Background

Structural and functional outcome of bone graft with first or second generation autologous chondrocyte implantation (ACI) in osteochondral defects has not been reported.

Abstract. Introduction. Osteoarthritis (OA) is one of the lead causes of pain and disability in adults. Bone marrow lesions (BMLs) are one feature of subchondral bone involvement in OA. MRI images suggest changes in tissue content and properties in the affected regions however, it is not known if this alters the mechanical behavior of the bone, which could in turn affect OA progression. The aim of this study was to characterize the mechanical properties of BMLs, using a combined experimental and computational approach. Methods. Six human cadaveric patellae from donors aged 56–76 were used in this study; all exhibited BML regions under MRI. Bone plugs were taken from non-BML (n = 6) and BML (n = 7) regions within the patellae, with guidance from the MRI. The plugs were imaged at 82µm resolution using micro computed tomography (µCT) and tested under uniaxial compression. Finite element (FE) models were created for each plug from the µCT scans and morphological properties such as bone volume fraction (BV/TV) were also determined. The relationship between bone volume fraction and apparent modulus was investigated for both sample groups. Results. The BV/TV range was similar for the BML and non-BML groups (0.25–0.46 and 0.18–0.44) From the experimental tests, a moderate positive correlation was found between BV/TV and apparent modulus in the no BML group (r= 0.57) while no correlation was found in the BML group (r = −0.02). From the FE results, a different relationship between BV/TV and element elastic modulus was found for the BML and non-BML groups. Conclusions. The results of this study show that in regions of bone containing BMLs, bone volume fraction does not predict overall apparent modulus and has different relationship to local modulus, suggesting the BML associated tissue structural changes affect mechanical behavior. Funders: EPSRC

Bone-patellar tendon-bone autografts, hamstring autografts or allografts are widely used grafts for ACL revision surgeries. Also use of quadriceps autograft for both primary and revision ACL surgeries is in an increasing popularity due to its biomechanical superior properties and less donor site morbidity. However, although several fixation techniques and devices for quadriceps tendon graft fixation on femoral side have been reported, literature lacks about biomechanical studies comparing properties of these different fixation techniques and devices. We aimed to investigate whether there is a difference between the fixation techniques of quadriceps tendon graft by using different fixation techniques and devices on the femoral side in terms of stiffness and amount of slippage in the tunnel. Full-thickness central parts of quadriceps tendons from paired knees of twenty five calf knees were fixed through a 10mm x 25mm tunnel in twenty five paired sheep femurs. Quadriceps tendon patellar side with soft tissue ending fixed with four different fixation devices (adjustable suspensory system (group 1), absorbable interference screw (group 2), titanium interference screw (group 3) and adjustable suspensory system + absorbable interference screw (group 4)) and quadriceps tendon with a patellar bone plug fixed with a titanium interference screw (group 5) were tested in a servohydraulic materials testing machine. 10 samples were included in each group. After applying a preload of 10 N, a cyclic force was applied for 20 cycles from 10N to 110N at a 1 hertz frequency. Amount of slippage in the tunnel was calculated as the difference measured in millimeters between length at 10 N after 20 cycles and starting length at 10 N (Graph 1). To determine the stiffness, a single load-to-failure cycle was performed at a strain rate of 20 mm/min as the last step (Figure 1). Rupture of the graft was not seen in any of the samples. Median values of amount of slippage in the tunnel were 6,41mm, 5,99mm, 3,01mm, 4,83mm, and 3,94mm respectively. Median values of maximum load at failure were 464N, 160N, 350N, 350N and 389N respectively. Amount of slippage in the tunnel was highest in the group 1 and was lowest in the group 3 (p<0.001). Group 1 was found to be most resistant group against load-to-failure test and group 2 was the weakest (p<0.001). However inter-group analyses between group 3 and 5 revealed that, although group 3 had the least slippage in the tunnel, group 5 was better in terms of stiffness, but there was no statistically significant difference (p=0,124 and 0,119 respectively). There was a significant difference between group 2 and 3 in both amount of slippage in the tunnel and stiffness (p=0,001 and 0.028 respectively)(Table 1). Our study revealed that, although quadriceps graft with a bone plug fixed with metal interference screws is widely presumed to be a stable fixation technique, there was no significant difference in terms of stiffness when compared with quadriceps graft with soft tissue ending fixed with a metal interference screw. Although adjustable suspensory device group was the best in the terms of resistance against load-to-failure, it was the worst in terms of amount of slippage from the tunnel. Thus, if a suspensory device is to be used, it must be kept in mind that a strong 20 cycles of intra-operative tension force must be applied to prevent further slippage of the graft in the tunnel which can result in failure of reconstruction. For any figures or tables, please contact the authors directly

Decellularised extracellular matrix scaffolds show great promise for the regeneration of damaged musculoskeletal tissues (cartilage, ligament, meniscus), however, adequate fixation into the joint remains a challenge. Here, we assess the osseo-integration of decellularised porcine bone in a sheep model. This proof-of-concept study supports the overall objective to create composite decellularised tissue scaffolds with bony attachment sites to enable superior fixation and regeneration. Porcine trabecular bone plugs (6mm diameter, 10mm long) were decellularised using a novel bioprocess incorporating low-concentration sodium dodecyl sulphate with protease inhibitors. Decellularised bone scaffolds (n=6) and ovine allograft controls (n=6) were implanted into the condyle of skeletally mature sheep for 4 and 12 weeks. New bone growth was visualised by oxytetracycline fluorescence and standard resin semi-quantitative histopathology. Scaffolds were found to be fully decellularised and maintained the native microarchitecture. Following 4-week implantation in sheep, both scaffold and allograft appeared well integrated. The trabecular spaces of the scaffold were filled with a fibro-mesenchymal infiltrate, but some areas showed a marked focal lymphocytic response, associated with reduced bone deposition. A lesser lymphocytic response was observed in the allograft control. After 12-weeks the lymphocytic reaction was minimised in the scaffold and absent in allografts. The scaffold showed a higher density of new mineralized bone deposition compared to allograft. New marrow had formed in both the scaffold and allografts. Following the demonstration of osteointegration this bioprocess can now be transferred to develop decellularised composite musculoskeletal tissue scaffolds and decellularised bone scaffolds for clinical regeneration of musculoskeletal tissues

Osteoarthritis is a debilitating disease affecting over 1.7 million people in the UK annually. Total ankle replacements are an increasingly sought option for repairing a late stage arthritic ankle, but result in the removal of significant portions of bone regardless of tissue quality. Hence, the mapping of bone quality would allow the use of targeted treatments at earlier stages of the disease. This study aims to develop characterisation methodologies using porcine tissue to investigate the mechanical properties of subchondral bone in the ankle. N=11 talar bone plugs (6mm diameter) were extracted from porcine ankles and embedded into Delrin endcaps using a thin layer of PMMA cement. These were scanned under micro-CT (16 microns) and subjected to quasi-static uniaxial compression to determine apparent stiffness for each specimen. Specimen-specific continuum FE models were developed, with material properties derived from the greyscale value of the underlying image. A python-based least squares regression (Opti4Abq, N=6) was used to minimise the difference between experimental and model stiffness values, to determine the coefficient linking greyscale and mechanical properties. Apparent stiffness, elastic modulus and compressive strength were compared to BV/TV, which was derived using BoneJ (a bone image plugin for the NIH ImageJ). The results show positive correlations between BV/TV and compressive strength, stiffness and Young's modulus. Average BV/TV across all samples was 0.45. Average experimental and computational stiffness were 986N/mm and 891 N/mm respectively. A 21.8% RMS error was found using the validation set (N=5), which was of similar order to the calibration set. Some specimens saw issues with misalignment of the specimen faces and the loading platens, likely causing overestimation of mechanical properties. This study has developed methods that can be translated for use with human ankle bone and will lead to the development of an accurate means of mapping arthritic bone in the ankle

Summary Statement. This study quantifies compositional differences in cartilage between CAM deformities of symptomatic FAI patients and normal cadaver controls. It shows a resemblance of CAM-FAI cartilage with those of osteoarthritic hips, objectively supporting previous hypothesis of abnormal contact stresses in CAM-FAI. Introduction. Degeneration of cartilage within articular joints is a pathological feature of osteoarthritis (OA). Femoroacetabular impingement (FAI), a condition of abnormal contact between the articular surfaces of the femur and acetabulum, has been widely associated with early onset OA of the hip. The purpose of this study was to quantitatively compare the proteoglycan (PG) content of the weight-bearing cartilage in surgical FAI patients versus those of cadavers without FAI. Patients and Methods. Osteochondral bone plugs were taken from the antero-superior weight-bearing surface of cam-deformities on the femoral heads of 11 surgical cam-FAI patients. These were compared to control specimens taken from 11 cadaveric hips (7 donors) at approximately the same location. The PG content of the specimens were then histologically compared using the model presented by Martin et al. In this method, Safranin-O binds to chondroitin sulfate, a PG abundant in cartilage, allowing it to be visualised and quantitatively compared. Specifically, the specimens were fixed in formalin, decalcified in EDTA and then sectioned to 7um thick. They were then stained with Safranin-O, which binds specifically and stoichiometrically with proteoglycan. This model allows for quantitative comparison of PG content whereby the red content (R. c. ) of the sample is linearly correlated with the amount of PG present in the sample when viewed under 4x microscopic magnification. Here, the red content was sampled by depth coordinate with superficial and deep zones analyzed. Results. In general, the R. C. in the cartilage of surgical patients was lower than that of the cadaveric controls in both the superficial and deep layers tested. This correlates to a decrease in the PG of the test subjects. In the surgical specimens, R. C. ranged from 0 – 31.9 in the superficial layer and 0 – 139.6 in the deep. When compared by layers, the R. C. of the superficial 30% specimens averaged an R. C. of 17.5 compared to 88.6 in the cadaveric controls. This represents an 80.2% depletion in the PG content. In the deep 70% layer, the average R. C. of the test subjects was 52.4, compared with 129.2 in the cadaveric controls. This represents a 59% depletion in the PG content of the deep layer. These results show large compositional change in the cartilage of surgical FAI versus control specimens that were statistically significant in all levels (superficial, deep, total yielding p<0.001, p=0.001, p<0.001, respectively). Discussion. The idea of abnormal cartilage at the cam deformity has been previously demonstrated through similar resection and staining techniques. Wagner et al showed cellular activity and qualitatively noted PG depletion in the cartilage on the Cam deformity, consistent with OA. However a quantitative assessment of PG content provides a better estimate of impingement severity and disease state. Results from this current study objectively corroborate previously obtained qualitative data, supporting existing hypothesis of abnormal contact stresses in cam-FAI while giving a more robust, objective quantification of cartilage breakdown at CAM sites

The ability to pre-clinically evaluate new cartilage substitution therapies in viable physiological biotribological models, such as the femoral-tibial joint would be advantageous. Methods for osteochondral (OC) plug culture have been developed and the aim of this study was to extend these methods to organ culture of whole femoral condylar and tibial osteochondral tissues. Porcine femoral condyles and tibial plateau were aseptically dissected. The majority of cancellous bone was removed leaving intact cartilage and a layer of cortical bone. OC plugs were from porcine knee condyles. “Whole joint” tissues and OC plugs were cultured in defined medium and the viability of the cartilage at day 0, 8 or 14 days of culture assessed by XTT assay and LIVE/DEAD staining. Histological analysis (H&E; alcian blue staining) was used to determine cell number and visualise glycosominoglycans (GAGs). GAG levels were quantified in the cartilage using the dimethylene blue assay. XTT conversion by OC plug cartilage reduced significantly between day 0 and day 8 with no further change between day 8 and 14. GAG levels did not change. “Whole joint” tissue behaved similarly with reduced XTT conversion between days 0 and 8 (femoral only) and days 0 and 14 (femoral and tibial). LIVE/DEAD staining showed the majority of cells remained alive in the mid and deep cartilage zones. There was a band of mainly dead cells in the surface zone, from day 0. There was no change in the GAG levels over the 14 day culture period. In conclusion, large cuts of femoral and tibial osteochondral tissues were maintained in organ culture for extended periods. Surface zone chondrocytes rapidly lost membrane integrity ex-vivo whereas mid- and deep zone chondrocytes remained viable. It is hypothesised that physiological loading in a novel physically interactive bioreactor will improve the viability and will be the focus of future studies

Objectives

We studied subchondral intraosseous pressure (IOP) in an animal model during loading, and with vascular occlusion. We explored bone compartmentalization by saline injection.

We evaluated two reconstruction techniques for a simulated posterolateral corner injury on ten pairs of cadaver knees. Specimens were mounted at 30° and 90° of knee flexion to record external rotation and varus movement. Instability was created by transversely sectioning the lateral collateral ligament at its midpoint and the popliteus tendon was released at the lateral femoral condyle. The left knee was randomly assigned for reconstruction using either a combined or fibula-based treatment with the right knee receiving the other. After sectioning, laxity increased in all the specimens. Each technique restored external rotatory and varus stability at both flexion angles to levels similar to the intact condition. For the fibula-based reconstruction method, varus laxity at 30° of knee flexion did not differ from the intact state, but was significantly less than after the combined method.

Both the fibula-based and combined posterolateral reconstruction techniques are equally effective in restoring stability following the simulated injury.

We report the effects of local administration of osteogenic protein-1 on the biomechanical properties of the overstretched anterior cruciate ligament in an animal model. An injury in the anterior cruciate ligament was created in 45 rabbits. They were divided into three equal groups. In group 1, no treatment was applied, in group II, phosphate-buffered saline was applied around the injured ligament, and in group III, 12.5 μg of osteogenic protein-1 mixed with phosphate-buffered saline was applied around the injured ligament. A control group of 15 rabbits was assembled from randomly-selected injured knees from among the first three groups. Each rabbit was killed at 12 weeks.

The maximum load and stiffness of the anterior cruciate ligament was found to be significantly greater in group III than either group 1 (p = 0.002, p = 0.014) or group II (p = 0.032, p = 0.025). The tensile strength and the tangent modulus of fascicles from the ligament were also significantly greater in group III than either group I (p = 0.002, p = 0.0174) or II (p = 0.005, p = 0.022).

The application of osteogenic protein-1 enhanced the healing in the injured anterior cruciate ligament, but compared with the control group the treated ligament remained lengthened. The administration of osteogenic protein-1 may have a therapeutic role in treating the overstretched anterior cruciate ligament.

Impacted morsellised allografts have been used successfully to address the problem of poor bone stock in revision surgery. However, there are concerns about the transmission of pathogens, the high cost and the shortage of supply of donor bone. Bone-graft extenders, such as tricalcium phosphate (TCP) and hydroxyapatite (HA), have been developed to minimise the use of donor bone. In a human cadaver model we have evaluated the surgical and mechanical feasibility of a TCP/HA bone-graft extender during impaction grafting revision surgery.

A TCP/HA allograft mix increased the risk of producing a fissure in the femur during the impaction procedure, but provided a higher initial mechanical stability when compared with bone graft alone. The implications of the use of this type of graft extender in impaction grafting revision surgery are discussed.