Thermoelectric Freeze-Casting of Biopolymer Blends: Fabrication and Characterization of Large-Size Scaffolds for Nerve Tissue Engineering Applications.

Peripheral nerve injuries (PNIs) are detrimental to the quality of life of affected individuals. Patients are often left with life-long ailments that affect them physically and psychologically. Autologous nerve transplant is still the gold standard treatment for PNIs despite limited donor site and partial recovery of nerve functions. Nerve guidance conduits are used as a nerve graft substitute and are efficient for the repair of small nerve gaps but require further improvement for repairs exceeding 30 mm. Freeze-casting is an interesting fabrication method for the conception of scaffolds meant for nerve tissue engineering since the microstructure obtained comprises highly aligned micro-channels. The present work focuses on the fabrication and characterization of large scaffolds (35 mm length, 5 mm diameter) made of collagen/chitosan blends by freeze-casting via thermoelectric effect instead of traditional freezing solvents. As a freeze-casting microstructure reference, scaffolds made from pure collagen were used for comparison. Scaffolds were covalently crosslinked for better performance under load and laminins were further added to enhance cell interactions. Microstructural features of lamellar pores display an average aspect ratio of 0.67 ± 0.2 for all compositions. Longitudinally aligned micro-channels are reported as well as enhanced mechanical properties in traction under physiological-like conditions (37 °C, pH = 7.4) resulting from crosslinking treatment. Cell viability assays using a rat Schwann cell line derived from sciatic nerve (S16) indicate that scaffold cytocompatibility is similar between scaffolds made from collagen only and scaffolds made from collagen/chitosan blend with high collagen content. These results confirm that freeze-casting via thermoelectric effect is a reliable manufacturing strategy for the fabrication of biopolymer scaffolds for future peripheral nerve repair applications.

Efficacy of Combining PRP and MMP Inhibitors in Treating Moderately Damaged Tendons Ex Vivo.

Platelet-rich plasma (PRP) and broad-spectrum matrix metalloproteinase inhibitors (MMPIs) have been used as therapeutic options for tendinopathy. However, mixed results have been reported regarding their efficacy. We posited that the combination of these two treatment strategies would be more beneficial for healing tendons than each treatment alone. Rat tail tendons were harvested and cultured without mechanical stress for 0, 4, or 10 days. Single and combination treatment with PRP and MMPIs with either broad- or narrow-spectrum (MMP-13 selective), was administered to 4-day stress-deprived (SD) tendons, an ex vivo model for moderate tendinopathy. This treatment was applied to the damaged tendons over 6 days. At the end of their culture time, the tendons were subjected to traction testing and pathohistology, immunohistochemistry, and viability assays. The results showed better histological features for the PRP + narrow-spectrum MMPI group compared with all individual treatment modalities. Moreover, higher fiber density, more elongated nucleus shape, smaller space between fibers, and a trend toward higher mechanical strength were noted for PRP + narrow-spectrum MMPI group compared with 10-day SD tendons. This study shows that the combination of PRP + narrow-spectrum MMPI is a potentially effective treatment approach for tendinopathy. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1838-1847, 2019.

Characterization of moderate tendinopathy in ex vivo stress-deprived rat tail tendons.

BACKGROUND: Stress deprivation (SD) has frequently been used as a model to study tendinopathy. Most of these studies have investigated either short-term (early tendinopathy) or long-term SD (advanced tendinopathy), while the transient mid-term SD has been given less attention. Therefore, the main objective of this study was to characterize mid-term SD. METHODS: To this end, live, healthy rat tail tendons (RTTs) were harvested and cultured without mechanical stress and then were divided into five groups based on their culture time (fresh, 2-day SD, 4-day SD, 6-day SD, and 10-day SD). For each group, the tendons were subjected to traction testing and pathohistology, immunohistochemistry, and viability assays. RESULTS: Our results showed that 4 days of SD resulted in moderate pathological changes in RTTs. These changes included increases in the space area between fibers, cell density, and fiber tortuosity as well as a decrease in collagen density and elongation of cell nuclei. No changes in the stress at failure of tendons were observed at this time point. CONCLUSIONS: This simple ex vivo model is expected to be useful for studying the progression of tendinopathy as well as for testing potential mechanobiological or pharmacological therapy strategies to stop or reverse the progression of the pathology.

Effect of manufacturing and experimental conditions on the mechanical and surface properties of silicone elastomer scaffolds used in endothelial mechanobiological studies.

BACKGROUND: Mechanobiological studies allow the characterization of cell response to mechanical stresses. Cells need to be supported by a material with properties similar to the physiological environment. Silicone elastomers have been used to produce various in vitro scaffolds of different geometries for endothelial cell studies given its relevant mechanical, optical and surface properties. However, obtaining defined and repeatable properties is a challenge as depending on the different manufacturing and processing steps, mechanical and surface properties may vary significantly between research groups. METHODS: The impact of different manufacturing and processing methods on the mechanical and surface properties was assessed by measuring the Young's modulus and the contact angle. Silicone samples were produced using different curing temperatures and processed with different sterilization techniques and hydrophilization conditions. RESULTS: Different curing temperatures were used to obtain materials of different stiffness with a chosen silicone elastomer, i.e. Sylgard 184®. Sterilization by boiling had a tendency to stiffen samples cured at lower temperatures whereas UV and ethanol did not alter the material properties. Hydrophilization using sulphuric acid allowed to decrease surface hydrophobicity, however this effect was lost over time as hydrophobic recovery occurred. Extended contact with water maintained decreased hydrophobicity up to 7 days. Mechanobiological studies require complete cell coverage of the scaffolds used prior to mechanical stresses exposure. Different concentrations of fibronectin and collagen were used to coat the scaffolds and cell seeding density was varied to optimize cell coverage. CONCLUSION: This study highlights the potential bias introduced by manufacturing and processing conditions needed in the preparation of scaffolds used in mechanobiological studies involving endothelial cells. As manufacturing, processing and cell culture conditions are known to influence cell adhesion and function, they should be more thoroughly assessed by research groups that perform such mechanobiological studies using silicone.

Histopathological, biomechanical, and behavioral pain findings of Achilles tendinopathy using an animal model of overuse injury.

Animal models of forced running are used to study overuse tendinopathy, a common health problem for which clear evidence for effective and accessible treatments is still lacking. In these models, pain evaluation is necessary to better understand the disease, help design and evaluate therapies, and ensure humane treatment of the animals. Therefore, the main objective of this study was to evaluate pain and pathologic findings in an animal model of moderate Achilles tendinopathy induced by treadmill running. Air puffs, instead of electrical shocks, were used to stimulate running so that pain associated with stimulation would be avoided. Pressure pain sensitivity was evaluated in vivo using a new instrumented plier, whereas spinal cord peptides were analyzed ex vivo with high-performance liquid chromatography tandem mass spectrometry. Tendon histologic slides were semiquantitatively evaluated, using the Bonar score technique and biomechanical properties, using the traction test. After 8 weeks of treadmill running (2 weeks for adaptation and 6 weeks for the lesion protocol), the protocol was stopped because the air puffs became ineffective to stimulate running. We, nevertheless, observed some histologic changes characteristic of overuse tendinopathy as well as decreased mechanical properties, increased Substance P and dynorphin A peptides but without pressure pain sensitivity. These results suggest that air-puffs stimulation is sufficient to induce an early stage tendinopathy to study new therapeutic drugs without inducing unnecessary pain. They also indicate that pain-associated peptides could be related with movement evoked pain and with the sharp breakdown of the running performance.

Button fixation technique for Achilles tendon reinsertion: a biomechanical study.

Chronic insertional tendinopathy of the Achilles tendon is a frequent and disabling pathologic entity. Operative treatment is indicated for patients for whom nonoperative management has failed. The treatment can consist of the complete detachment of the tendon insertion and extensive debridement. We biomechanically tested a new operative technique that uses buttons for fixation of the Achilles tendon insertion on the posterior calcaneal tuberosity and compared it with 2 standard bone anchor techniques. A total of 40 fresh-frozen cadaver specimens were used to compare 3 fixation techniques for reinserting the Achilles tendon: single row anchors, double row anchors, and buttons. The ultimate loads and failure mechanisms were recorded. The button assembly (median load 764 N, range 713 to 888) yielded a median fixation strength equal to 202% (range 137% to 251%) of that obtained with the double row anchors (median load 412 N, range 301 to 571) and 255% (range 213% to 317%) of that obtained with the single row anchors (median load 338 N, range 241 to 433N). The most common failure mechanisms were suture breakage with the buttons (55%) and pull out of the implant with the double row (70%) and single row (85%) anchors. The results of the present biomechanical cadaver study have shown that Achilles tendon reinsertion fixation using the button technique provides superior pull out strength than the bone anchors tested.

Low amplitude characterization tests conducted at regular intervals can affect tendon mechanobiological response.

In bioreactor studies of tissue mechanobiology, characterizing changes in tissue quality is essential for understanding and predicting the response to mechanical stimuli. Unfortunately, current methods are often destructive and cannot be used at regular intervals on the same sample to characterize progression over time. Non-destructive methods such as low amplitude stress relaxation tests could be used, but then, the following dilemma comes into play: how can we accurately measure live tissue progression over time if the tissue is reacting to our measurement methods? In this study, we investigated the hypothesis that stress relaxation tests at physiological amplitudes conducted at regular intervals between stimulation periods do not modify tissue progression over time. Live, healthy tendons were subjected to mechanical stimuli inside a bioreactor for 3 days. The tendons were grouped based on the daily characterization protocol (24 or 0 stress relaxation tests) and their progression over time were compared. Stress relaxation tests at physiological amplitudes modified the tendon response to mechanical stimulation as observed through mechanical and histologic analyses. Possible solutions to eliminate or minimize the effect of stress relaxation tests are to use the mechanical stimuli to characterize tissue progression or to limit the number of stress relaxation tests.

A sit-ski design aimed at controlling centre of mass and inertia.

This article introduces a sit-ski developed for the Canadian Alpine Ski Team in view of the Vancouver 2010 Paralympic games. The design is predominantly based on controlling the mass distribution of the sit-ski, a critical factor in skiing performance and control. Both the antero-posterior location of the centre of mass and the sit-ski moment of inertia were addressed in our design. Our design provides means to adjust the antero-posterior centre of mass location of a sit-ski to compensate for masses that would tend to move the antero-posterior centre of mass location away from the midline of the binding area along the ski axis. The adjustment range provided is as large as 140 mm, thereby providing sufficient adaptability for most situations. The suspension mechanism selected is a four-bar linkage optimised to limit antero-posterior seat movement, due to suspension compression, to 7 mm maximum. This is about 5% of the maximum antero-posterior centre of mass control capacity (151 mm) of a human participant. Foot rest inclination was included in the design to modify the sit-ski inertia by as much as 11%. Together, these mass adjustment features were shown to drastically help athletes' skiing performance.

Low stress tendon fatigue is a relatively rapid process in the context of overuse injuries.

To stimulate healing and prevent tendinosis through optimized physical exercise, it is important to elucidate the tendon response to repetitive mechanical loading. However, the study of this response is challenging due to complex cell-matrix interactions. In an initial approximation, the authors examined tendon mechanical response only, and did not consider cellular activity. The authors investigated the hypothesis that mechanical degradation occurs relatively rapidly (< 24 h) even at very low stress levels. The authors subjected rat tail tendons to mechanical loadings oscillating between 0 and 1.5 MPa up to one of three fatigue levels: 4% strain, 8% strain, or rupture. Using non-destructive mechanical tests, changes in tendon strain and compliance over the entire fatigue testing period were evaluated. Using microscopy techniques, the structural evidence of mechanical degradation was examined. The changes in tendon strain and compliance progressed nonlinearly and accelerated before rupture which took place around the 15-h mark. Histological analyses revealed a higher degree of alteration in the collagen network at increased fatigue levels. At rupture, local zones of damage with low fibril density were evident. These results imply that a repair process must act rapidly at critical sites; otherwise, enzymatic degradation could cause further damage in the manner of a vicious cycle.

Preparation of rat tail tendons for biomechanical and mechanobiological studies.

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation. Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber. At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor. Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

Relative contributions of mechanical degradation, enzymatic degradation, and repair of the extracellular matrix on the response of tendons when subjected to under- and over- mechanical stimulations in vitro.

Tendon response to mechanical loading results in either homeostasis, improvement, or degeneration of tissue condition. In an effort to better understand the development of tendinopathies, this study investigated the mechanical and structural responses of tendons subjected to under- and over-stimulations (1.2% and 1.8% strain respectively, 1 Hz). The objective was to examine three sub-processes of tendon response: mechanical degradation, enzymatic degradation, and repair of the extracellular matrix. We subjected rat tail tendons to a 10-day stimulation protocol with four periods of 6 h each day: 30 min of stimulation and 5 h 30 min of rest. To investigate the contribution of the three sub-processes, we controlled the contribution of the cells through variations in the nutrient and protease inhibitor content in the in vitro solutions. Using nondestructive cyclic tests, we evaluated the daily changes in the peak stress. To assess structural changes, we carried out microscopic analyses at the end of the study period. We observed that the relative contributions of the sub-processes differed according to the stimulation amplitude. With over-stimulation of tendons immersed in DMEM, we succeeded in reducing enzymatic degradation and increasing peak stress. In under-stimulation, the addition of protease inhibitors was required to obtain the same result.

Tissue reorganization in response to mechanical load increases functionality.

In the rapidly growing field of tissue engineering, the functional properties of tissue substitutes are recognized as being of the utmost importance. The present study was designed to evaluate the effects of static mechanical forces on the functionality of the produced tissue constructs. Living tissue sheets reconstructed by the self-assembly approach from human cells, without the addition of synthetic material or extracellular matrix (ECM), were subjected to mechanical load to induce cell and ECM alignment. In addition, the effects of alignment on the function of substitutes reconstructed from these living tissue sheets were evaluated. Our results show that tissue constructs made from living tissue sheets, in which fibroblasts and ECM were aligned, presented higher mechanical resistance. This was assessed by the modulus of elasticity and ultimate strength as compared with tissue constructs in which components were randomly oriented. Moreover, tissue-engineered vascular media made from a prealigned living tissue sheet, produced with smooth muscle cells, possessed greater contractile capacity compared with those produced from living tissue sheets that were not prealigned. These results show that the mechanical force generated by cells during tissue organization is an asset for tissue component alignment. Therefore, this work demonstrates a means to improve the functionality (mechanical and vasocontractile properties) of tissues reconstructed by tissue engineering by taking advantage of the biomechanical forces generated by cells under static strain.

Cross-sectional profiles and volume reconstructions of soft tissues using laser beam measurements.

Precise geometric reconstruction is a valuable tool in the study of soft tissues biomechanics. Optical methods have been developed to determine the tissue cross section without mechanical contact with the specimen. An adaptation of the laser micrometer developed by Lee and Woo [ASME J. Biomech. Eng., 110 (2), pp. 110-114]. is proposed in which the laser-collimated beam rotates around and moves along a fixed specimen to reconstruct its cross sections and volume. Beam motion is computer controlled to accelerate data acquisition and improve beam positioning accuracy. It minimizes time-dependent shape modifications and increases global reconstruction precision. The technique is also competent for the measurement of immersed collagen matrices.

Increasing strain and strain rate strengthen transient stiffness but weaken the response to subsequent compression for articular cartilage in unconfined compression.

Strain amplitude and strain rate dependent nonlinear behavior and load-induced mechanical property alterations of full-thickness bovine articular cartilage attached to bone were investigated in unconfined compression. A sequence of test compressions of finite deformation (ranging from 0.9% to 34.5% nominal strain) was performed at strain rates ranging from approximately 0.053%/s to 5.8%/s. Peak and equilibrium loads were analyzed to determine strain amplitude and strain rate dependence of linear versus nonlinear responses. The test protocol was designed to reveal changes in mechanical properties due to these finite deformations by interspersing small-amplitude witness ramps of approximately 1.1% deformation and approximately 0.44%/s strain rate between the test ramps ("witness" meaning to assess any mechanical property changes). We found that peak loads displayed high nonlinearity, stiffening with both increasing compression amplitude and more so with increasing strain rate. The response to witness ramps suggested that mechanical weakening occurred when compression amplitude reached 1.9-2.9% strain and beyond, and that weakening was much more significant at higher strain rate. These findings delineate regimes of linear versus nonlinear behavior of cartilage, and indicate the types of loads which can cause mechanical property alterations. Biological implications of this study are that strain amplitude and strain rate dependent stiffening may be essential to bear physiological loads and to protect cells and matrix from mechanical damage. Structural changes reflected by mechanical weakening at small compression could also initiate remodeling or disease processes.