In Vitro Engineering of High Modulus Cartilage-Like Constructs.

To date, the outcomes of cartilage repair have been inconsistent and have frequently yielded mechanically inferior fibrocartilage, thereby increasing the chances of damage recurrence. Implantation of constructs with biochemical composition and mechanical properties comparable to natural cartilage could be advantageous for long-term repair. This study attempted to create such constructs, in vitro, using tissue engineering principles. Bovine synoviocytes were seeded on nonwoven polyethylene terephthalate fiber scaffolds and cultured in chondrogenic medium for 4 weeks, after which uniaxial compressive loading was applied using an in-house bioreactor for 1 h per day, at a frequency of 1 Hz, for a further 84 days. The initial loading conditions, determined from the mechanical properties of the immature constructs after 4 weeks in chondrogenic culture, were strains ranging between 13% and 23%. After 56 days (sustained at 84 days) of loading, the constructs were stained homogenously with Alcian blue and for type-II collagen. Dynamic compressive moduli were comparable to the high end values for native cartilage and proportional to Alcian blue staining intensity. We suggest that these high moduli values were attributable to the bioreactor setup, which caused the loading regime to change as the constructs developed, that is, the applied stress and strain increased with construct thickness and stiffness, providing continued sufficient cell stimulation as further matrix was deposited. Constructs containing cartilage-like matrix with response to load similar to that of native cartilage could produce long-term effective cartilage repair when implanted.

Enamel matrix derivative enhances tissue formation around scaffolds used for tissue engineering of ligaments.

The following in vitro translational study investigated whether enamel matrix derivative (EMD), an approved biomimetic treatment for periodontal disease (Emdogain) and hard-to-heal wounds (Xelma), enhanced synovial cell colonization and protein synthesis around a scaffold used clinically for in situ tissue engineering of the torn anterior cruciate ligament (ACL). Synovial cells were enzymatically extracted from bovine synovium and dynamically seeded onto polyethylene terephthalate (PET) scaffolds. The cells were cultured in low-serum medium (0.5% FBS) for 4 weeks with either a single administration of EMD at the start of the 4 week period or multiple administrations of EMD at regular intervals throughout the 4 weeks. Samples were harvested and evaluated using the Hoechst DNA assay, BCA protein assay, cresolphthalein complexone calcium assay, SDS-PAGE, ELISA and electron microscopy. A significant increase in cell number (DNA) (p < 0.01), protein content (p < 0.01) and TGFbeta1 synthesis (p < 0.01) was observed with multiple administrations of EMD. Additionally, SDS-PAGE showed an increase in high molecular weight proteins, characteristic of the fibril-forming collagens. Electron microscopy supported these findings, showing that scaffolds treated with multiple administrations of EMD were heavily coated with cells and extracellular matrix (ECM) that enveloped the fibres. Multiple administrations of EMD to synovial cell-seeded scaffolds enhanced the formation of tissue in vitro. Additionally, it was shown that EMD enhanced TGFbeta1 synthesis of synovial cells, suggesting a potential mode of action for EMD's capacity to stimulate tissue regeneration.

Cyclic straining of cell-seeded synthetic ligament scaffolds: development of apparatus and methodology.

Cyclic tensile strains acting along a ligament implant are known to stimulate cells that colonize it to proliferate and to synthesize an extracellular matrix (ECM), which will then remodel and form a new ligament structure. However, this process of tissue induction is poorly understood. As a first step toward elucidating this process, we aimed to investigate the effect of cyclic tensile strain on the proliferation of, and possible ECM synthesis by, cells colonizing ligament scaffolds. Because there was no commercially available apparatus to undertake such investigation the objectives of this study were to develop an apparatus for the application of cyclic tensile strains on cell-seeded synthetic ligament scaffolds and to develop and validate (through preliminary data obtained using the apparatus) methodology for studying the effect of cyclic strain on cell proliferation. We designed a multi-station test apparatus that operated inside an incubator. It allowed the application of tensile cyclic strains of between 0.5% and 5% at a frequency of 1 Hz on cell-seeded polyester ligament scaffolds immersed in culture medium. Test stations with windows in their bases could be easily de-coupled from the apparatus. This allowed monitoring of cell proliferation and morphology, with inverted light microscopy, through the transparent glass bases of the culture wells. Preliminary experiments lasting for 1 day or 9 weeks examined the effect of selected aspects of the cyclic strain on proliferation of cells seeded onto ligament scaffolds. Tests lasting for 1 day showed that the application of cyclic tensile strain of 5% for 4 h increased cell proliferation 24% above that observed in unstrained controls (p < .05). Scanning electron microscopy data from tests lasting for 9 weeks demonstrated further the dependency of cell proliferation and possible ECM synthesis on the magnitude of the strain. The larger the amplitude, the greater was the coverage of the scaffold with cells and ECM. Transmission electron microscopy of the ECM observed at 9 weeks showed evidence of collagen fibrils aligned in the direction of load in strained scaffolds, whereas the tissue on the control scaffolds was random.

The potential use of enamel matrix derivative for in situ anterior cruciate ligament tissue engineering: a translational in vitro investigation.

Polyester scaffolds have been used as an alternative to autogenous tissues for the reconstruction of the anterior cruciate ligament (ACL). They are biocompatible and encourage tissue infiltration, leading to neoligament formation. However, rupture can occur, caused by abrasion of the scaffold against the bone tunnels through which it is implanted. Good early tissue induction is therefore considered essential to protect the scaffold from this abrasion. Enamel matrix derivative (EMD) is used clinically in the treatment of periodontal disease. It is a complex mix of proteins with growth factor-like activity, which enhances periodontal ligament fibroblast attachment, proliferation, and differentiation, leading to the regeneration of periodontal bone and ligament tissues. We hypothesized that EMD might, in a similar manner, enhance tissue induction around scaffolds used in ACL reconstruction. This preliminary investigation adopted a translational approach, modelling in vitro 3 possible clinical modes of EMD administration, to ascertain the suitability of each protocol for application in an animal model or clinically. Preliminary investigations in monolayer culture indicated that EMD had a significant dose-dependent stimulatory effect (p < 0.05, n = 6) on the proliferation of bovine primary synovial cells. However, pre-treating culture plates with EMD significantly inhibited cell attachment (p < 0.01, n = 6). EMD's effects on synovial cells, seeded onto ligament scaffolds, were then investigated in several in vitro experiments modelling 3 possible modes for clinical EMD administration (pre-, intra-, and post-operative). In the pre-operative model, EMD was adsorbed onto scaffolds before the addition of cells. In the intra-operative model, EMD and cells were added simultaneously to scaffolds in the culture medium. In the post-operative model, cells were pre-seeded onto scaffolds before EMD was administered. EMD significantly inhibited cell adhesion in the pre-operative model (p < 0.05, n = 6) and had no significant benefit in the intra-operative model. In the post-operative model, the addition of EMD to previously cell-seeded scaffolds significantly increased their total deoxyribonucleic acid content (p < 0.01, n = 5). EMD's stimulative effect on cell proliferation in vitro suggests that it may accelerate scaffold colonization by cells (and in turn tissue induction) in situ. However, its inhibitory effect on synovial cell attachment in vitro implies that it may only be suited to post-operative administration.

Deformation and failure of cartilage in the tensile mode.

The aim of this study was to visualize, at the ultrastructural level, the deformation and failure mechanism of cartilage matrix in the tensile mode. Full-thickness dumbbell-shaped specimens were prepared from adult bovines. There were two specimen groups; in the 'parallel' group the specimen axis was parallel to the split lines defining the preferential orientation of the collagen in the articular surface, and in the 'perpendicular' group the specimen axis was perpendicular to the split lines. Specimens were placed with the articular surface uppermost and subjected to a graded series of strain within individual mini-tension devices, while observed with stereomicroscopy and confocal laser scanning microscopy. Thereafter, the changes in the ultrastructure were observed with both scanning and transmission electron microscopy. The mechanism of cartilage failure in the tensile mode comprised the following stages, whether the strain was applied parallel or perpendicular to the split line. (1) At 0% strain a fibrillar meshwork within the articular surface was predominantly orientated in the direction of the split line. (2) As strain increased, the fibrillar meshwork became more orientated in the parallel group and reorientated in the perpendicular group in the direction of the applied strain. (3) After complete reorientation of the fibrillar meshwork in the direction of the applied strain, the initial sign of failure was rupture of the fibrillar meshwork within the articular surface. (4) Subsequently, the rupture rapidly propagated into the deeper layers. Greater strains were required for fibrillar reorientation and complete rupture in the 'perpendicular group' than in the parallel group.

Effect of patellar tendon shortening on tracking of the patella.

BACKGROUND: Although 10% postoperative patellar tendon shortening after bone-patellar tendon-bone autograft reconstruction of the anterior cruciate ligament has been reported, there are no published studies assessing the effect of shortening on patellofemoral joint biomechanics under physiological loading conditions. PURPOSE: To investigate the influence of patellar tendon shortening on patellofemoral joint biomechanics. STUDY DESIGN: Controlled laboratory study. METHODS: The authors evaluated the patellofemoral contact area, the location of contact, and the patellofemoral joint reaction force and contact stresses in 7 cadaveric knees before and after 10% patellar tendon shortening. Shortening was achieved using a specially designed device. Experimental conditions simulating those occurring during level walking were employed: physiological quadriceps loads and corresponding angles of tibial rotation were applied at 15 degrees , 30 degrees , and 60 degrees flexion of the knee. Patellofemoral joint contact areas were measured before and after shortening using the silicone oil-carbon black powder suspension squeeze technique. RESULTS: After patellar tendon shortening, patellofemoral joint contact areas were displaced proximally on the patellar surface and distally on the femoral surface. Although the contact area increased by 18% at 15 degrees of knee flexion (P = .04), no significant change occurred at 30 degrees or 60 degrees of knee flexion (P > .05). Patellofemoral contact stress remained unchanged after patellar tendon shortening (P > .05) at each flexion angle. CONCLUSION: Our results suggest that a 10% shortening of the patellar tendon does not alter patellar contact stresses during locomotion. It is not clear whether apparent changes in contact location in all positions and contact area at 15 degrees would have clinical consequences.

Effect of cyclic tensile strain on proliferation of synovial cells seeded onto synthetic ligament scaffolds--an in vitro simulation.

OBJECTIVE: Cyclic tensile strain is pivotal to the remodeling of tissue induced in implants used in reconstruction of anterior cruciate ligaments whether these implants were of autogenous tissues or synthetic materials. However, this process is poorly understood. The objective of this study was to investigate the short and medium-term effect of cyclic tensile strain on the proliferation of synovial cells seeded on ligament scaffolds. METHODS: 206 ligament scaffolds made from plasma treated polyester with an ultimate tensile strength of 320 N were used in this study. Synovial cells were obtained from the metatarsophalangeal joints of 2 years old bovines. After expansion of these, they were seeded onto the scaffolds which were subjected, in a specialized apparatus, to a cyclic tensile strain of 4.5% at a frequency of 1 Hz. Initially, the strain was applied for a period of 4 h, which was subsequently reduced in further experiments to 1.0 h and 0.5 h. In further tests, strains of approximately 2.5%, 1% and 0.6% were applied for 1 h at the same frequency. In all the above tests, which were short-term tests (lasting for approximately 1 day), cell proliferation was investigated by the uptake of thymidine with which cells were labeled according to prescribed protocols. Cell proliferation was further examined with light microscopy after 5 weeks and the degree of fill of inter-yarn spaces was quantified for strain amplitudes of 1, 2.5 and 4.5%. Equal number of control (not strained) specimens was used at each time point. RESULTS: In the 1-day experiments, for all durations of application of cyclic strain (exercise), the effect of strain on cell proliferation was inhibitory during the period of exercise and up to 18 h from its commencement, but was stimulatory 22-24 h afterwards. This stimulatory effect was maximal at an exercise period of 1 h. The study has also shown that there is a threshold for the amplitude of the strain (1%), at and below which cell proliferation was not significantly different from that observed in control specimens (P was <0.05 for all data). After 5 weeks of cyclic strain application, it was shown that the higher the amplitude of strain the larger was the area occupied by cells of the intra-yarn space. CONCLUSION: Both the amplitude of cyclic strain and duration of its application affect the proliferation of synovial cells seeded on ligament scaffolds. The data should be useful when selecting or designing an implant, and when prescribing a postoperative exercise regime.

Double-band reconstruction of the ACL using a synthetic implant: a cadaveric study of knee laxity.

In this study, the anterior laxity and internal rotation of five cadaveric knee joints were compared when the anterior cruciate ligament (ACL) was intact, after its reconstruction with the anteromedial band (AMB) only, then after its reconstruction with the double band, with the posterolateral band (PLB) tensioned first at 20 degrees and then at 90 degrees, and finally with the ACL resected. The tests were performed using a mechanical apparatus that allowed the joint 6 degrees of freedom and also the application of external loads and torques on the tibia. The loads used were 50, 90, and 130 N for the anterior laxity test, and a torque of 2, 3, and 4 Nm in the internal rotation test. In all cases, laxity with double-band reconstruction was closer to the natural value than when it was constructed with the AMB only. In some cases, double-band reconstruction imposed a higher constraint on the joint than did the natural ACL. Measurement of the residual tension on the PLB after its final anchoring was also performed during passive flexion. This test revealed a high tension on this band with the knee in hyperextension, followed by a decrease in value through to 45 degrees and a slight increase at 90 degrees, thus following a similar trend to that of the natural PLB.

Comparative study of the covered area of Leeds-Keio (LK) artificial ligament and radio frequency generated glow discharge treated Leeds-Keio (Bio-LK) ligament with synovial cells.

The Leeds-Keio artificial ligament (LK), which was developed not only as a ligament substitute but also as a scaffold for tissue induction in knee ligament reconstruction, has been in clinical use since 1982 in Europe and Japan. Recently, we have developed radio frequency generated glow discharge (RFGD)-treated LK ligament (Bio-LK) to expedite the process of tissue induction and its maturation. In this study of cell adhesion to the scaffold, we report the difference in the covered area with synovial cells when using scaffolds made from treated and untreated materials. Plasma clot methods were used in this study. The covered area on LK and Bio-LK by cells was stained by 0.1% toluidine blue and analyzed using NIH image. The covered area of Bio-LK was about three times higher than that of LK (untreated) at 3 weeks. In scanning electron microscopy, more cells were observed on fibers of Bio-LK, and these filled the space among the fibers more extensively. The spreading of covered area means that cell attachment, cell proliferation, and cell migration on the fibers are likely to be improved. Our experimental study indicates that Bio-LK will possibly speed up the process of induction of autogenous tissue from synovium.

Hydrostatic pressure modulates proteoglycan metabolism in chondrocytes seeded in agarose.

OBJECTIVE: To investigate the effect of isolated hydrostatic pressure on proteoglycan metabolism in chondrocytes. METHODS: Bovine articular chondrocytes cultured in agarose gels were subjected to 5 MPa hydrostatic pressure for 4 hours in either a static or a pulsatile (1 Hz) mode, and changes in glycosaminoglycan (GAG) synthesis, hydrodynamic size, and aggregation properties of proteoglycans and aggrecan messenger RNA (mRNA) levels were determined. RESULTS: The application of 5 MPa static pressure caused a significant increase in GAG synthesis of 11% (P < 0.05). Column chromatography showed that this increase in GAG synthesis was associated with large proteoglycans. In addition, semiquantitative reverse transcriptase-polymerase chain reaction showed a 4-fold increase in levels of aggrecan mRNA (P < 0.01). CONCLUSION: Hydrostatic pressure in isolation, which does not cause cell deformation, can affect proteoglycan metabolism in chondrocytes cultured in agarose gels, indicating an important role of hydrostatic pressure in the regulation of extracellular matrix turnover in articular cartilage.

Upregulation of aggrecan and type II collagen mRNA expression in bovine chondrocytes by the application of hydrostatic pressure.

The aim of this study was to investigate the effect of hydrostatic pressure on the expression of messenger ribonucleic acid (mRNA) for specific extracellular matrix proteins in chondrocytes. Chondrocytes obtained from bovine metatarsophalangeal joints were embedded in cylindrical 2% agarose gels. A novel experimental system was used to apply 5 MPa of static hydrostatic pressure to these chondrocytes for 4 hours. The application of hydrostatic pressure caused a significant increase in the level of aggrecan mRNA by almost four fold (p<0.01) as well as a 50% increase in the level of type II collagen mRNA (p<0.05). However, there was no significant change in the level of TIMP-1 mRNA. It was suggested that the application of hydrostatic pressure, in the absence of cell deformation, can bring about changes in the matrix components which may play an important role in the homeostasis and mechanical properties of articular cartilage.

Estimation of forces at the interface between an artificial limb and an implant directly fixed into the femur in above-knee amputees.

This article describes the method used for estimating the forces and moments, acting during locomotion, at the interface between an artificial leg and an implant directly fixed into the femur, in above-knee amputees. Twelve transfemoral amputees completed a predefined gait assessment during which kinetic (ground reaction loads and torque) and kinematic (limb orientation) gait data were recorded. A developed mathematical model enabled the ground reaction forces to be translated to the level of amputation. It is assumed that the loads calculated at the stump-socket interface would approximate those experienced by the proposed implant. The longitudinal force and the moment in the sagittal plane were the two most significant loads at the stump. These data were essential to obtain to facilitate the analysis of stress arising at the implant-bone interface that has been subsequently undertaken.

Tensile fatigue behaviour of articular cartilage.

Although fatigue has been implicated in cartilage failure there are only two studies by the same author, and in both of which cartilage was tested in the direction parallel to the collagen orientation in the surface layer. In the present work articular cartilage was tested also along the perpendicular direction, being the direction in which cartilage possesses lower tensile strength. Specimens were tested under cyclic tensile load. Number of cycles at failure was recorded as well as elongation of the specimen. To date 72 specimens have been tested all from one knee joint. The number of cycles to failure ranged between two and 1.5 million. The surface and deep layers have better fatigue properties whether tested in the parallel or the perpendicular direction, while the middle layer was far weaker. Better fatigue behaviour was observed with specimens tested in parallel than in perpendicular direction to the fibres.

The measurement of muscle stiffness in anterior cruciate injuries -- an experiment revisited.

OBJECTIVE: To investigate the reported changes in angular stiffness of the knee due to the hamstrings muscles in the anterior cruciate ligament (ACL) deficient knee. DESIGN: The flexors of the knee were modelled as springs and the model tested on patients and controls. BACKGROUND: A previous study had used a similar model but showed no difference between an ACL deficient and an uninjured knee. METHODS: A modification of a recently described method for measuring the stiffness of the hamstrings was used. ACL deficient patients were investigated and also normal controls. RESULTS: We found that stiffness not only varied between patients, but also between legs, with the injured leg having significantly stiffer flexor muscles (P < 0.001). We also showed that the stiffness of the knee due to the flexors is the same in normal subjects as that in the uninjured contralateral knee of the ACL injured patient. CONCLUSIONS: The measurement of the stiffness of the hamstrings following ACL injury may be a valid method of assessing patients with such injuries. Further investigation is required.