Terri-Ann N. Kelly

Silk microfiber-reinforced silk hydrogel composites for functional cartilage tissue repair

Cartilage tissue lacks an intrinsic capacity for self-regeneration due to slow matrix turnover, a limited supply of mature chondrocytes and insufficient vasculature. Although cartilage tissue engineering has achieved some success using agarose as a scaffolding material, major challenges of agarose-based cartilage repair, including non-degradability, poor tissue-scaffold integration and limited processing capability, have prompted the search for an alternative biomaterial. In this study, silk fiber-hydrogel composites (SF-silk hydrogels) made from silk microfibers and silk hydrogels were investigated for their potential use as a support material for engineered cartilage. We demonstrated the use of 100% silk-based fiber-hydrogel composite scaffolds for the development of cartilage constructs with properties comparable to those made with agarose. Cartilage constructs with an equilibrium modulus in the native tissue range were fabricated by mimicking the collagen fiber and proteoglycan composite architecture of native cartilage using biocompatible, biodegradable silk fibroin from Bombyx mori. Excellent chondrocyte response was observed on SF-silk hydrogels, and fiber reinforcement resulted in the development of more mechanically robust constructs after 42 days in culture compared to silk hydrogels alone. Thus, we demonstrate the versatility of silk fibroin as a composite scaffolding material for use in cartilage tissue repair to create functional cartilage constructs that overcome the limitations of agarose biomaterials, and provide a much-needed alternative to the agarose standard.

Tissue-engineered articular cartilage exhibits tension–compression nonlinearity reminiscent of the native cartilage

The tensile modulus of articular cartilage is much larger than its compressive modulus. This tension-compression nonlinearity enhances interstitial fluid pressurization and decreases the frictional coefficient. The current set of studies examines the tensile and compressive properties of cylindrical chondrocyte-seeded agarose constructs over different developmental stages through a novel method that combines osmotic loading, video microscopy, and uniaxial unconfined compression testing. This method was previously used to examine tension-compression nonlinearity in native cartilage. Engineered cartilage, cultured under free-swelling (FS) or dynamically loaded (DL) conditions, was tested in unconfined compression in hypertonic and hypotonic salt solutions. The apparent equilibrium modulus decreased with increasing salt concentration, indicating that increasing the bath solution osmolarity shielded the fixed charges within the tissue, shifting the measured moduli along the tension-compression curve and revealing the intrinsic properties of the tissue. With this method, we were able to measure the tensile (401±83kPa for FS and 678±473kPa for DL) and compressive (161±33kPa for FS and 348±203kPa for DL) moduli of the same engineered cartilage specimens. These moduli are comparable to values obtained from traditional methods, validating this technique for measuring the tensile and compressive properties of hydrogel-based constructs. This study shows that engineered cartilage exhibits tension-compression nonlinearity reminiscent of the native tissue, and that dynamic deformational loading can yield significantly higher tensile properties.

Analysis of radial variations in material properties and matrix composition of chondrocyte-seeded agarose hydrogel constructs

OBJECTIVE To examine the radial variations in engineered cartilage that may result due to radial fluid flow during dynamic compressive loading. This was done by evaluating the annuli and the central cores of the constructs separately. METHOD Chondrocyte-seeded agarose hydrogels were grown in free-swelling and dynamic, unconfined loading cultures for 42 days. After mechanical testing, constructs were allowed to recover for 1-2h, the central 3mm cores removed, and the cores and annuli were retested separately. Histological and/or biochemical analyses for DNA, glycosaminoglycan (GAG), collagen, type I collagen, type II collagen, and elastin were performed. Multiple regression analysis was used to determine the correlation between the biochemical and material properties of the constructs. RESULTS The cores and annuli of chondrocyte-seeded constructs did not exhibit significant differences in material properties and GAG content. Annuli possessed greater DNA and collagen content over time in culture than cores. Dynamic loading enhanced the material properties and GAG content of cores, annuli, and whole constructs relative to free-swelling controls, but it did not alter the radial variations compared to free-swelling culture. CONCLUSION Surprisingly, the benefits of dynamic loading on tissue properties extended through the entire construct and did not result in radial variations as measured via the coring technique in this study. Nutrient transport limitations and the formation of a fibrous capsule on the periphery may explain the differences in DNA and collagen between cores and annuli. No differences in GAG distribution may be due to sufficient chemical signals and building blocks for GAG synthesis throughout the constructs.

Amino acids supply in culture media is not a limiting factor in the matrix synthesis of engineered cartilage tissue

Summary.Increased amino acid supplementation (0.5×, 1.0×, and 5.0× recommended concentrations or additional proline) was hypothesized to increase the collagen content in engineered cartilage. No significant differences were found between groups in matrix content or dynamic modulus. Control constructs possessed the highest compressive Young’s modulus on day 42. On day 42, compared to controls, decreased type II collagen was found with 0.5×, 1.0×, and 5.0× supplementation and significantly increased DNA content found in 1.0× and 5.0×. No effects were observed on these measures with added proline. These results lead us to reject our hypothesis and indicate that the low collagen synthesis in engineered cartilage is not due to a limited supply of amino acids in media but may require a further stimulatory signal. The results of this study also highlight the impact that culture environment can play on the development of engineered cartilage.

Spatially varying material properties of the rat caudal intervertebral disc.

Study Design. The use of a microscopy based material testing technique to assess the local material properties of rat caudal intervertebral discs under uniaxial compression. Objectives. To better understand the cell environment of rat caudal intervertebral discs during mechanical loading and elucidate better the role of the nucleus pulposus to the overall disc material properties. Summary of Background Data. Rat tail models of disc degeneration have been widely used for their similarity with the degeneration phenomena in human beings. Degenerative patterns in the disc are often inhomogeneous, however, only average material properties of rodent discs have been studied. Knowledge of the spatially varying properties within the disc is necessary to understand the disc cell milieu during tissue loading. Methods. Rat caudal motion segments were tested intact, sectioned, and with alterations of nucleus pulposus using microscopy based techniques. Local displacements and strains were obtained using digital image correlation. Strains and load measurements were used to get the average apparent Young’s modulus, peak stress, local Young’s modulus, and local Poisson’s ratio. Results. There was no difference observed in the average apparent Young’s modulus among experimental groups. Peak stresses decreased significantly when the nucleus pulposus was replaced with extremely fluid-like materials. The axial displacement field showed 3 distinct linear distributions in samples which were sectioned. The center region in all groups had significantly smaller axial strain and showed a higher local Young’s modulus. Conclusions. The average equilibrium Young’s modulus may be dependent on short-range ultrastructural organization. Spatially varying material properties within the intervertebral disc may be caused by orientation of fiber bundles in the different regions of the anulus fibrosus. The fiber bundles are better able to resist compressive loads when oriented parallel rather than perpendicular to the loading direction. At equilibrium, the anulus fibrosus also appears to have a shielding effect independent of the material filling up the nucleus pulposus space.

Chondroitinase-ABC Digestion and Dynamic Loading Increase Tension-Compression Nonlinearity in Tissue-Engineered Cartilage

Native articular cartilage exhibits tension-compression nonlinearity (TCN), where the compressive modulus is lower than its relatively high tensile modulus [1–2]. TCN produces in restricted lateral expansion of the tissue upon axial compression. We previously demnostrated that osmotic swelling can be used to measure the TCN of engineered cartilage by placing the tissue in an initial state of tensile strain. Incremental application of compression can be used to study the tissue’s mechanical properties as it transitions from tension to compression [3]. Although engineered cartilage is able to achieve the Young’s modulus (EY) and glycosaminoglycan (GAG) content of native tissue, the collagen content and dynamic modulus (G*) consistently underperform the native tissue. Removing GAG with chondroitinase ABC (cABC) has been shown to significantly decrease the tissue properties immediately after digestion but the properties rebound, with improved collagen content and G* compared to undigested controls [4]. Furthermore, we have previously shown that cABC digestion significantly increases TCN in engineered cartilage [3]. Dynamic loading (DL) has been shown to significantly increase the mechanical properties without significantly altering biochemical composition of engineered cartilage, however the mechanism through which DL modulates the mechanical strength of engineered cartilage may be due in part to improved extracellular matrix (ECM) organization [5]. We therefore hypothesize that cABC digestion and DL will improve the tensile properties of engineered cartilage.Copyright