healing MCL.

Ligaments were examined at 6 and 12 weeks of healing. We found healing of all ligaments with hypercellularity and fibroblast elongation along the axis of loading, as expected. Unexpected, however, was the finding of multiple osteophytes in both the repaired and nonrepaired specimens at the medial borders of the joint and at the MCL insertions. These were felt to affect possibly the biomechanics of the MCL by causing stress risers at the point where they undermine the ligament. Biochemically, we demonstrated a correlation between collagen content and hydroxypyridinium crosslinks and modulus of elasticity. While this implies that the modulus is dependent on collagen content and hydroxypyridinium crosslink density, modulus is also probably dependent on other factors such as collagen organization, type and internal structure. Overall, the detailed characterization and correlation between the histological, biochemical, and biomechanical properties of the healing MCL in the severe knee injury model provide insight into the functional behavior of the healing MCL.     

Enhanced histologic repair in a central wound in the anterior cruciate ligament with a collagen-platelet-rich plasma scaffold.

The anterior cruciate ligament (ACL) of the knee is an intra-articular ligament that fails to heal after primary repair. The medial collateral ligament (MCL) of the knee is an extra-articular ligament that heals uneventfully in the majority of cases. Why these two ligaments have such different responses to injury remains unclear. In this article, we address two hypotheses: first, that the histologic response to injury is different in intra-articular and extra-articular ligaments, and second, that the response of the intra-articular ligaments can be altered by placing a collagen-platelet-rich plasma (collagen-PRP) hydrogel in the wound site. Wounds were created in extra-articular ligaments (MCL and/or patellar ligament) and an intra-articular ligament (ACL) in canine knees, and the histologic response to injury evaluated at 3 days (n = 3), 7 days (n = 4), 3 weeks (n = 5), and 6 weeks (n = 5). In the 3-week (n = 5) and 6-week (n = 5) animals, bilateral central wounds were made in the ACLs and the wounds in one knee of each animal treated with a collagen-PRP hydrogel while the contralateral side was untreated. Extra-articular ligament wounds had greater filling of the wound site and increased presence in the wound site of fibrinogen, fibronectin, PDGF-A, TGF-beta1, FGF-2, and von Willebrand's factor when compared to intra-articular ligament wounds. Treatment of the intra-articular wound with a collagen-PRP hydrogel resulted in increased filling of the wound site with repair tissue that had similar profiles of growth factor and protein expression to the extra-articular ligament wounds. The use of a collagen-PRP scaffold can ameliorate histologic differences noted between healing extra-articular ligamentous wounds and nonhealing intra-articular ligamentous wounds. This study supports the hypothesis that premature scaffold failure may play a key role in the normally expected failure of the ACL to heal after injury.     

α-Smooth muscle actin and TGF-β receptor I expression in the healing rabbit medial collateral and anterior cruciate ligaments

The aim of our study was to advance the knowledge about the biological differences in the healing of the anterior cruciate ligament (ACL) versus the medial collateral ligament (MCL). We quantified α-smooth muscle actin (α-SMA) expression and TGF-β receptor I (TGF-βRI) expression in experimentally injured rabbit ligaments (from day 3 to 12 weeks post-injury). Myofibroblasts (α-SMA positive cells) were identified as early as the third day post-injury in MCL and their density increased steadily up to day 21. Myofibroblasts were also detected in injured ACL but their density remained very low at all time points. The percentage of positive TGF-βRI area significantly increased in both injured ligaments compared to controls, with a peak expression at day 21; however, it remained constantly lower in ACL compared to MCL. A significant correlation was found between the percentage of TGF-βRI positive cells and the percentage of α-SMA expression only in injured MCL. These results provide evidence that myofibroblasts are important players in MCL remodelling after injury. The combined presence of myofibroblasts and TGF-βRI in the first 3 weeks post-MCL injury may partially explain the difference in the MCL and ACL healing process.     

Valgus medial collateral ligament rupture causes concomitant loading and damage of the anterior cruciate ligament

This study tested the hypothesis that application of a valgus force necessary to create a complete medial collateral ligament (MCL) injury causes damage to the anterior cruciate ligament (ACL). Twelve cadaveric knees were used to measure concomitant loading and damage to the ACL in valgus knee loading sufficient to cause a grade III MCL injury. Displacement sensors were placed on the anteromedial bundle of the ACL and posterior oblique ligament to monitor tensile strain during creation of the MCL injury. A valgus moment was applied to knees flexed at 30 degrees, displacing the joint into valgus rotation beyond MCL rupture. Following valgus loading and MCL injury, femur-ACL-tibia specimens were tested to failure to compare ACL mechanical integrity to noninjured control specimens. Average ACL strength in MCL ruptured knees (1250 +/- 90 N) was statistically lower (P < or = .05) than that for control knees (2110 +/- 50 N). Strain measurements exhibited concomitant posterior oblique ligament strain during valgus loading, whereas ACL strain increased substantially only after MCL rupture. These data indicate that the ACL can be compromised in isolated grade III MCL injuries.     

Effects of cigarette smoking on early medial collateral ligament healing in a mouse model.

Cigarette smoking delays the healing process and increases morbidity associated with many common musculoskeletal disorders such as medial collateral ligament (MCL) injury. In the current study, a murine model of MCL healing was used to test the hypothesis that smoking impairs extracellular matrix synthesis after injury. Mice were divided into two groups, a nonsmoking control group and a group exposed to smoke for 2 months prior to surgical MCL injury. Mice were euthanized at 3 and 7 days after surgery. Subsequently, propidium iodine staining was used to quantify cellular density of injured and sham ligaments. Immunohistochemical staining and in situ hybridization to mRNA were used to detect proliferation, apoptosis, and type I collagen gene expression at the site of injury. Cell density increased significantly from baseline to 7 days after injury in control mice. In mice exposed to cigarette smoke, there was a significantly lower cellular density compared to controls at this time point (p=0.01). There was no difference in proliferation between groups at the site of injury, and the low level of proliferation observed was not sufficient to account for the large increase in cell density by day 7. No evidence of apoptosis was observed in any of the groups at the site of injury. Type I collagen gene expression was higher in controls compared to smokers at day 7. Almost all of the cells in the substance of the injured MCL at day 7 were spindle-shaped and expressed type I collagen, suggesting that increased cell density from day 3 to day 7 represented an increase in ligament cells rather than an increased inflammatory response. We conclude that the decreased cellular density and type I collagen expression in the injured ligament of mice exposed to smoke begin to provide a cellular and molecular basis for delayed or deficient early healing in these animals.