Metabolic Labeling to Probe the Spatiotemporal Accumulation of Matrix at the Chondrocyte-Hydrogel Interface.

Hydrogels are engineered with biochemical and biophysical signals to recreate aspects of the native microenvironment and to control cellular functions such as differentiation and matrix deposition. This deposited matrix accumulates within the pericellular space and likely affects the interactions between encapsulated cells and the engineered hydrogel; however, there has been little work to study the spatiotemporal evolution of matrix at this interface. To address this, metabolic labeling is employed to visualize the temporal and spatial positioning of nascent proteins and proteoglycans deposited by chondrocytes. Within covalently crosslinked hyaluronic acid hydrogels, chondrocytes deposit nascent proteins and proteoglycans in the pericellular space within 1 d after encapsulation. The accumulation of this matrix, as measured by an increase in matrix thickness during culture, depends on the initial hydrogel crosslink density with decreased thicknesses for more crosslinked hydrogels. Encapsulated fluorescent beads are used to monitor the hydrogel location and indicate that the emerging nascent matrix physically displaces the hydrogel from the cell membrane with extended culture. These findings suggest that secreted matrix increasingly masks the presentation of engineered hydrogel cues and may have implications for the design of hydrogels in tissue engineering and regenerative medicine.

3D bioprinting via an in situ crosslinking technique towards engineering cartilage tissue.

3D bioprinting is a promising approach for the repair of cartilage tissue after damage due to injury or disease; however, the design of 3D printed scaffolds has been limited by the availability of bioinks with requisite printability, cytocompatibility, and bioactivity. To address this, we developed an approach termed in situ crosslinking that permits the printing of non-viscous, photocrosslinkable bioinks via the direct-curing of the bioink with light through a photopermeable capillary prior to deposition. Using a norbornene-modified hyaluronic acid (NorHA) macromer as a representative bioink and our understanding of thiol-ene curing kinetics with visible light, we varied the printing parameters (e.g., capillary length, flow rate, light intensity) to identify printing conditions that were optimal for the ink. The printing process was cytocompatible, with high cell viability and homogenous distribution of mesenchymal stromal cells (MSCs) observed throughout printed constructs. Over 56 days of culture in chondrogenic media, printed constructs increased in compressive moduli, biochemical content (i.e., sulfated glycosaminoglycans, collagen), and histological staining of matrix associated with cartilage tissue. This generalizable printing approach may be used towards the repair of focal defects in articular cartilage or broadly towards widespread biomedical applications across a range of photocrosslinkable bioinks that can now be printed.

Influence of hyaluronic acid modification on CD44 binding towards the design of hydrogel biomaterials.

Hyaluronic acid (HA) is a linear polysaccharide of d-glucuronic acid and N-acetyl-d-glucosamine that is native to many tissues and interacts with cells via cell-surface receptors (e.g., CD44). HA has been extensively explored as a chemically-modified macromer for crosslinking into biomaterials, such as hydrogels and macroporous scaffolds. However, the influence of the extent and type of HA modification on its binding to CD44 is not well understood or quantified. To address this, we modified HA at either the carboxylic acid or the primary alcohol with various chemical groups (e.g., norbornenes, methacrylates) and magnitudes (~10, 20, or 40% of disaccharides) and then characterized binding in both soluble and hydrogel forms. HA binding to CD44 immobilized on plates or presented by cells was influenced by the extent and type of its modification, where increased modification (i.e., ~40%) generally decreased binding. The adhesion of CD44-modified beads to hydrogels as measured by atomic force microscopy revealed a similar trend, particularly with decreased adhesion with hydrophobic modifications to the carboxylic acid. Further, the chondrogenesis of mesenchymal stromal cells when encapsulated in hydrogels fabricated from modified HA macromers was reduced at high modification, behaving similarly to inert hydrogel controls. This work suggests that the types and extents of modification of polysaccharides are important factors that should be considered in preserving their biological function when processed as hydrogels.

Gallol-derived ECM-mimetic adhesive bioinks exhibiting temporal shear-thinning and stabilization behavior.

3D bioprinting is an attractive technique to fabricate well-organized, cell-laden constructs for tissue repair and disease modeling. Although numerous hydrogel bioinks have been developed, materials are still needed that mimic the cellular microenvironment, have the appropriate viscosity and stabilization for printing, and are cytocompatible. Here, we present a unique gallol-modified extracellular matrix (ECM) hydrogel ink that is inspired by rapid fruit browning phenomena. The gallol-modification of ECM components (e.g., hyaluronic acid, gelatin) allowed (i) immediate gelation and shear-thinning properties by dynamic hydrogen bonds on short time-scales and (ii) further auto-oxidation and covalent crosslinking for stabilization on longer time-scales. The gallol ECM hydrogel ink was printable using an extrusion-based 3D printer by exploiting temporal shear-thinning properties, and further showed cytocompatibility (∼95% viability) and on-tissue printability due to adhesiveness of gallol moieties. Printed cell-laden filaments degraded and swelled with culture over 6 days, corresponding to increases in cell density and spreading. Ultimately, this strategy is useful for designing hydrogel inks with tunable properties for 3D bioprinting. STATEMENT OF SIGNIFICANCE: 3D bioprinting is a promising technique for the fabrication of cell-laden constructs for applications as in vitro models or for therapeutic applications. Despite the previous development of numerous hydrogel bioinks, there still remain challenging considerations in the design of bioinks. In this study, we present a unique cytocompatible hydrogel ink with gallol modification that is inspired by rapid fruit browning phenomena. The gallol hydrogel ink has three important properties: i) it shows immediate gelation by dynamic, reversible bonds for shear-thinning extrusion, ii) it allows spontaneous stabilization by subsequent covalent crosslinking after printing, and iii) it is printable on tissues by adhesive properties of gallol moieties. As such, this work presents a new approach in the design of hydrogel inks.

Combinatorial hydrogels with biochemical gradients for screening 3D cellular microenvironments.

3D microenvironmental parameters control cell behavior, but can be challenging to investigate over a wide range of conditions. Here, a combinatorial hydrogel platform is developed that uses light-mediated thiol-norbornene chemistry to encapsulate cells within hydrogels with biochemical gradients made by spatially varied light exposure. Specifically, mesenchymal stem cells are photoencapsulated in norbornene-modified hyaluronic acid hydrogels functionalized with gradients (0-5 mM) of peptides that mimic cell-cell or cell-matrix interactions, either as single or orthogonal gradients. Chondrogenesis varied spatially in these hydrogels based on the local biochemical formulation, as indicated by Sox9 and aggrecan expression levels. From 100 combinations investigated, discrete hydrogels are formulated and early gene expression and long-term cartilage-specific matrix production are assayed and found to be consistent with screening predictions. This platform is a scalable, high-throughput technique that enables the screening of the effects of multiple biochemical signals on 3D cell behavior.

Dose and Timing of N-Cadherin Mimetic Peptides Regulate MSC Chondrogenesis within Hydrogels.

The transmembrane glycoprotein N-cadherin (NCad) mediates cell-cell interactions found during mesenchymal condensation and chondrogenesis. Here, NCad-derived peptides (i.e., HAV) are incorporated into hyaluronic acid (HA) hydrogels with encapsulated mesenchymal stem cells (MSCs). Since the dose and timing of NCad signaling are dynamic, HAV peptide presentation is tuned via alterations in peptide concentration and incorporation of an ADAM10-cleavable domain between the hydrogel and the HAV motif, respectively. HA hydrogels functionalized with HAV result in dose-dependent increases in early chondrogenesis of encapsulated MSCs and resultant cartilage matrix production. For example, type II collagen and glycosaminoglycan production increase ≈9- and 2-fold with the highest dose of HAV (i.e., 2 × 10-3 m), respectively, when compared to unmodified hydrogels, while incorporation of an efficient ADAM10-cleavable domain between the HAV peptide and hydrogel abolishes increases in chondrogenesis and matrix production. Treatment with a small-molecule ADAM10 inhibitor restores the functional effect of the HAV peptide, indicating that timing and duration of HAV peptide presentation is crucial for robust chondrogenesis. This study demonstrates a nuanced approach to the biofunctionalization of hydrogels to better emulate the complex cell microenvironment during embryogenesis toward stem-cell-based cartilage production.

* Thermosensitive Poly(N-vinylcaprolactam) Injectable Hydrogels for Cartilage Tissue Engineering.

Injectable hydrogels have gained prominence in the field of tissue engineering for minimally invasive delivery of cells for tissue repair and in the filling of irregular defects. However, many injectable hydrogels exhibit long gelation times or are not stable for long periods after injection. To address these concerns, we used thermosensitive poly(N-vinylcaprolactam) (PNVCL) hydrogels due to their cytocompatibility and fast response to temperature stimuli. Changes in the PNVCL molecular weight and concentration enabled the development of hydrogels with tunable mechanical properties and fast gelation times (<60 s when the temperature was raised from room temperature to physiologic temperature). Chondrocytes (CHs) and mesenchymal stem cells were encapsulated in PNVCL hydrogels and exhibited high viability (∼90%), as monitored by Live/Dead staining and Alamar Blue assays. Three-dimensional constructs of CH-laden PNVCL hydrogels supported cartilage-specific extracellular matrix production both in vitro and after subcutaneous injection in nude rats for up to 8 weeks. Moreover, biochemical analyses of constructs demonstrated a time-dependent increase in glycosaminoglycans (GAGs) and collagen, which were significantly augmented in the implants cultured in vivo. Histological analyses also demonstrated regular distribution of synthesized cartilage components, including abundant GAGs and type II collagen. The findings from this study demonstrate thermosensitive PNVCL as a candidate injectable biomaterial to deliver cells for cartilage tissue engineering.

Three-dimensional transesophageal echocardiography for determination of the mitral valve area after mitral valve repair surgery for mitral stenosis.

BACKGROUND: Pressure half-time (PHT) method is usually unreliable for accurate determination of mitral valve area (MVA) immediately after surgical intervention of mitral stenosis (MS). The planimetry method using three-dimensional (3D) transesophageal echocardiography (3D-planimetery method) could enhance accurate determination of the intraoperative MVA. Authors investigated the efficacy of 3D-planimetry method in determining MVA immediately after mitral valve repair procedure (MVRep) for severe mitral stenosis (MS). METHODS: In severe MS patients undergoing elective MVRep (N.=41), intraoperative MVAs were determined by using PHT-method and 3D-planimetry method before and immediately after cardiopulmonary bypass (pre- and post-MVAPHT, and -MVA3D-planimetry). MVAs were also determined by using multi-detector computed tomographic scan (MDCT) before MVRep and within 7 days after MVRep (pre- and post-MVACT). MVAs determined by using three different methods were analysed. RESULTS: Mitral inflow pressure gradient (median [25th-75th percentile]) was significantly reduced after MVRep (3.0 [2.0-4.0] vs. 7.0 [6.0-9.0] mmHg; P<0.001). Pre-MVAPHT, pre-MVA3D-planimetry and preop-MVACT (mean [95% confidence interval]) did not differ significantly (1.08 [1.00-1.05], 1.08 [0.98-1.08], and 1.14 [1.07-1.22] cm2, respectively), but post-MVA3D-planimetry and post-MVACT (2.22 [2.07-2.36] and 2.31 [2.07-2.36] cm2, respectively) were significantly larger than post-MVAPHT (1.98 [1.83-2.13] cm2; P=0.007 and P<0.001, respectively). The correlation coefficient between post-MVA3D-planimetry and post-MVACT (0.59, P<0.01) was greater than that between post-MVAPHT and post-MVACT (0.39, P=0.01). CONCLUSIONS: These results support the clinical efficacy of 3D-planimetry for accurate evaluation of the MVA immediately after MVRep for severe MS, as a valuable alternative to PHT-method which usually underestimates MVA during this period.

Fibrous Scaffolds with Varied Fiber Chemistry and Growth Factor Delivery Promote Repair in a Porcine Cartilage Defect Model.

Current clinically approved methods for cartilage repair are generally based on either endogenous cell recruitment (e.g., microfracture) or chondrocyte delivery (e.g., autologous chondrocyte implantation). However, both methods culminate in repair tissue with inferior mechanical properties and the addition of biomaterials to these clinical interventions may improve their efficacy. To this end, the objective of this study was to investigate the ability of multipolymer acellular fibrous scaffolds to improve cartilage repair when combined with microfracture in a large animal (i.e., minipig) model. Composite scaffolds were formulated from a combination of hyaluronic acid (HA) fibers and poly(ɛ-caprolactone) (PCL) fibers, either with or without transforming growth factor-ß3 (TGFß3). After 12 weeks in vivo, material choice and TGFß3 delivery had a significant impact on outcomes; specifically, PCL scaffolds without TGFß3 had inferior gross appearance and reduced mechanical properties, whereas HA scaffolds that released TGFß3 resulted in improved histological scores and increased type 2 collagen content. Importantly, analysis of the overall dataset revealed that histology, but not gross appearance, was a better predictor of mechanical properties. This study highlights the importance of scaffold properties on in vivo cartilage repair as well as the need for numerous quantitative outcome measures to fully evaluate treatment methods.