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.

Instrument-assisted cross fiber massage increases tissue perfusion and alters microvascular morphology in the vicinity of healing knee ligaments.

BACKGROUND: Ligament injuries are common clinical problems for which there are few established interventions. Instrument-assisted cross fiber massage (IACFM) was recently shown to accelerate the restoration of biomechanical properties in injured rodent knee medial collateral ligaments (MCL). The current study aimed to investigate the influence of IACFM on regional perfusion and vascularity in the vicinity of healing rodent knee MCL injuries. METHODS: Bilateral knee MCL injuries were induced in female Sprague-Dawley rats. Commencing 1 week post-injury, 1 minute of IACFM was introduced unilaterally 3 times/week for 3 weeks. The contralateral injured MCL served as an internal control. Regional tissue perfusion was assessed in vivo throughout healing using laser Doppler imaging, whereas regional microvascular morphology was assessed ex vivo via micro-computed tomography of vessels filled with contrast. RESULTS: IACFM had no effect on tissue perfusion when assessed immediately, or at 5, 10, 15 or 20 min following intervention (all p > 0.05). However, IACFM-treated hindlimbs had enhanced tissue perfusion when assessed 1 day following the 4th and 9th (last) treatment sessions (all p < 0.05). IACFM-treated hindlimbs also had greater perfusion when assessed 1 wk following the final treatment session (32 days post-injury) (p < 0.05). Subsequent investigation of microvascular morphology found IACFM to increase the proportion of arteriole-sized blood vessels (5.9 to <41.2 µm) in the tibial third of the ligament (p < 0.05). CONCLUSIONS: These findings suggest IACFM alters regional perfusion and vascularity in the vicinity of healing rodent knee MCL injuries. This effect may contribute to the beneficial effect of IACFM observed on the recovery of knee ligament biomechanical properties following injury.

The effects of plasma rich in growth factors (PRGF-Endoret) on healing of medial collateral ligament of the knee.

PURPOSE: Platelet-rich plasma (PRP) has been increasingly used in sports-related injuries for therapeutic applications. However, there are numerous manufacturing procedures and treatment protocols of PRP use, which make difficult to assess its real efficacy for tissue healing. This study addressed to evaluate the therapeutic effects of locally delivered plasma rich in growth factors (PRGF-Endoret) on the early healing of medial collateral ligament (MCL) in rabbit knees. METHODS: Thirty-one Japanese white rabbits were subjected to a mop-end tear in the MCL of the left knee. PRGF-Endoret was prepared using Anitua's technique. Two groups were set up. In 17 knees, prepared 1.0 ml of PRGF-Endoret after clotting was applied on the tear site, while in 14 knees the tear site was untreated serving as a control. Quantitative aspects of PRGF-Endoret, the concentration of platelets, leukocytes and erythrocytes and therapeutic growth factors such as PDGF-BB and TGF-ß1 were measured. Rabbits were sacrificed at 3 and 6 weeks after the operation and histological and biomechanical evaluation were performed. RESULTS: No leukocytes were measured and certain amount of growth factors such as PDGF-BB and TGF-ß1 were confirmed in the PRGF-Endoret. PRGF-Endoret stimulated proliferation of fibroblasts and neovascularization, and induced statistically better structural properties in repaired MCL. CONCLUSIONS: Our findings provide evidence that local administration of PRGF-Endoret promotes early steps in ligament healing and the repair of structural properties in a rabbit model. PRGF-Endoret would be a useful product in clinical treatment of ligament injuries.

Role of angiogenesis after muscle derived stem cell transplantation in injured medial collateral ligament.

We performed this study to investigate the therapeutic role of vascular endothelial growth factor (VEGF) in medial collateral ligament (MCL) healing. Murine muscle derived stem cells (MDSCs) obtained via the preplate technique were retrovirally transduced to express: (1) VEGF and nLacZ (MDSC-VEGF), (2) soluble fms-like tyrosine kinase-1 (sFLT1, a VEGF-specific antagonist) and nLacZ (MDSC-sFLT1), and (3) nLacZ (MDSC-nLacZ). After transecting the MCL of immunodeficient rats, 5 × 10(5) cells of each of the transduction groups list above were transplanted into the MCL injury site. A control group was injected with phosphate-buffered saline (PBS) only. Immunohistochemical staining demonstrated that there were more Isolectin B4 and ß-galactosidase double positive cells in the rats transplanted with MDSC-VEGF transduced cells than the other groups at week 1. Capillary density was significantly higher in the MDSC-VEGF group than the other groups at week 2; however, there were no significant differences in the biomechanical assessment between the MDSC-VEGF and MDSC-nLacZ groups. On the other hand, the MDSC-sFLT1 group revealed a lower capillary density than the other two groups and the functional ligament healing of the MDSC-sFLT1 group was significantly decreased compared to the other groups when assessed biomechanically. The findings of the present study suggest that angiogenesis plays a critical role in the healing process of injured MCL.

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.

Neural stimulation does not mediate attenuated vascular response in ACL-deficient knees: potential role of local inflammatory mediators.

Chronic inflammation associated with osteoarthritis (OA) alters normal responses and modifies the functionality of the articular vasculature. Altered responsiveness of the vasculature may be due to excessive neural activity associated with chronic pain and inflammation, or from the production of inflammatory mediators which induce vasodilation. Using laser speckle perfusion imaging (LSPI), blood flow to the medial collateral ligament (MCL) of adult rabbits was measured in denervated ACL transected knees (n = 6) and compared to unoperated control (n = 6) and 6-week anterial cruciate ligament (ACL)-transected knees (n = 6). Phenylephrine and neuropeptide Y were applied to the MCL vasculature in topical boluses of 100 microL (dose range 10(-14) to 10(-8) mol and 10(-14) to 10(-9) mol, respectively). Denervation diminished vasoconstrictive responsiveness to phenylephrine compared to both control and ACL-transected knees. Denervation minimally enhanced vascular responses to neuropeptide Y (NPY) compared to ACL deficiency alone, which nevertheless remained significantly diminished from control responses. To evaluate the potential role of inflammatory dilators in the diminished contractile responses, phenylephrine was coadministered with histamine, substance P, and prostaglandin E(2). High-dose histamine, and low-dose substance P and PGE(2) were able to inhibit contractile responses in the MCL of control knees. Excessive neural input does not mediate diminished vasoconstrictive responses in the ACL transected knee; inflammatory mediators may play a role in the deficient vascular responsiveness of the ACL transected knee.

Injury-induced changes in mRNA levels differ widely between anterior cruciate ligament and medial collateral ligament.

BACKGROUND: The drastic difference in healing capacity between the anterior cruciate ligament and the medial collateral ligament is still largely unexplained. Few studies have compared the profiles of messenger ribonucleic acid expression for healing-associated molecules in ligaments during the course of healing. HYPOTHESIS: Injury responses of the injured anterior cruciate ligament and medial collateral ligament are characterized by very different profiles of angiogenesis-promoting and repair-associated gene expression during the healing process. STUDY DESIGN: Controlled laboratory study. METHODS: Reverse-transcriptase polymerase chain reaction was used to assay expression of messenger ribonucleic acid for 11 healing- and angiogenesis-associated molecules at 3 days and 2, 6, and 16 weeks after anterior cruciate ligament or medial collateral ligament injury in adult female New Zealand White rabbits. RESULTS: Marked differences were found in the postinjury changes in messenger ribonucleic acid levels in the anterior cruciate ligament compared to the medial collateral ligament. Notably, messenger ribonucleic acid levels for the important repair-associated growth factor transforming growth factor-beta1 did not increase in injured anterior cruciate ligament at any time point. Similarly, unlike the injured medial collateral ligament, no statistically significant increases in messenger ribonucleic acid levels for the important scar matrix protein collagen III were detected in injured anterior cruciate ligament. In contrast, matrix metalloproteinase messenger ribonucleic acid levels were markedly elevated in injured anterior cruciate ligament but only modestly increased in medial collateral ligament. CONCLUSION: The results suggest that injury leads to an antifibrotic, catabolic response in the rabbit anterior cruciate ligament, possibly to prevent fibrosis and diminish the risk for loss of joint motion. CLINICAL RELEVANCE: The development of effective biologically based treatments for anterior cruciate ligament injuries will need to incorporate strategies to deal with the significant differences in the molecular responses to injury of these tissues.

Nerve growth factor improves ligament healing.

Previous work has shown that innervation participates in normal ligament healing. The present study was performed to determine if exogenous nerve growth factor (NGF) would improve the healing of injured ligament by promoting reinnervation, blood flow, and angiogenesis. Two groups of 30 Sprague-Dawley rats underwent unilateral medial collateral ligament transection (MCL). One group was given 10 microg NGF and the other was given PBS via osmotic pump over 7 days after injury. After 7, 14, and 42 days, in vivo blood flow was measured using laser speckle perfusion imaging (LSPI). Morphologic assessments of nerve density, vascularity, and angiogenesis inhibitor production were done in three animals at each time point by immunohistochemical staining for the pan-neuronal marker PGP9.5, the endothelial marker vWF, and the angiogenesis inhibitor thrombospondin-2 (TSP-2). Ligament scar material and structural mechanical properties were assessed in seven rats at each time point. Increased nerve density was promoted by NGF at both 14 and 42 days. Exposure to NGF also led to increased ligament vascularity, as measured by histologic assessment of vWF immunohistochemistry, although LSPI-measured blood flow was not significantly different from controls. NGF treatment also led to decreased expression of TSP-2 at 14 days. Mechanical testing revealed that exposure to NGF increased failure load by 40%, ultimate tensile strength by 55%, and stiffness by 30% at 42 days. There were no detectable differences between groups in creep properties. The results suggest that local application of NGF can improve ligament healing by promoting both reinnervation and angiogenesis, and results in scars with enhanced mechanical properties.

Endothelial dysfunction and decreased vascular responsiveness in the anterior cruciate ligament-deficient model of osteoarthritis.

Chronic inflammation associated with osteoarthritis (OA) may alter normal vascular responses and contribute to joint degradation. Vascular responses to vasoactive mediators were evaluated in the medial collateral ligament (MCL) of the anterior cruciate ligament (ACL)-deficient knee. Chronic joint instability and progressive OA were induced in rabbit knees by surgical transection of the ACL. Under halothane anesthesia, laser speckle perfusion imaging (LSPI) was used to measure MCL blood flow in unoperated control (n = 12) and 6-wk ACL-transected knees (n = 12). ACh, bradykinin, histamine, substance P (SP), and prostaglandin E(2) (PGE(2)) were applied to the MCL vasculature in topical boluses of 100 microl (dose range 10(-14) to 10(-8) mol). In normal joints, ACh, bradykinin, histamine, and PGE(2) evoked a dilatory response. Substance P caused a biphasic response that was dilatory from 10(-14) to 10(-11) mol and constricting at higher doses. In ACL-deficient knees, ACh, bradykinin, histamine, and SP decreased perfusion, whereas PGE(2) had a biphasic response that decreased perfusion at 10(-14) to 10(-11) mol and was dilatory at higher concentrations. Sodium nitroprusside increased perfusion in resting and phenylephrine-precontracted vessels with no significant differences between ACL-transected and control knees. Femoral artery occlusion and release increased perfusion by 74.3 +/- 11.1% in control knees but only by 25.8 +/- 4.4% in ACL-deficient knees. The altered responsiveness of the MCL vasculature to these inflammatory mediators may indicate endothelial dysfunction in the MCL, which may contribute to the progression and severity of OA and to the adaptation of the joint in an altered mechanical environment.

Correlation of healing capacity with vascular response in the anterior cruciate and medial collateral ligaments of the rabbit.

In clinical terms, functional recovery after anterior cruciate ligament (ACL) injury is generally poorer than after medial collateral ligament (MCL) injury. In experimental studies of injury, the early phases of ligament healing require an augmented blood supply. We hypothesized that the differences in healing properties of the ACL and MCL would be reflected in the magnitude of their vascular responses to partial injury. This study is the first to quantify and define the time course of changes in blood flow and vascular volume following hemisection of the rabbit ACL and MCL. Adult female rabbits were assigned to control, sham operation, ACL hemisection or MCL hemisection groups. Standardized ACL or MCL injuries were surgically induced. About 2, 6 or 16 weeks later, blood flow and vascular volume of the ACL and MCL were measured. The MCL of the rabbit responded to hemisection with a large significant increase in blood flow and a substantial angiogenic response associated with inflammation and scar formation. During subsequent matrix remodelling, blood flow and vascular volume returned towards control values. In contrast, the ACL showed only a 2-fold increase in vascular volume, no increase in blood flow and atrophied after hemisection. The superior capacity of the MCL to increase its blood supply through angiogenesis and increased flow is essential for ligament healing to occur, and may be the major difference in healing potential between the ACL and MCL.

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.

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.

Denervation impairs healing of the rabbit medial collateral ligament.

Little is known about the contribution of innervation to ligament healing after traumatic disruption, although there is good evidence of an important role for the peripheral nervous system in the healing of fractures and skin injuries. Tissues such as ligament, with a low resting blood supply, are dependent on substantial increases in blood flow and vascular volume during the initial stages of repair. We hypothesized that this initial healing response would be strongly promoted by neurogenic inflammation. Since the saphenous nerve (a major sensory branch of the femoral nerve) supplies the medial half of the knee joint, we elected to use femoral nerve transection as a model to determine the role of sensory and autonomic innervation in the initial outcome of repair of the injured medial collateral ligament. Twelve adult, female NZW rabbits underwent right medial collateral ligament transection. Of these, six rabbits underwent right femoral nerve transection to disrupt the somatic sensory and autonomic nerve supply to the knee joint and six were kept neurologically intact (controls). At six weeks post-injury, the animals were assessed by laser Doppler perfusion imaging (LDI) to determine the local blood flow, at both the injury site and at the uninjured contralateral ligament. The animals were then killed, the knee joints were removed and the biomechanical characteristics of the healing bone-median collateral ligament (MCL)-bone complexes assessed. In a separate cohort of 16 rabbits, vascular volumes of the injured ligaments were measured by infusion of a carmine red/gelatin solution. At six weeks post-injury, in vivo measurement of perfusion with LDI revealed that normally innervated ligaments had an almost three-fold higher average blood flow. Carmine red/gelatin infusion revealed a 50% higher density of blood vessels as compared to denervated ligaments. The force required for ultimate failure was found to be 50% higher in normally innnervated MCL's as compared to denervated MCL's: 153.14 +/- 20.71 N versus 101.29 +/- 17.88 N (p < 0.05). Static creep was increased by 66% in denervated MCL's: 2.83 +/- 0.45% versus 1.70 +/- 0.12% (p < 0.05). Total creep was increased by 45% in denervated MCL's: 5.29 +/- 0.62% compared to 3.64 +/- 0.31% in innervated MCL's (p < 0.05). We conclude that intact innervation makes a critical contribution to the early healing responses of the MCL of adult rabbits.

Adaptation of post-traumatic angiogenesis in the rabbit knee by apposition of torn ligament ends.

Apposition of torn ligament ends has been shown to have a beneficial effect on healing of the medial collateral ligament; however, the mechanism underlying this improved recovery is unclear. Excessive post-traumatic angiogenesis, an inherent component of soft-tissue regeneration, may be functionally detrimental in relatively hypovascular tissues such as ligaments. The present study therefore examined the relationship between contact of transected ligament ends and vascular remodeling. Female New Zealand White rabbits were subjected to a gap injury, Z-plasty apposition, or sham operation to the midsubstance of the medial collateral ligament. Six weeks after treatment, the volume of vessels supplying the healing zone of the medial collateral ligament, as well as the ipsilateral lateral collateral ligament, posterior cruciate ligament. menisci, and medial capsule, was quantified by carmine red vascular casting. The volume of vessels supplying the neoligamentous scar formed by gap injury to the medial collateral ligament was found to be twice that of ligaments that had undergone the sham operation, and lateral collateral ligament and meniscal vascularity was also augmented in the injured joint. The medial collateral ligaments that underwent Z-plasty apposition exhibited a level of vascularity comparable with that of the control ligaments that had undergone the sham procedure, whereas meniscal and lateral collateral ligament vascularities remained elevated in this group. Capsular and posterior cruciate ligament vascularities were unaffected by gap injury or Z-plasty to the ipsilateral medial collateral ligament. These findings indicate that injury to the medial collateral ligament not only stimulates angiogenesis in the healing ligament, but other ipsilateral soft tissues also undergo vascular remodeling. Furthermore, apposition of an injured medial collateral ligament modifies these pro-angiogenic events, and this may partly explain why contact of torn ligament ends is beneficial for post-traumatic recovery of an injured joint.

Late gestational changes in sympathomimetic sensitivity in primagravid rabbit ligaments.

The adrenoceptor profile of blood vessels supplying the medial collateral ligament (MCL) of virgin and primagravid (day 29 of pregnancy) rabbit knees was examined. Topical bolus administration of the alpha1-adrenoceptor agonists methoxamine and phenylephrine, and the alpha2-adrenoceptor agonist clonidine, to exposed knee joints resulted in a dose-dependent vasoconstriction of ligamentous vessels. The rank order of potency for the alpha-agonists was found to be phenylephrine > methoxamine > clonidine. Dobutamine, which acts on beta1-adrenoceptors, and ritodrine, which is a beta2-agonist, imparted a mild vasodilatatory effect on MCL blood vessels with the efficacy of ritodrine being greater than that of dobutamine. In primagravid rabbits, the constrictor effects of methoxamine and phenylephrine were significantly attenuated compared with virgin control animals (P < 0.0001), whereas clonidine-mediated vasoconstriction was unaltered in the gravid animal (P = 0.3957). With respect to beta-adrenoceptor activity, the dilatational effect of dobutamine was the same as in controls (P = 0.5294), while ritodrine vasoactivity was completely abolished in primagravid knees (P < 0.005). These findings suggest that the adrenergic control of rabbit MCL blood flow is predominantly mediated by postjunctional alpha1 and beta2-adrenoceptors. Pregnancy leads to either downregulation and (or) desensitisation of alpha1 and beta2-adrenoceptors in the ligament which could disrupt normal joint homeostasis.

[Microangiographic evaluation of vascular supply of the medial collateral ligament of the knee joint]. / Mikroangiographische Darstellung der Gefässversorgung des medialen Kollateralbandes am Kniegelenk.

Because the vascularization of ligaments is an important factor in ligament healing we wanted to examine the vascularization status of the medial collateral ligament of the knee. The type of vascularization was examined by microangiographie of 21 postmortem preparations. We could observe a vascularization from the proximal end of the ligament by one or two major vessels in all cases. These vessels were mostly localized in the ventral third of the ligament. From the proximal to the distal part of the ligament we found a decreasing density of blood vessels. We could not observe vessel free ligament areas. Due to this vascularization status we would advise the differentiation of two ligament areas respecting the following therapy. Because of the good vascularization in the proximal part of the ligament we can accept good conditions concerning complete healing after a non-operative treatment and an operative therapy by suture or refixation. In the case of interligamentous and distal ruptures operative reconstruction by sutures often in difficult and a conservative therapy leads to a high rate of poor results. Therefore we advise fibrin bonding in these ligament areas if a surgical procedure is indicated because of the high risk of vessel strangulation after suture.

Pregnancy-induced changes in rabbit medial collateral ligament vasoregulation.

The ligaments of weight-bearing joints are known to become mechanically inferior during pregnancy, and it has been postulated that this may be due to changes in tissue perfusion. Calcitonin gene-related peptide (CGRP) and epinephrine exert a tonic influence on the vasculature of the medial collateral ligament (MCL), and the present study examined whether these vasoactive influences were altered by pregnancy. Ligament perfusion experiments were performed on primigravid New Zealand White rabbits with the use of laser Doppler perfusion imaging. In pregnant animals (day 29), MCL basal perfusion fell significantly compared with control; however, values returned to normal 5 days postpartum. In normal joints, topical application of CGRP resulted in a dose-dependent increase in MCL perfusion, whereas epinephrine administration caused a dose-dependent fall in blood flow. During pregnancy, the vasodilator effect of CGRP was completely abolished, whereas adrenergic vasoconstriction was greater than normal. Both responses returned postpartum. Pregnancy in the rabbit produces hypoemia in the MCL, and this phenomenon may be effected by a tempering of CGRP dilator responses and an augmentation of alpha-adrenoceptor-mediated vasoconstriction.

Vascular volume determination of articular tissues in normal and anterior cruciate ligament-deficient rabbit knees.

The vasculature of diarthroidal joints has been well documented; however, the volume of vessels supplying different articular tissues is unknown. Angiogenesis, the formation of new vessels from preexisting ones, is difficult to quantify in joints due to the unavailability of a suitable technique. Although angiogenesis is known to occur in rheumatoid arthritis, the development of new vessels following joint injury has not been ascertained. A vascular casting technique was developed using carmine red dye to measure the vascular volume of the medial collateral ligament (MCL), lateral collateral ligament (LCL), menisci, medial capsule, and infrapatellar fat pad of the rabbit knee joint. Vascular volume determinations were repeated at 4 weeks in a group of anterior cruciate ligament (ACL)-transected animals and in a sham-operated control group. The volume of vessels supplying the MCL was estimated to be 0.22 +/- 0.07 microliter (mean +/- S.E.M.), the LCL volume was 0.25 +/- 0.05 microliter, the medial meniscus volume was 0.19 +/- 0.03 microliter, the lateral meniscus volume was 0.40 +/- 0.08 microliter, the medial capsule volume was 0.14 +/- 0.05 microliter, and the infrapatellar fat pad volume was 1.90 +/- 0.62 microliters. Following ACL transection, angiogenesis was found to occur in the MCL only. All other tissue vascularities were not significantly different from sham-operated controls. A quantifiable method for measuring vascular volume of knee joint tissues has been described. Joint instability stimulates angiogenesis in the ipsilateral MCL; however, the absence of angiogenic activity in other articular tissues might help explain the lack of posttraumatic healing associated with these joints.

Physiological and mechanical adaptations of rabbit medial collateral ligament after anterior cruciate ligament transection.

Progressive physiological and mechanical changes in the medial collateral ligament of the adult rabbit were investigated for as long as 48 weeks after disruption of the anterior cruciate ligament. Eighty-one New Zealand White rabbits were separated into experimental, sham-operated control, and normal control groups. The experimental group underwent unilateral transection of the right anterior cruciate ligament, sham-operated animals served as controls for comparison, and normal animals were evaluated as age-matched, undisturbed (no surgery) controls. Blood flow to the medial collateral ligament (as a physiological measure) and mechanical function (structural and material properties) were assessed at 6, 14, and 48 weeks. The results indicated that loss of the anterior cruciate ligament leads to early mechanical deterioration of the medial collateral ligament with a corresponding loss of physiological homeostasis. Six to 14 weeks after the transection, values for cross-sectional area of the medial collateral ligaments rapidly increased to 1.5 times control values. The ligament became twice as large as the control ligament by 48 weeks. Concomitantly, medial collateral ligament stress at failure of the medial collateral ligament complex decreased rapidly 6-14 weeks after the transection and eventually fell to one-half that of controls by 48 weeks. In terms of low-load behaviour, laxity and load relaxation were significantly greater than that of controls 6 weeks after transection and were further increased by 14 weeks. By 48 weeks, laxity values had recovered somewhat and load-relaxation measures had recovered to near control values. At both 6 and 14 weeks, a statistically significant elevation in blood flow was demonstrated compared with controls. By 48 weeks, however, blood flow was no different from that of the sham-operated control. Thus, early after transection of the anterior cruciate ligament, both low-load and high-load mechanical properties of the medial collateral ligament deteriorated and the rate of blood flow was temporarily elevated. By 48 weeks, blood flow declined to near control values, with a corresponding recovery in viscoelastic behaviour. These findings suggest that, after transection of the anterior cruciate ligament, viscoelastic behaviour of the medial collateral ligament may be related to changes in blood flow and that restoration of normal flow patterns and vascular responses may be linked to the recovery of some low-load mechanical properties in the anterior cruciate ligament-deficient medial collateral ligament.

Normal and healing ligament vascularity: a quantitative histological assessment in the adult rabbit medial collateral ligament.

Normal and healing adult rabbit medial collateral ligaments (MCL) have been assessed for microvascular anatomy using a quantitative image analysis methodology. MCL preparation by ink-gelatin perfusion enabled acceptable visualisation of microvascular channels within the tissue. Fifteen adult rabbits were studied; 3 normal rabbits formed an external time-zero control group; 12 animals received a standardised gap injury to the right MCL. The ligament injury was permitted to heal for 3, 6, 17 or 40 wk (3 animals in each healing group). Results confirmed that the normal MCL is hypovascular (about 1.46% vascularity by area) and that microvascular channels are highly organised and oriented longitudinally deep within the tissue. Healing MCL scar becomes twice as vascular as normal ligament early on, but returns to near normal values by 40 wk. Microvascular channels appear less organised in scar than in contralateral controls, but remodel with time. The directional scatter and spatial extent of ligament microvascular channels is quantifiable in normal and healing tissues.