Optimizing the decellularization process of an upper limb skeletal muscle; implications for muscle tissue engineering.

Upper limb muscle reconstruction is required following cancer resection, trauma, and congenital deformities. Current surgical reconstruction of the muscle involves local, regional and free flaps. However, muscle reconstruction is not always possible due to the size of the defect and functional donor site morbidity. These challenges could be addressed with the production of scaffolds composed of an extracellular matrix (ECM) derived from decellularized human skeletal muscle. This study aimed to find an optimal technique to decellularize a flexor digitorum superficialis muscle. The first two protocols were based on a detergent only (DOT) and a detergent-enzymatic protocol (DET). The third protocol avoided the use of detergents and proteolytic enzymes (NDNET). The decellularized scaffolds were characterized using qualitative techniques including histological and immunofluorescent staining and quantitative techniques assessing deoxyribonucleic acid (DNA), glycosaminoglycan (GAG), and collagen content. The DOT protocol consisting of 2% SDS for 4 hours was successful at decellularizing human FDS, as shown by DNA content assay and nuclei immunofluorescence staining. The DOT protocol maintained the microstructure of the scaffolds as shown by Masson's trichrome staining and collagen and GAG content. DET and NDNET protocols maintained the ECM, but were unsuccessful in removing all DNA content after two cycles of decellularization. Decellularization of skeletal muscle is a viable option for muscle reconstruction using a detergent only technique for upper limb defects. Further testing in vivo will assess the effectiveness of decellularized scaffolds for upper limb muscle skeletal tissue engineering.

Optimizing the decellularization process of human maxillofacial muscles for facial reconstruction using a detergent-only approach.

Trauma, congenital diseases, and cancer resection cause muscle deformities of the human facial muscle. Muscle defects are either treated with local or distal flaps if direct closure is not possible. However, such surgical interventions are limited by donor morbidity and limited tissue availability. Decellularized scaffolds provide alternative strategies for replacing and restoring missing facial muscle by creating scaffolds that mimic the native tissue. This study aimed to develop a protocol to decellularize human zygomaticus major muscle (ZMM) and masseter muscle (MM). Three protocols were assessed including a detergent-only treatment (DOT), detergent-enzymatic treatment (DET) protocol, and a third nondetergent nonenzymatic treatment protocol. Scaffolds were then characterized via histological, immunofluorescent, and quantitative techniques to assess which protocol provided optimal decellularization and maintenance of the extracellular matrix (ECM). The results demonstrated three cycles of DOT protocol consisting of 2% sodium dodecyl sulfate for 4 hr was optimal for decellularization for both ZMM and MM. After three cycles, DNA content was significantly reduced compared with native ZMM and MM (p < .05) with preservation of collagen and glycosaminoglycan content and ECM on histological analysis. DET and nondetergent nonenzymatic treatment protocols were unsuccessful in decellularizing the ZMM and MM with residual DNA content after four cycles and caused ECM disruption on histological analysis. All protocols did not impair the mechanical properties and supported human fibroblast growth. In conclusion, the DOT protocol is effective in producing human decellularized muscle scaffolds that maintain the ECM. Further investigation of detergent only decellurization techniques should be explored as a first step to create effective scaffolds for muscle tissue engineering.

Neurovascular anatomy around the knee: Relevance of the dangers of self-drilling external fixator pin tips.

INTRODUCTION: With external fixation of the femur and tibia, iatrogenic injury to neurovasculature from self-drilling tips of fixation pins is an important consideration in pin placement. Precise knowledge of the neurovascular anatomy in the distal femur and proximal tibia is important to limit potential pin misplacement. METHOD: Six pin placement sites on six cadaveric legs were used in accordance with current placement techniques. After pin placement, the soft tissue around each pin was dissected and the distances between the pin tips and the surrounding neurovasculature were measured. RESULTS: The resultant data allow for a description of safe and unsafe corridors which can be used for external fixator pin placement. Safe sagittal insertion into the distal femur should consist of two pins: (1) 90 mm ± proximal from the proximal pole of the patella and 3 mm ± medially, (2) 55 mm ± proximal from the proximal pole of the patella and 2 mm ± laterally. Safe coronal insertion into the distal femur should consist of two pins: (1) 30 mm ± proximal to the lateral epicondyle, (2) 100 mm ± proximal to the lateral epicondyle. Safe proximal tibial pin placement should consist of two pins and be placed at an oblique angle: (1) 20 mm ± distal to the tibial tuberosity and 2 mm ± medially, (2) 55 mm ± distal to the tibial tuberosity and 2 mm ± medially. DISCUSSION: This study forms an investigation into the safe areas for placement of external fixator pins, within the distal femur and proximal tibia, specifically, detailing the best practice for pin placement in relation to the tips of the external fixation pins.

Optimising the decellularization of human elastic cartilage with trypsin for future use in ear reconstruction.

Decellularized scaffolds can induce chondrogenic differentiation of stem cells. This study compares different methods to optimise the decellularization of auricular cartilage. The process consisted of an initial 12 hour dry freeze thaw which froze the cartilage specimens in an empty tube at -20 °C. Samples were allowed to thaw at room temperature followed by submersion in phosphate buffer solution in which they were frozen at -20 °C for a 12 hour period. They were then allowed to thaw at room temperature as before. Protocol A subsequently involved subjecting specimens to both deoxyribonuclease and sodium deoxycholate. Protocol B and C were adaptations of this using 0.25% trypsin (7 cycles) and a 0.5 molar solution of ethylenediaminetetraacetic acid (3 hours for each cycle) respectively as additional steps. Trypsin accelerated the decellularization process with a reduction in DNA content from 55.4 ng/µL (native) to 17.3 ng/µL (P-value < 0.05) after 14 days. Protocol B showed a faster reduction in DNA content when compared with protocol A. In comparison to protocol C after 14 days, trypsin also showed greater decellularization with a mean difference of 11.7 ng/µL (P-value < 0.05). Histological analysis with H&E and DAPI confirmed depletion of cells at 14 days with trypsin.

An In Vitro Biomechanical Study on Ovine Rib Flexibility With Increasing Deconstruction-As an Alternative to Rib Resection for Costoplasty.

STUDY DESIGN: An in vitro biomechanical study into the effect of rib deconstruction on the flexibility in ovine ribs, using three-point bending. OBJECTIVE: To examine the feasibility and possible effectiveness of a more conservative costoplasty using an in vitro animal model. SUMMARY OF BACKGROUND DATA: Costoplasty remains useful in the treatment of adolescent idiopathic scoliosis, rib hump, and associated chest wall deformities. However, traditional costoplasty increases morbidity and blood loss. METHODS: Ribs 2-10 were dissected from four fresh half ovine rib cages. The ribs were randomly allocated to Groups 1 to 4. The ribs underwent deconstruction, 10 mm from the lateral tubercle and 30 mm long, according to their group: Group 1 = control; Group 2 = convex cortical bone removed; Group 3 = convex cortical and cancellous bone removed; Group 4 = all but the ventral cortex is removed. Flexibility was tested by loading the concave side of each rib while fixed at the rib head and equidistant from the center of the resected area. The ribs were deformed at 0.5 mm/s up to a maximum load of 9.99 kg or fracturing. Load was plotted against displacement to find the load/displacement coefficient for each group. Statistical analysis was by an analysis of variance with Tukey's honestly significant difference post hoc testing. RESULTS: The load/displacement coefficients were as follows: Group 1 = 131.93 (±27.52) N/mm, Group 2 = 93.36 (±40.71) N/mm, Group 3 = 88.66 (±25.84) N/mm, and Group 4 = 29.69 (±29.11) N/mm. Group 4 was significantly less stiff than Groups 1, 2, and 3 (p < .01). No ribs in Groups 1, 2, and 4 fractured. Five of 8 ribs in Group 3 fractured during loading. CONCLUSIONS: Deconstructing the rib down to the concave side significantly increases the flexibility by approximately 4.5 times. Despite large removal of bone, it retains the ability to withstand 10 kg of load without fracture.

Injection pressure mapping of intraneural vs. perineural injections: further lessons from cadaveric studies.

BACKGROUND: The aim of the study was to investigate the difference between intraneural and perineural injection pressures in human cadavers. Targeted nerves included the cervical roots, the supraclavicular and infraclavicular brachial plexus, the sciatic-subgluteal nerve and the common peroneal and tibial nerves. METHODS: Ten readings were obtained for each nerve location. Over ten seconds, 1 mL of 0.9% NaCl was injected - deliberately slower than in clinical practice to eliminate the risk of aberrant readings relating to the speed of injection. Perineural injections occurred at least 1 mm outside the epineurium. After pressure recordings were completed 0.1mL of dye was injected, and dissection performed to confirm needle placement. Ultrasound and dissection images were matched with light microscopy pictures for all locations. RESULTS: The average pressure for intraneural injections was 24.1±5.7 psi and 6.1±2.1 psi for perinereural. The average injection pressure generated for the cervical trunk, supraclavicular, infraclavicular, sciatic subgluteal, peroneal and tibial nerves respectively were 31.2±6.0 psi, 24±15.0 psi, 23.4±9.5 psi, 22.6±8.8 psi 19.7±6 psi, 17±7.3 psi intraneurally and 6.1±2.0 psi, 9.1±5.5 psi, 10±4.9 psi, 6±2.4 psi, 6±2.4 psi and 7±2.5 psi perineurally. For intraneural injections statistically significant differences were demonstrated between the peroneal and tibial nerves compared to cervical roots/trunks/division/cords of brachial plexus. CONCLUSIONS: The study has consistently demonstrated statistically significant differences between intraneural and perineural injection pressures. It effectively created a "map" of intraneural injection pressures for the most common peripheral nerves blocks and demonstrated a pattern between proximal and distal locations. The study also revealed limitations of either techniques, ultrasound and injection pressure monitoring reinforcing the concept of their simultaneous application.

Effects of bone damage on creep behaviours of human vertebral trabeculae.

A subgroup of patients suffering with vertebral fractures can develop progressive spinal deformities over time. The mechanism underlying such clinical observation, however, remains unknown. Previous studies suggested that creep deformation of the vertebral trabeculae may play a role. Using the acoustic emission (AE) technique, this study investigated effects of bone damage (modulus reduction) on creep behaviours of vertebral trabecular bone. Thirty-seven human vertebral trabeculae samples were randomly assigned into five groups (A to E). Bones underwent mechanical tests using similar experimental protocols but varied degree of bone damage was induced. Samples first underwent creep test (static compressive stress of 0.4MPa) for 30min, and then were loaded in compression to a specified strain level (0.4%, 1.0%, 1.5%, 2.5%, and 4% for group A to E, respectively) to induce different degrees of bone damage (0.4%, no damage control; 1.0%, yield strain; 1.5%, beyond yield strain, 2.5% and 4%, post-ultimate strains). Samples were creep loaded (0.4MPa) again for 30min. AE techniques were used to monitor bone damage. Bone damage increased significantly from group A to E (P<0.05), with >30% of modulus reduction in group D and E. Before compressive loading, creep deformation was not different among the five groups and AE hits in creep test were rare. After compressive loading, creep deformation was significantly greater in group D and E than those in other groups (P<0.05). The number of AE hits and other AE measurements during creep test were significantly greater in group D and E than in group A, B, and C (P<0.05 for all). Data suggested that with the increase of vertebral trabecular bone damage, substantial creep deformation may occur even when the vertebra was under physiological loads. The boosted creep deformation observed may be attributed to newly created trabecular microfractures. Findings provide a possible explanation as to why some vertebral fracture patients develop progressive spinal deformity over time.

Quantification of the articular view of the elbow afforded by standard portals using 30° arthroscopy.

BACKGROUND: The present study quantifies the field of view (FOV) from standard arthroscopy portals and aims to identify anatomical regions where the FOV is limited. METHODS: Eleven cadaveric elbows were examined through standard anteromedial, anterolateral and posterior portals. The FOV was marked with dye using a spinal needle. The articular surfaces were then exposed and the percentage FOV seen was calculated. FOV percentage areas were compared using a Student's t-test (JMP, version 10, SAS Institute Inc., Cary, NC, USA). P < 0.05 was considered statistically significant. RESULTS: The mean (SD) FOV percentage area seen from the anteromedial portal and anterolateral portal was 91.69% (3.63%) and 92.03% (3.93%), respectively, for the anterior humerus articular surface. There was no significant difference in the mean FOV percentage area seen from the anteromedial and anterolateral portals (p = 0.99). The mean (SD) FOV percentage area of the posterior humerus articular surface was 84.69% (2.28%). The mean (SD) FOV percentage area seen of the radial head and trochlear was 16.05% (2.66%) and 4.14% (1.76%), respectively. CONCLUSIONS: The present study is the first to quantify the FOV of elbow arthroscopy. The majority of the anterior and posterior humerus articular surface can be seen through standard portals. The limitations in FOV are primarily confined to the radial head and trochlear notch.

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing.

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions. This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Pressure Monitoring of Intraneural an Perineural Injections Into the Median, Radial, and Ulnar Nerves; Lessons From a Cadaveric Study.

BACKGROUND: Nerve damage after regional anesthesia has been of great concern to anesthetists. Various modalities have been suggested to recognize and prevent its incidence. An understudied area is the measurement of intraneural pressure during peripheral nerve blockade. Previous investigations have produced contradicting results with only one study being conducted on human cadavers. OBJECTIVES: The purpose of this investigation was to systematically record intraneural and perineural injection pressures on the median, ulnar, and radial nerves exclusively as a primary outcome. MATERIALS AND METHODS: Ultrasonography-guided injections of 1 mL of 0.9% NaCl over ten seconds were performed on phenol glycerine embalmed cadaveric median, ulnar, and radial nerves. A total of 60 injections were performed, 30 intraneural and 30 perineural injections. The injections pressure was measured using a controlled disc stimulation device. Anatomic dissection was used to confirm needle placement. RESULTS: Intraneural needle placement produced significantly greater pressures than perineural injections did. The mean generated pressures in median, radial, and ulnar nerves were respectively 29.4 ± 9.3, 27.3 ± 8.5, and 17.9 ± 7.0 pound per square inch (psi) (1 psi = 51.7 mmHg) for the intraneural injections and respectively 7.2 ± 2.5, 8.3 ± 2.5, and 6.7 ± 1.8 psi for perineural injections. Additionally the intraneural injection pressures of the ulnar nerve were lower than those of the median and radial nerves. CONCLUSIONS: Obtained results demonstrate significant differences between intraneural and perineural injection pressures in the median, ulnar, and radial nerves. Intraneural injection pressures show low specificity but high sensitivity suggesting that pressure monitoring might be a valuable tool in improving the safety and efficacy of peripheral nerve blockade in regional anesthesia. Peripheral nerves "pressure mapping" hypothetically might show difference amongst various nerves depending on anatomic location, histologic structure, and ultrasonographic appearance.

Odontoid process fractures: the role of the ligaments in maintaining stability. A biomechanical, cadaveric study.

AIMS: We wished to investigate the role of the cervical ligaments in maintaining atlantoaxial stability after fracture of the odontoid process. METHODS: We dissected eight fresh-frozen cadaveric cervical spines to prepare the C1 and C2 vertebrae for biomechanical analysis. The C1 and C2 blocks were mounted and biomechanical analysis was performed to test the stability of the C1-C2 complex after cutting the odontoid process to create an Anderson and D'Alonzo type II fracture then successive division of the atlantoaxial ligaments. Biomechanical analysis of stiffness, expressed as Young's modulus, was performed under right rotation, left rotation and anterior displacement. RESULTS: The mean Young's modulus in anterior displacement decreased by 37% when the odontoid process was fractured (p = 0.038, 95% confidence interval 0.04-1.07). The mean Young's modulus in anterior displacement decreased proportionally (compared to the previous dissection) by the following percentages when the structures were divided: facet joint capsules (bilateral) 16%, ligamentum flavum 27%, anterior longitudinal ligament 10%. These differences did not reach statistical significance (p > 0.05). DISCUSSION: We have found that the odontoid process itself may account for up to 37% of the stiffness of the C1-C2 complex and that soft tissue structures account for further resistance to movement. We suggest magnetic resonance imaging (MRI) of the soft tissues in the acute setting of a minimally displaced odontoid process fracture to plan management of the injury. If the MRI determines that there is associated ligament injury it is likely that the fracture is unstable and we would suggest operative management.

An anatomical and cadaveric study examining the risk of sural nerve injury in percutaneous Achilles tendon repair using the Achillon device.

BACKGROUND: Percutaneous Achilles tendon repairs are gaining in popularity. This study aims to quantify the risk of sural nerve injury when using the Achillon device. METHODS: The Achillon device was instrumented into 15 cadaveric specimens and through dissection the rate of sural nerve puncture and the position of the sural nerve in relation to the Achilles tendon was documented. RESULTS: The sural nerve was found lateral to the Achilles tendon insertion point over a range of 14.3mm and crossed the lateral border of the Achilles tendon over a range of 57.7mm. The sural nerve was punctured a total of 6 times and in 4 out of 15 cadaveric specimens (27%). Four out of the 6 punctures occurred when the Achillon device was instrumented distally. CONCLUSIONS: The sural nerve displays a highly variable anatomical course and there is a risk of puncture during percutaneous Achilles tendon repair using the Achillon device.

Ankle position affects dorsalis pedis artery exposure in anterior ankle arthroscopy.

BACKGROUND: In anterior ankle arthroscopy, the anterior working area (AWA) is restricted by the presence of the dorsalis pedis artery (DPA) and tendons. Pseudoaneurysms caused by iatrogenic damage to the DPA are difficult to identify intraoperatively. In knee arthroscopy, risk of popliteal artery damage is reduced in the flexed position [1]. This study investigates how DPA movement is affected by dorsiflexion and plantarflexion with the aim of identifying the positions providing the greatest AWA. METHODS: Twelve cadaveric ankles were dissected to access the DPA. While distracted, ankles were progressively dorsiflexed at 5° intervals from maximum plantarflexion. DPA and tibialis anterior tendon (TA) movement at each 5° interval was measured by their respective distances from the inferior border of the medial malleolus. RESULTS: Mean ankle dorsiflexion was 24.58±1.30° with all specimens showing anterior DPA and TA movement as dorsiflexion increased. Mean DPA and TA movement at maximum dorsiflexion was 3.58±0.29mm and 2.92±0.34mm respectively. A ratio of 1:1.23 relates TA and DPA movement (inmm), and a ratio of 10:1.46 relates dorsiflexion angle to DPA movement (inmm). CONCLUSION: Anterior movement of the dorsalis pedis artery during dorsiflexion increases the AWA for anterior arthroscopy. Increasing the AWA with maximal dorsiflexion may prove to be a valuable method for lowering the risk of iatrogenic DPA damage. Additionally, increased AWA may allow the use of larger diameter surgical instruments allowing greater control and a reduction in operation time.

The biomechanical and histological sequelae of common skin banking methods.

Human skin allografts are used worldwide as an adjunct for the healing of burns when autograft skin is not available or not indicated. Allograft skin comes from human cadaveric donors, and so must be preserved until use. This study forms the first investigation to compare the mechanical and histological integrity of human split-thickness skin grafts preserved by either glycerolisation or cryopreservation (with or without the cryoprotectant DMSO). Stress relaxation was used to assess mechanical properties, whilst histological analysis allowed for evaluation of structural integrity. Preservation of tissue, whether by freezing or glycerolisation, altered the relaxation behaviours of skin. Young's modulus upon initial loading significantly decreased for skin frozen without cryoprotectant, but remained unchanged for skin frozen with cryoprotectant and skin preserved with glycerol. After 1.5h of stress relaxation, both fresh skin and skin frozen without DMSO displayed similar relaxation rates. Samples frozen with DMSO or preserved with glycerol had increased relaxation rate and had not reached load equilibrium within this time. To understand the structural basis for the biomechanical changes, samples were histologically assessed. All preservation protocols resulted in a similar degree of visible damage, but cryopreservation appeared particularly damaging to the extracellular matrix, whereas glycerolisation caused dramatic separation of the epidermis from the underlying dermis. The mechanical property alterations reveal that preservation results in laxity, which clinically could hinder contact dependent healing properties, but alternatively may increase capacity for coverage. The structural changes confirm that preservation techniques do not conserve grafts in an in vivo state.

Gait characteristics over the course of a race in recreational marathon competitors.

We analyzed gait and function of the supporting limb in participants of a marathon race at three stages: prerace, midrace (18 km), and near the end of the race (36 km). We confirmed that the most successful runners were able to maintain running speed for the duration of the race with little change in speed or gait. Speed slowed progressively during the race for those with slower race times, but stride frequency-stride length relationships remained normal for the speed they ran. These findings differ from most lab-based studies of fatigue, in which runners are forced to match a constant preset treadmill speed. Small changes in maximum ground force were seen in both slow- and fast-running participants as race end approached.

Anisotropic properties of bovine nasal cartilage.

To investigate the structural anisotropy in bovine septal cartilage, quantitative procedures in microscopic magnetic resonance imaging (µMRI), polarized light microscopy (PLM), and mechanical indentation were used to measure the tissue in three orthogonal planes: vertical, medial, and caudocephalic. The quantitative T2 imaging experiments in µMRI found strong anisotropy in the images of both vertical and caudocephalic planes but little anisotropy in the images from the medial plane. The PLM birefringent experiments found that the retardation values in the medial section were only about 10% of these in the vertical and caudocephalic sections and that the angle values in all three sections followed the rotation of the tissue section in the microscope stage. The stress relaxation experiments in mechanical indentation showed reduced stiffness in the medial plane compared to stiffness in either the vertical or caudocephalic planes. Collectively, the results in this project coherently indicate a marked structural anisotropy in cartilage from the nasal septum, where the long axis of the collagen fibrils is oriented in parallel with the medial axis.

Direct Visualisation of the Depth-Dependent Mechanical Properties of Full-Thickness Articular Cartilage.

OBJECTIVE: The structural anisotropy of articular cartilage controls its deformation response. As proteoglycans and collagen vary with depth, simple uniaxial compression results in inhomogeneous deformation with distinct depth-dependent mechanical properties. Investigations into depth-dependent mechanical properties of articular cartilage have previously required tissue modification after specimen isolation. Such modifications include histological processes, freezing, subchondral bone removal, and fluorescent staining that may alter the tissue, limiting in vivo applicability. DESIGN: Using a custom tissue-sectioning device, 0.1 mm thick unfixed, unstained, osetochondral samples were obtained. A customized apparatus loaded samples to 12.5, 24, and 29% compression in under a microscope with 10× magnification. Equilibrium load was measured after stress relaxation. Intra-tissue displacement was measured by tracing groups of cells between the different compression levels using a digital imaging program. Cell distance from the subchondral bone was measured to identify intra-tissue displacement and calculate strain. RESULTS: The results reveal that stress levels and intra-tissue displacement increased with greater tissue compression (p <0.05). Intra-tissue displacement decreased as depth from the articular surface increased (p<0.01). This occurred for each level of tissue compression. Overall compressive resistance is seen to increase with depth from the articular surface. CONCLUSIONS: The current study identifies a method directly visualising and assessing the depth-dependent structural response to compression. The ability to avoid tissue modification after specimen isolation, allows this procedure to more closely approximate in vivo conditions and may provide an important method for analyzing the coordinated changes in cartilage composition and function due to ageing and disease.

Depth-dependent anisotropies of amides and sugar in perpendicular and parallel sections of articular cartilage by Fourier transform infrared imaging.

Full thickness blocks of canine humeral cartilage were microtomed into both perpendicular sections and a series of 100 parallel sections, each 6 µm thick. Fourier transform infrared (IR) imaging was used to image each tissue section eleven times under different IR polarizations (from 0° to 180° polarization states in 20° increments and with an additional 90° polarization), at a spatial resolution of 6.25 µm and a wavenumber step of 8 cm⁻¹. With increasing depth from the articular surface, amide anisotropies increased in the perpendicular sections and decreased in the parallel sections. Both types of tissue sectioning identified a 90° difference between amide I and amide II in the superficial zone (SZ) of cartilage. The fibrillar distribution in the parallel sections from the SZ was shown to not be random. Sugar had a weak but recognizable anisotropy in the upper part of the radial zone (RZ) in the perpendicular sections. The depth-dependent anisotropic data were fitted with a theoretical equation that contained three signature parameters, which illustrate the arcade structure of collagens with the aid of a fibril model. Fourier-transform IR imaging of both perpendicular and parallel sections provides the possibility of determining the three-dimensional macromolecular structures in articular cartilage. Being sensitive to the orientation of the macromolecular structure in healthy articular cartilage aids the prospect of detecting the early onset of the tissue degradation that may lead to pathological conditions such as osteoarthritis.

Freeze-thaw treatment effects on the dynamic mechanical properties of articular cartilage.

BACKGROUND: As a relatively non-regenerative tissue, articular cartilage has been targeted for cryopreservation as a method of mitigating a lack of donor tissue availability for transplant surgeries. In addition, subzero storage of articular cartilage has long been used in biomedical studies using various storage temperatures. The current investigation studies the potential for freeze-thaw to affect the mechanical properties of articular cartilage through direct comparison of various subzero storage temperatures. METHODS: Both subzero storage temperature as well as freezing rate were compared using control samples (4°C) and samples stored at either -20°C or -80°C as well as samples first snap frozen in liquid nitrogen (-196°C) prior to storage at -80°C. All samples were thawed at 37.5°C to testing temperature (22°C). Complex stiffness and hysteresis characterized load resistance and damping properties using a non-destructive, low force magnitude, dynamic indentation protocol spanning a broad loading rate range to identify the dynamic viscoelastic properties of cartilage. RESULTS: Stiffness levels remained unchanged with exposure to the various subzero temperatures. Hysteresis increased in samples snap frozen at -196°C and stored at -80°C, though remained unchanged with exposure to the other storage temperatures. CONCLUSIONS: Mechanical changes shown are likely due to ice lens creation, where frost heave effects may have caused collagen damage. That storage to -20°C and -80°C did not alter the mechanical properties of articular cartilage shows that when combined with a rapid thawing protocol to 37.5°C, the tissue may successfully be stored at subzero temperatures.