Tuning the properties of elastin mimetic hybrid copolymers via a modular polymerization method.

We have synthesized elastin mimetic hybrid polymers (EMHPs) via the step-growth polymerization of azide-functionalized poly(ethylene glycol) (PEG) and alkyne-terminated peptide (AKAAAKA)(2) (AK2) that is abundant in the cross-linking domains of the natural elastin. The modular nature of our synthesis allows facile adjustment of the peptide sequence to modulate the structural and biological properties of EMHPs. Therefore, EMHPs containing cell-binding domains (CBDs) were constructed from α,ω-azido-PEG and two types of alkyne-terminated AK2 peptides with sequences of DGRGX(AKAAAKA)(2)X (AK2-CBD1) and X(AKAAAKA)(2)XGGRGDSPG (AK2-CBD2, X = propargylglycine) via a step-growth, click coupling reaction. The resultant hybrid copolymers contain an estimated five to seven repeats of PEG and AK2 peptides. The secondary structure of EMHPs is sensitive to the specific sequence of the peptidic building blocks, with CBD-containing EMHPs exhibiting a significant enhancement in the α-helical content as compared with the peptide alone. Elastomeric hydrogels formed by covalent cross-linking of the EMHPs had a compressive modulus of 1.06 ± 0.1 MPa. Neonatal human dermal fibroblasts (NHDFs) were able to adhere to the hydrogels within 1 h and to spread and develop F-actin filaments 24 h postseeding. NHDF proliferation was only observed on hydrogels containing RGDSP domains, demonstrating the importance of integrin engagement for cell growth and the potential use of these EMHPs as tissue engineering scaffolds. These cell-instructive, hybrid polymers are promising candidates as elastomeric scaffolds for tissue engineering.

Sprifermin treatment enhances cartilage integration in an in vitro repair model.

Cartilage integration remains a clinical challenge for treatment of focal articular defects. Cartilage exhibits limited healing capacity that declines with tissue maturation. Many approaches have been investigated for their ability to stimulate healing of mature cartilage or integration of repair tissue or tissue-engineered constructs with native cartilage. Growth factors present in immature tissue may enhance chondrogenesis and promote integrative repair of cartilage defects. In this study, we assessed the role of one such factor, fibroblast growth factor 18 (FGF18). Studies using FGF18 have shown a variety of positive effects on cartilage, including stimulation of chondrocyte proliferation, matrix biosynthesis, and suppression of proteinase activity. To explore the role of FGF18 on cartilage defect repair, we hypothesized that treatment with recombinant human FGF18 (sprifermin) would increase matrix synthesis in a defect model, thus improving integration strength. To test this hypothesis, 6 mm cartilage cylinders were harvested from juvenile bovine knees. A central 3 mm defect was created in each explant, and this core was removed and replaced. Resulting constructs were cultured in control or sprifermin-containing medium (weekly 24-h exposure of 100 ng/ml sprifermin) for 4 weeks. Mechanical testing, biochemical analysis, micro-CT, scanning electron microscopy, and histology were used to assess matrix production, adhesive strength, and structural properties of the cartilage-cartilage interface. Results showed greater adhesive strength, increased collagen content, and larger contact areas between core and annular cartilage in the sprifermin-treated group. These findings present a novel treatment for cartilage injuries that have potential to enhance defect healing and lateral cartilage-cartilage integration. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2648-2656, 2018.

Time-dependent functional maturation of scaffold-free cartilage tissue analogs.

One of the most critical parameters in cartilage tissue engineering which influences the clinical success of a repair therapy is the ability to match the load-bearing capacity of the tissue as it functions in vivo. While mechanical forces are known to positively influence the development of cartilage matrix architecture, these same forces can induce long-term implant failure due to poor integration or structural deficiencies. As such, in the design of optimal repair strategies, it is critical to understand the timeline of construct maturation and how the elaboration of matrix correlates with the development of mechanical properties. We have previously characterized a scaffold-free method to engineer cartilage utilizing primary chondrocytes cultured at high density in hydrogel-coated culture vessels to promote the formation of a self-aggregating cell suspension that condenses to form a cartilage-like biomass, or cartilage tissue analog (CTA). Chondrocytes in these CTAs maintain their cellular phenotype and deposit extracellular matrix to form a construct that has characteristics similar to native cartilage; however, the mechanical integrity of CTAs had not yet been evaluated. In this study, we found that chondrocytes within CTAs produced a robust matrix of proteoglycans and collagen that correlated with increasing mechanical properties and decreasing cell-matrix ratios, leading to properties that approached that of native cartilage. These results demonstrate a unique approach to generating a cartilage-like tissue without the complicating factor of scaffold, while showing increased compressive properties and matrix characteristics consistent with other approaches, including scaffold-based constructs. To further improve the mechanics of CTAs, studies are currently underway to explore the effect of hydrodynamic loading and whether these changes would be reflective of in vivo maturation in animal models. The functional maturation of cartilage tissue analogs as described here support this engineered cartilage model for use in clinical and experimental applications for repair and regeneration in joint-related pathologies.

Design and characterization of a dynamic vibrational culture system.

To engineer a functional vocal fold tissue, the mechanical environment of the native tissue needs to be emulated in vitro. We have created a dynamic culture system capable of generating vibratory stimulations at human phonation frequencies. The novel device is composed of a function generator, a power amplifier, an enclosed loudspeaker and a circumferentially-anchored silicone membrane. The vibration signals are translated to the membrane aerodynamically by the oscillating air pressure underneath. The vibration profiles detected on the membrane were symmetrical relative to the centre of the membrane as well as the resting position over the range of frequencies (60-300 Hz) and amplitudes tested (1-30 µm). The oscillatory motion of the membrane gave rise to two orthogonal, in-plane strain components that are similar in magnitude (0.47%) and are strong functions of membrane thickness. Neonatal foreskin fibroblasts (NFFs) attached to the membrane were subjected to a 1 h vibration at 60, 110 and 300 Hz, with the displacement at the centre of the membrane varying in the range 1-30 µm, followed by a 6 h rest. These regimens did not cause morphological changes to the cells. An increase in cell proliferation was detected when NFFs were driven into oscillation at 110 Hz with a normal displacement of 30 µm. qPCR results showed that the expression of genes encoding some extracellular matrix proteins was altered in response to changes in vibratory frequency and amplitude. The dynamic culture device provides a potentially useful in vitro platform for evaluating cellular responses to vibration.

High-frequency viscoelastic shear properties of vocal fold tissues: implications for vocal fold tissue engineering.

The biomechanical function of the vocal folds (VFs) depends on their viscoelastic properties. Many conditions can lead to VF scarring that compromises voice function and quality. To identify candidate replacement materials, the structure, composition, and mechanical properties of native tissues need to be understood at phonation frequencies. Previously, the authors developed the torsional wave experiment (TWE), a stress-wave-based experiment to determine the linear viscoelastic shear properties of small, soft samples. Here, the viscoelastic properties of porcine and human VFs were measured over a frequency range of 10-200 Hz. The TWE utilizes resonance phenomena to determine viscoelastic properties; therefore, the specimen test frequency is determined by the sample size and material properties. Viscoelastic moduli are reported at resonance frequencies. Structure and composition of the tissues were determined by histology and immunochemistry. Porcine data from the TWE are separated into two groups: a young group, consisting of fetal and newborn pigs, and an adult group, consisting of 6-9-month olds and 2+-year olds. Adult tissues had an average storage modulus of 2309±1394 Pa and a loss tangent of 0.38±0.10 at frequencies of 36-200 Hz. The VFs of young pigs were significantly more compliant, with a storage modulus of 394±142 Pa and a loss tangent of 0.40±0.14 between 14 and 30 Hz. No gender dependence was observed. Histological staining showed that adult porcine tissues had a more organized, layered structure than the fetal tissues, with a thicker epithelium and a more structured lamina propria. Elastin fibers in fetal VF tissues were immature compared to those in adult tissues. Together, these structural changes in the tissues most likely contributed to the change in viscoelastic properties. Adult human VF tissues, recovered postmortem from adult patients with a history of smoking or disease, had an average storage modulus of 756±439 Pa and a loss tangent of 0.42±0.10. Contrary to the results of some other investigators, no significant frequency dependence was observed. This lack of observable frequency dependence may be due to the modest frequency range of the experiments and the wide range of stiffnesses observed within nominally similar sample types.

Effects of matrix composition, microstructure, and viscoelasticity on the behaviors of vocal fold fibroblasts cultured in three-dimensional hydrogel networks.

Vocal fold diseases and disorders are difficult to treat surgically or therapeutically. Tissue engineering offers an alternative strategy for the restoration of functional vocal folds. As a first step toward vocal fold tissue engineering, we investigated the responses of primary vocal fold fibroblasts (PVFFs) to two types of collagen and hyaluronic acid (HA)-based hydrogels that are compositionally similar, but structurally variable and mechanically different. Type A hydrogels were composed of mature collagen fibers reinforced by oxidized HA, whereas type B hydrogels contained immature collagen fibrils interpenetrated in an amorphous, covalently cross-linked HA matrix. PVFFs encapsulated in either matrix adopted a fibroblastic morphology and expressed genes related to important extracellular matrix proteins. DNA analysis indicated a linear growth profile for cells encapsulated in type B gels from day 0 to 21, in contrast to an initial dormant, nonproliferative period from day 0 to 3 experienced by cells in type A gels. At the end of the culture, similar DNA content was detected in both types of constructs. A reduction in collagen content was observed for both types of constructs after 28 days of culture, with type A constructs generally retaining higher amounts of collagen than type B constructs. The HA content in the constructs decreased steadily throughout the culture, with type A constructs consistently exhibiting less HA than type B constructs. Using the torsional wave analysis, we found that the elastic moduli for type A constructs decreased sharply during the first week of culture, followed by 2 weeks of matrix stabilization without significant changes in matrix stiffness. Conversely, the elastic modulus for type B constructs increased moderately over time. It is postulated that PVFFs residing in gels alter the matrix organization, chemical compositions, and viscoelasticity through cell-mediated remodeling processes.

Synthesis and Characterization of Elastin-Mimetic Hybrid Polymers with Multiblock, Alternating Molecular Architecture and Elastomeric Properties.

We are interested in developing elastin-mimetic hybrid polymers (EMHPs) that capture the multiblock molecular architecture of tropoelastin as well as the remarkable elasticity of mature elastin. In this study, multiblock EMHPs containing flexible synthetic segments based on poly(ethylene glycol) (PEG) alternating with alanine-rich, lysine-containing peptides were synthesized by step-growth polymerization using α,ω-azido-PEG and alkyne-terminated AKA(3)KA (K = lysine, A = alanine) peptide, employing orthogonal click chemistry. The resulting EMHPs contain an estimated three to five repeats of PEG and AKA(3)KA and have an average molecular weight of 34 kDa. While the peptide alone exhibited α-helical structures at high pH, the fractional helicity for EMHPs was reduced. Covalent cross-linking of EMHPs with hexamethylene diisocyanate (HMDI) through the lysine residue in the peptide domain afforded an elastomeric hydrogel (xEMHP) with a compressive modulus of 0.12 MPa when hydrated. The mechanical properties of xEMHP are comparable to a commercial polyurethane elastomer (Tecoflex SG80A) under the same conditions. In vitro toxicity studies showed that while the soluble EMHPs inhibited the growth of primary porcine vocal fold fibroblasts (PVFFs) at concentrations ≥0.2 mg/mL, the cross-linked hybrid elastomers did not leach out any toxic reagents and allowed PVFFs to grow and proliferate normally. The hybrid and modular approach provides a new strategy for developing elastomeric scaffolds for tissue engineering.