Synovial and cartilage responsiveness to peri-operative hyaluronic acid ± dexamethasone administration following a limited injury to the rabbit stifle joint.

Posttraumatic osteoarthritis (PTOA) can develop after an injury to the knee. Previous studies have indicated that an intra-articular (IA) injection of the potent glucocorticoid dexamethasone (DEX) may significantly prevent induction of PTOA. The aim of the present study was to investigate the effectiveness of a single IA injection of hyaluronic acid (HA), alone and in combination with DEX following a localized intra-articular injury as a PTOA-preventing treatment option. An established rabbit model of surgical injury consisting of dual intra-articular (IA) drill holes in a non-cartilaginous area of the femoral notch near the origin of the anterior cruciate ligament (ACL) to allow for bleeding into the joint space was used. Immediately following surgery, subjects were treated with HA, HA + DEX, or received no treatment. An uninjured control group was used for comparison (N = 5/group). Rabbits were sacrificed and investigated at 9 weeks post-injury. At 9 weeks post-injury, there was a significant protective capacity of the single IA treatment of DEX + HA on the histological grade of the synovial tissue, and some variable location-specific effects of HA alone and HA + DEX interactions on cartilage damage. Thus, it is possible that co-treatment with HA may interfere with the effectiveness of the DEX. In vitro friction testing indicated that DEX did not interfere with the lubricating ability of HA or synovial fluid on cartilage. These results suggest that a single IA administration of HA in combination with DEX following an IA injury is not recommended for inhibition of PTOA progression in this model.

Analysis of change in gait in the ovine stifle: normal, injured, and anterior cruciate ligament reconstructed.

BACKGROUND: Many patients who undergo anterior cruciate ligament (ACL) reconstructive surgery develop post-traumatic osteoarthritis (PTOA). ACL reconstructive surgery may not fully restore pre-injury joint biomechanics, thereby resulting in further joint damage and contributing to the development of PTOA. In an ovine model of idealized ACL reconstruction (ACL-R), it has been shown that signs of PTOA develop within surgical joints by 20 weeks post-surgery. The aim of the present study was to investigate whether altered kinematics contribute to early PTOA development within ACL-R joints of the ovine injury model by comparing the gait of these surgical animals to the gait of a stable normal control group, and an unstable injury group in which the ACL and medial collateral ligament (MCL) had been transected. METHODS: Fifteen skeletally mature female sheep were allocated evenly into 3 treatment groups: normal control, ACL-R, and ACL/MCL Tx (each group n = 5). Each animal's gait was recorded at baseline, 4 weeks post injury, and 20 weeks post injury. Principal component analysis (PCA) was used to identify the kinematic patterns that may be discriminant between treatment groups. Results from previous studies were referenced to present the amount of gross PTOA-like changes that occurred in the joints. RESULTS: ACL-R and ACL/MCL transected (Tx) animals developed a similar amount of early PTOA-like changes within the surgical joints, but differed significantly in the amount of kinematic change present at 20 weeks post-surgery. We showed that the stifle joint kinematics of ACL/MCL Tx differed significantly from those of CTRL and the majority of ACL-R animals, while no significant differences in joint kinematic changes were found between ACL-R and CTRL animals. CONCLUSIONS: These results suggest that the early PTOA-like changes reported in the ACL-R model cannot be attributed exclusively to post-surgical kinematic changes, and therefore biologic components in the post-injury environment must be contributing significantly to PTOA development.

Embryonic stem cell-derived osteocytes are capable of responding to mechanical oscillatory hydrostatic pressure.

Osteoblasts can be derived from embryonic stem cells (ESCs) by a 30 day differentiation process, whereupon cells spontaneously differentiate upon removal of LIF and respond to exogenously added 1,25α(OH)2 vitamin D3 with enhanced matrix mineralization. However, bone is a load-bearing tissue that has to perform under dynamic pressure changes during daily movement, a capacity that is executed by osteocytes. At present, it is unclear whether ESC-derived osteogenic cultures contain osteocytes and whether these are capable of responding to a relevant cyclic hydrostatic compression stimulus. Here, we show that ESC-osteoblastogenesis is followed by the generation of osteocytes and then mechanically load ESC-derived osteogenic cultures in a compression chamber using a cyclic loading protocol. Following mechanical loading of the cells, iNOS mRNA was upregulated 31-fold, which was consistent with a role for iNOS as an immediate early mechanoresponsive gene. Further analysis of matrix and bone-specific genes suggested a cellular response in favor of matrix remodeling. Immediate iNOS upregulation also correlated with a concomitant increase in Ctnnb1 and Tcf7l2 mRNAs along with increased nuclear TCF transcriptional activity, while the mRNA for the repressive Tcf7l1 was downregulated, providing a possible mechanistic explanation for the noted matrix remodeling. We conclude that ESC-derived osteocytes are capable of responding to relevant mechanical cues, at least such that mimic oscillatory compression stress, which not only provides new basic understanding, but also information that likely will be important for their use in cell-based regenerative therapies.

Cartilage boundary lubrication of ovine synovial fluid following anterior cruciate ligament transection: a longitudinal study.

OBJECTIVE: To assess ovine synovial fluid (oSF) from different post-injury time points for (1) proteoglycan-4 (PRG4) and hyaluronan (HA) concentration, (2) HA molecular weight (MW) distribution, (3) cartilage boundary lubrication function, and (4) lubricant composition-function relationships. The association between cartilage boundary lubrication and gross cartilage changes after injury was also examined. METHODS: oSF was collected 2, 4, 10, and 20 weeks post anterior cruciate ligament (ACL) transection in five skeletally mature sheep. PRG4 and HA concentrations were measured using sandwich enzyme-linked immunosorbent assay, and HA MW distribution by agarose gel electrophoresis. Cartilage boundary lubrication of oSF was assessed using a cartilage-cartilage friction test. Gross damage to articular cartilage was also quantified at 20 weeks using modified Drez scoring protocol. RESULTS: Early (2-4 weeks) after ACL injury, PRG4 concentrations were significantly higher (P = 0.045, P = 0.037), and HA concentrations were substantially lower (P = 0.005, P = 0.005) compared to 20 weeks. The HA MW distribution also shifted towards lower ranges in the early post-injury stage. The kinetic friction coefficients were significantly higher 2-4 weeks post injury (P = 0.008 and P = 0.049) compared to 20 weeks. Poor cartilage boundary lubricating ability early after injury was associated with cartilage damage at 20 weeks. CONCLUSION: Altered composition and diminished boundary lubrication of oSF early after ACL transection may pre-dispose the articular cartilage to degenerative changes and initiate osteoarthritis (OA). These observations also provide potential motivation for biotherapeutic interventions at earlier time points post injury.

Changes of early post-traumatic osteoarthritis in an ovine model of simulated ACL reconstruction are associated with transient acute post-injury synovial inflammation and tissue catabolism.

The study described here tested the hypothesis that early intra-articular inflammation is associated with the development of post-traumatic osteoarthritis (PTOA) in a sheep model. We extended previously published work in which we investigated joint gross morphology and synovial mRNA expression of inflammatory and catabolic molecules 2 weeks after anatomic Anterior cruciate ligament (ACL) autograft reconstructive surgery (ACL-R). The same variables have been analyzed at 20 weeks post surgery together with new experimental variables at both time points. Animals were sacrificed at 20 weeks post ACL-R surgery and their joints graded for signs of PTOA. Synovial samples were harvested for histological grading plus mRNA and protein analysis for a panel of inflammatory and catabolic molecules. The mRNA expression levels for this panel plus connective tissue matrix turnover molecules were also investigated in cartilage samples. Results of gross morphological assessments at 20 weeks post surgery showed some changes consistent with early OA, but indicated little progression of damage from the 2 week time point. While significant alterations in mRNA levels for synovial inflammatory and catabolic molecules were detected at 2 weeks, values had normalized by 20 weeks. Similarly, all mRNA expression levels for inflammatory and catabolic molecules in articular cartilage had returned to normal levels by 20 weeks post ACL-R surgery. We conclude that synovial inflammatory processes are initiated very early after ACL-R surgery and may instigate events that lead to the gross cartilage and joint abnormalities observed as early as 2 weeks. However, the absence of sustained inflammation and joint instability may prevent OA progression.

There is significant load sharing and physical interaction between the anteromedial and posterolateral bundles of the ovine ACL under anterior tibial loads.

INTRODUCTION: The ovine stifle joint is an ideal preclinical model to test the ability of double-bundle reconstruction techniques in providing superior joint stability and less osteoarthritis (OA) compared with single-bundle techniques. However, knowledge of the normal ovine ACL and more specifically the load sharing and physical interaction between the two main bundles of the ovine ACL is currently limited. METHODS: Six ovine stifle joints were tested using a robotic testing system. Each joint was cyclically loaded to 200N in the anterior tibial direction between 30° and 90° flexion and the force-displacement data were recorded for both the intact ACL, and when the anteromedial (AM, n=3) or the posterolateral bundle (PL, n=3) was transected. RESULTS: The load shared by the AM bundle increased from 30° to 75° of flexion at all loading levels (25-200N); whereas, the load shared by the PL bundle decreased between full extension and mid flexion (60°) and then increased again. The load borne by the AM bundle did not change in response to increasing Anterior Tibial Loads (ATL) at each flexion angle, in contrast to the PL bundle (P=0.004). Physical interaction between bundles was greatest at 60° and under 50N ATL. CONCLUSION: These data will help create double-bundle ACL reconstructions in sheep which are functionally similar to intact native ACL. In turn, this model can be used to examine the success of anatomically accurate double-bundle reconstructions to prevent the development of OA. LEVEL OF EVIDENCE: III.

Technical issues in using robots to reproduce joint specific gait.

Reproduction of the in vivo motions of joints has become possible with improvements in robot technology and in vivo measuring techniques. A motion analysis system has been used to measure the motions of the tibia and femur of the ovine stifle joint during normal gait. These in vivo motions are then reproduced with a parallel robot. To ensure that the motion of the joint is accurately reproduced and that the resulting data are reliable, the testing frame, the data acquisition system, and the effects of limitations of the testing platform need to be considered. Of the latter, the stiffness of the robot and the ability of the control system to process sequential points on the path of motion in a timely fashion for repeatable path accuracy are of particular importance. Use of the system developed will lead to a better understanding of the mechanical environment of joints and ligaments in vivo.

New understanding of the complex structure of knee menisci: implications for injury risk and repair potential for athletes.

Menisci help maintain the structural integrity of the knee. However, the poor healing potential of the meniscus following a knee injury can not only end a career in sports but lead to osteoarthritis later in life. Complete understanding of meniscal structure is essential for evaluating its risk for injury and subsequent successful repair. This study used novel approaches to elucidate meniscal architecture. The radial and circumferential collagen fibrils in the meniscus were investigated using novel tissue-preparative techniques for light and electron microscopic studies. The results demonstrate a unique architecture based on differences in the packaging of the fundamental collagen fibrils. For radial arrays, the collagen fibrils are arranged in parallel into ∼10 µm bundles, which associate laterally to form flat sheets of varying dimensions that bifurcate and come together to form a honeycomb network within the body of the meniscus. In contrast, the circumferential arrays display a complex network of collagen fibrils arranged into ∼5 µm bundles. Interestingly, both types of architectural organization of collagen fibrils in meniscus are conserved across mammalian species and are age and sex independent. These findings imply that disruptions in meniscal architecture following an injury contribute to poor prognosis for functional repair.

Strength of medial structures of the knee joint are decreased by isolated injury to the medial collateral ligament and subsequent joint immobilization.

Past studies of the healing of the medial collateral ligament (MCL) in animal models have been conducted over a variety of healing intervals, some as early as 1 week. One concern with testing at early healing intervals is the difficulty in identifying and isolating the tissues that carry load. The purpose of this study was to determine if isolation of the MCL and healing time are critical factors in the assessment of structural strength in this model. Furthermore, the effect of immobilization on these critical factors was investigated. Our approach was to calculate the load-sharing ratio between the MCL and the MCL plus capsule. A 4 mm gap was created in the midsubstance of both hindlimb MCLs of 52 female New Zealand White rabbits (n=104). Of these, 29 rabbits had their right hindlimb pin immobilized (immobilized group), leaving the left hindlimb non-immobilized. Testing was performed at 3 (n=12), 6 (n=22), and 14 (n=24) weeks. The remaining 23 rabbits, which had both limbs non-immobilized (non-immobilized group), were tested at 3 (n=10), 6 (n=12), 14 (n=12), and 40 (n=12) weeks. For both groups, half of the specimens at each healing interval were used to test the MCL alone and half to test the MCL plus capsule, except for 3 week immobilized joints where only the MCL plus capsule was tested. Additionally, MCL (n=12), MCL plus capsule (n=6), and capsule alone (n=5) were tested from normal animals. The load-sharing ratio at MCL failure for the normal joint was 89%, suggesting an MCL-dominated response. For the non-immobilized group, the load-sharing ratio was 24% at 3 weeks of healing, suggesting a capsule-dominated response. At and after 6 weeks of healing, an MCL-dominated response was observed, with the ratio being 68% or greater. Thus, at less than 6 weeks of healing, the structural strength capabilities of the joint may be better represented by the medial structures rather than the isolated MCL. Immobilization delayed the transition from a capsule-dominated response to an MCL-dominated response in this model.

Biomechanical study using fuzzy systems to quantify collagen fiber recruitment and predict creep of the rabbit medial collateral ligament.

In normal daily activities, ligaments are subjected to repeated loads, and respond to this environment with creep and fatigue. While progressive recruitment of the collagen fibers is responsible for the toe region of the ligament stress-strain curve, recruitment also represents an elegant feature to help ligaments resist creep. The use of artificial intelligence techniques in computational modeling allows a large number of parameters and their interactions to be incorporated beyond the capacity of classical mathematical models. The objective of the work described here is to demonstrate a tool for modeling creep of the rabbit medial collateral ligament that can incorporate the different parameters while quantifying the effect of collagen fiber recruitment during creep. An intelligent algorithm was developed to predict ligament creep. The modeling is performed in two steps: first, the ill-defined fiber recruitment is quantified using the fuzzy logic. Second, this fiber recruitment is incorporated along with creep stress and creep time to model creep using an adaptive neurofuzzy inference system. The model was trained and tested using an experimental database including creep tests and crimp image analysis. The model confirms that quantification of fiber recruitment is important for accurate prediction of ligament creep behavior at physiological loads.

Influence of mechanical and biological signals on gene expression in human MG-63 cells: evidence for a complex interplay between hydrostatic compression and vitamin D3 or TGF-beta1 on MMP-1 and MMP-3 mRNA levels.

Biological mediators can influence the activity and differentiation of bone cells. 1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3) is known to induce differentiation of precursors into mature osteoblasts, and transforming growth factor-beta1 (TGF-beta1) can modulate the activity of bone cells leading to alterations in proliferation and gene expression patterns. Bone-derived cells were loaded via intermittent cyclic hydrostatic pressure (icHP) on cells under basal conditions and in the presence of 1,25-(OH)2D3 or TGF-beta1. Evaluating the effects of loading on the cells allowed for a comparison to be made between responsiveness to biomechanical and biochemical stimuli and their potential interplay. The effects of icHP on mRNA levels for the specific genes involved in bone remodelling and differentiation were measured in MG-63 cells using reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels for matrix metalloproteinase-1 and -3 (MMP-1 and MMP-3) were significantly, and uniquely, increased (p < 0.001) in cells exposed to icHP under serum-free conditions for 4-12 h. However, mRNA levels for MMP-3, but not MMP-1, were significantly enhanced in cells subjected to static hydrostatic pressure (HP). Treatment of cells with 1,25-(OH)2D3 resulted in increased (p < 0.001) mRNA levels for osteocalcin and decreased (p < 0.001) mRNA levels for both MMP-1 and MMP-3. In cells exposed to icHP and 1,25-(OH)2D3, the mRNA levels for both MMP-1 and MMP-3 were elevated (p < 0.001) compared with hormone alone, but not to the same degree (p < 0.01) as cells subjected to icHP alone. Addition of TGF-beta1 to cells led to increases in cell proliferation and expression of collagen I, as well as decreases in expression of osteocalcin and MMP-1 and MMP-3. Exposure of cells to icHP and TGF-beta1 again led to unique and significant increases in expression of MMP-1 and MMP-3. No changes in mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or any of the other 9 genes assessed, including those for MMP-2 and MMP-13, were detected under any of the conditions described. Therefore, icHP can induce alterations in mRNA levels for a specific subset of genes in both premature and mature osteoblasts. Such stimuli can modulate the impact of potent biological mediators in defining patterns of gene expression by bone cells and potentially modify function in vivo.

New perspectives on bioengineering of joint tissues: joint adaptation creates a moving target for engineering replacement tissues.

The current paradigm in tissue engineering is that "full regeneration" or "total replacement" of normal tissue is required in order to restore joint function. However, there is considerable evidence that suggests that targets other than "normality" may actually be required for tissue substitutes. Sometimes "less than normal" tissue properties of substitutes may be required following an injury, and sometimes "more than normal" may be required (following tissue degradation, damage, and failure). Diarthrodial joints function as "organs" in a physiological sense and normal individual joint tissues work together to share the mechanical requirements demanded by internal and external forces. Each tissue has some genetic and biological ability to adapt and/or remodel, to accommodate to the changing biomechanical needs invoked by injury and each tissue changes with age. This dynamic genetic and environmentally driven situation affecting the (uninjured) tissues in both injured and uninjured joints suggests that there is a "moving target" for bioengineered replacement tissues. After degeneration, damage, and failure of adaptation of other joint components, the mechanical requirements of replacement tissues likely increases dramatically beyond those of their normal counterparts. These concepts have important implications to designs of tissue bioengineering experiments and to their mechanical targets.

Healing ligaments have decreased cyclic modulus compared to normal ligaments and immobilization further compromises healing ligament response to cyclic loading.

Ligaments help maintain joint stability by resisting excessive strain during the repetitive loading experienced during daily activity. Healing ligaments may be less able to fulfill this role, straining more under equivalent loading than normal ligaments. We examined the cyclic stress-strain response of normal and healing ligaments to repetitive low loads (<10% of the normal ligament failure strength). Rabbit medial collateral ligaments (MCLs) were surgically gapped in either a unilateral (right MCL; n=23) or bilateral (right and left MCLs; n=17) fashion with immobilization of the right hindlimb in the bilateral group. These MCL scars were allowed to heal for 3, 6, and 14 weeks and were cyclic creep tested at 2.2, 4.1, and 7.1 MPa, respectively. Creep test stresses were a constant 30% of the failure strength of non-immobilized scars at the different healing intervals. Normal MCLs were creep tested at 4.1 and 7.1 MPa (n=13). The cyclic modulus of the non-immobilized scars was less than that of normal ligaments. The percent increase in modulus during cycling was greater for scars than for normal ligaments, likely related to increased viscous dissipation or material inferiorities in scars. Furthermore, immobilization significantly decreased the ability of scars to resist strain, with a majority of immobilized scars failing during repetitive loading. Such failures were preceded by a reduction in cyclic modulus indicating damage to the healing ligaments that was predictive of eventual total failure. The implications of this study are that joints with healing ligaments may have increased strain in joint structures while they are under stress, potentially leading to joint instability. Although immobilization could be used temporarily to maintain joint stability, remobilization would likely lead to total failure of the healing ligament.

Ligament creep recruits fibres at low stresses and can lead to modulus-reducing fibre damage at higher creep stresses: a study in rabbit medial collateral ligament model.

Ligaments are subjected to a range of loads during different activities in vivo, suggesting that they must resist creep at various stresses. Cyclic and static creep tests of rabbit medial collateral ligament were used as a model to examine creep over a range of stresses in the toe- and linear-regions of the stress-strain curve: 4.1 MPa (n = 7), 7.1 MPa (n = 6), 14 MPa (n = 9) and 28 MPa (n = 6). We quantified ligament creep behaviour to determine if, at low stresses, modulus would increase in a cyclic creep test and collagen fibres would be recruited in a static creep test. At higher creep stresses, a decrease in measured modulus was expected to be a potential marker of damage. The increase in modulus during cyclic creep and the increase in strain during static creep were similar between the three toe-region stresses (4.1, 7.1, 14 MPa). However, at the linear-region stress (28 MPa), both these parameters increased significantly compared to the increases at the three toe-region stresses. A concurrent crimp analysis revealed that collagen fibres were recruited during creep, evidenced by decreased area of crimped fibres at the end of the static creep test. Interestingly, a predominance of straightened fibres was observed at the end of the 28 MPa creep test, suggesting a limited potential for fibre recruitment at higher, linear-region stresses. An additional 28 MPa (n = 6) group had mechanically detectable discontinuities in their stress-strain curves during creep that were related to reductions in modulus and suggested fibre damage. These data support the concept that collagen fibre recruitment is a mechanism by which ligaments resist creep at low stresses. At a higher creep stress, which was still only about a third of the failure capacity, damage to some ligaments occurred and was marked by a sudden reduction in modulus. In the cyclic tests, with continued cycling, the modulus increased back to original values obtained before the discontinuity suggesting that other fibres were being recruited to bear load. These results have important implications for our understanding of how fibre recruitment and stress redistribution act in normal ligament to minimize creep and restore modulus after fibre damage.

Medial collateral ligament autografts have increased creep response for at least two years and early immobilization makes this worse.

Recent evidence has shown that 10-40% of knee joints reconstructed with soft-tissue autografts have a recurrence of abnormal joint laxity over time. One possible explanation is the "stretching out" (or unrecovered creep) of the graft tissue. To test in vitro creep and creep recovery of fresh anatomic ligament autografts in an extra-articular environment, 16 rabbits underwent an orthotopic medial collateral ligament (MCL) autograft procedure to one hindlimb. Three subgroups of animals had either unrestricted cage activity for 1 year (n = 5) or 2 years (n = 5) or pin-immobilization for the first 6 weeks followed by cage activity for the remainder of 1 year (n = 6). Following laxity measurements, to test their creep response, isolated MCL grafts were cyclically and then statically creep tested in vitro at 4.1 MPa, allowed to recover at zero load for 20 min, and finally elongated to failure. Due to differences in cross-sectional area between the grafts and normal MCLs, two normal control groups were tested: stress-matched tested at 4.1 MPa (16.2 N; n = 7) and force-matched tested at 29.1 N (7.1 MPa; n = 6). Ligament grafts had normal laxity but significantly increased creep and decreased creep recovery compared to normal MCLs after I and 2 years of healing (p < 0.0004). Graft failure stress was also significantly less than normal (p < 0.0001). Immobilized grafts had significantly greater creep compared to non-immobilized grafts at 1 year of healing (p < 0.05). These results support previous observations concerning material inferiority of fresh anatomic rabbit MCL autografts, but add the concept that such grafts also have increased potential to creep with either slower or incomplete recovery when subjected to low stresses in vitro. Joint and ligament laxities in situ were normal in this model, however, suggesting either that in vivo MCL graft stresses are lower than those used here in vitro or that these tissues have other mechanisms by which they can recover their functional length in vivo.

Early healing processes of free tendon grafts within bone tunnels is bone-specific: a morphological study in a rabbit model.

In order to function as effective ligament replacements, free tendon grafts must become firmly healed into bone tunnels as soon as possible. We hypothesized that graft incorporation would be bone-specific. Free semitendinosus tendon grafts were inserted into drill holes in a lapine medial collateral ligament reconstruction model; thus, creating tibial and femoral bone-specific incorporation sites. Femur-semitendinosus tendon-tibia complexes were harvested from 26 rabbits for histological analysis at various healing times: 0, 6, 12, or 24 weeks post-surgery. Incorporation and remodeling of the graft in the chondral callus was much more extensive at the cancellous-filled femoral insertion than within the marrow-dominated tibial insertion, suggesting that tendon graft healing may depend on the cancellous bone architecture at the graft site.

Altering ligament water content affects ligament pre-stress and creep behaviour.

The water content of a ligament can be altered by injury and surgical intervention in vivo, and inadvertently or purposely during in vitro tests. We investigated how altering the water content of the rabbit medial collateral ligament (MCL) affected its resulting creep behaviour (defined as an increase in strain from sequential cyclic and static creep tests). The water content of normal MCLs 4) was compared to that of MCLs soaked for 1 h in a sucrose solution (n = 4) or phosphate buffered saline (PBS; n = 8). Sucrose exposure decreased hydration and PBS exposure increased hydration. In addition, soaking in PBS caused a shift in ligament zero (the position where there was 0.1 N of tension on the ligament). Following the same single solution treatment, additional MCLs were creep tested at 4.1 MPa using a load based on the ligament cross-sectional area measured before solution treatment: sucrose (n = 4), PBS new "ligament zero" (n = 5). and PBS old "ligament zero" (n = 6). Normal MCLs were also tested at 4.1 MPa (n = 7) in a humidity chamber that maintained normal ligament water content. Additional MCLs were treated with both solutions in series (n = 12) to examine the reversibility of the mechanical changes caused by single solution treatment. This was the first investigation to show that ligament creep behaviour was clearly affected by the initial state of hydration: creep decreased with decreased hydration and creep increased with increased hydration. Another unique finding was that ligaments with increased hydration had decreased ligament functional length and increased ligament pre-stress. The creep behaviour of these ligaments was decreased if they were loaded from the pre-stressed state compared to the unloaded state. These results suggest that maintenance of physiological water content is important for in vitro mechanical testing of ligaments and controlling the low-load stress state of ligaments in situ.

Efficient transfer of intact oligonucleotides into the nucleus of ligament scar fibroblasts by HVJ-cationic liposomes is correlated with effective antisense gene inhibition.

The efficacy of two different cationic liposomes, Lipofectin and hemagglutinating virus of Japan (HVJ)-cationic liposomes, on nuclear uptake of fluorescence-labeled phosphorothioate oligodeoxyribonucleotide (S-ODN) by ligament scar fibroblasts and suppression of decorin mRNA expression when antisense decorin S-ODN was transferred was investigated. There was no significant difference in nuclear uptake of fluorescent ODN between the two methods. However, only HVJ-cationic liposomes had a significant effect on suppression of decorin mRNA expression levels. To address the discrepancy, the molecular integrity of the transferred ODN in the cells was assessed by analysis of fluorescence resonance energy transfer (FRET) within double-fluorescence-labeled S-ODN. More than 70% of the ODN transfected by HVJ-cationic liposomes remained intact within the nucleus at 20 h after transfection, while the majority of the ODN transferred by Lipofectin was degraded at this point. These results suggest a strong relationship between the nuclear integrity of transfected antisense ODN and its suppression of target mRNA expression.

A 2D finite element model of the interventricular septum under normal and abnormal loading.

The interventricular septum is the structure that separates the left and right ventricles of the heart. Under normal loading conditions, it is concave to the left ventricle, but under abnormal loading the septum flattens and occasionally inverts. In the past, the septum has frequently been modelled as integral to the left ventricle with the effects of pressure from the right ventricle being ignored. Under abnormal loading, the septum has been described as behaving equivalent to a "flapping sail". There has been no consideration of structural behaviour under these conditions. A 2-D plane stress FE model of the septum was used to investigate the difference in structural behaviour of the septum during diastole between normal and abnormal loading. The biaxial stress patterns that develop are distinctively disparate. Under normal loading, the septum behaves much like a thick-walled cylinder subject to internal and external pressure, with the resulting stresses being circumferential tension and radial compression, both varying with radius. These stresses are very low throughout most of diastole. However, under abnormal loading, the septum behaves in an arch-like fashion, with high compressive stresses almost circumferential in direction, combined with radial compression. We conclude that right ventricular pressures cause bending effects in the wall of the heart, and that under abnormal loading, the compressive stresses that develop in the septum may lead to an understanding of certain, previously unexplained, pathological conditions.

Compression of interventricular septum during right ventricular pressure loading.

The interventricular septum, which flattens and inverts in conditions such as pulmonary hypertension, is considered by many to be an unstressed membrane, in that its position is assumed to be determined solely by the transseptal pressure gradient. A two-dimensional finite element model was developed to investigate whether compression and bending moments (behavior incompatible with a membrane) exist in the septum during diastole under abnormal loading, i.e., pulmonary artery (PA) constriction. Hemodynamic and echocardiographic data were obtained in six open-chest anesthetized dogs. For both control and PA constriction, the measured left ventricular and right ventricular pressures were applied to a residually stressed mesh. Adjustments were made to the stiffness and end-bending moments until the deformed and loaded residually stressed mesh matched the observed configuration of the septum. During PA constriction, end-bending moments were required to obtain satisfactory matches but not during control. Furthermore, substantial circumferential compressive stresses developed during PA constriction. Such stresses might impede septal blood flow and provoke the unexplained ischemia observed in some conditions characterized by abnormal septal motion.