A novel 3D vascular assay for evaluating angiogenesis across porous membranes.

Microfluidic technology has been extensively applied to model the functional units of human organs and tissues. Since vasculature is a key component of any functional tissue, a variety of techniques to mimic vasculature in vitro have been developed to address complex physiological and pathological processes in 3D tissues. Herein, we developed a novel, in vitro, microfluidic-based model to probe microvasculature growth into and across implanted porous membranes. Using ePTFE and polycarbonate as examples, we characterize the vascularization potential of these thin porous membranes using this device. This tool will allow for the assessment of porous materials early in their development, prior to their use for encapsulating implants or drugs, while minimizing the need for animal studies. Employing quantitative morphometric analysis and measurements of vascular permeability, we demonstrate our model to be an effective platform for evaluation of angiogenic potential of an implanted membrane biomaterial. Results show that endothelial cells can either migrate as single cells or form continuous sprouts across porous membranes, which is a material structure-dependent behavior. Our model is advantageous over conventional Transwell assays as it is amenable to quantitative assessment of vascular sprouting in 3D, and in contrast to animal models it can be employed more efficiently and with real-time assessment capabilities. This new tool could be applied either to test the suitability of a wide range of biomaterials for implantation or to screen different pro-angiogenic factors for therapeutic applications, and will advance the design of new biomaterials.

In situ formation of poly(vinyl alcohol)-heparin hydrogels for mild encapsulation and prolonged release of basic fibroblast growth factor and vascular endothelial growth factor.

Heparin-based hydrogels are attractive for controlled growth factor delivery, due to the native ability of heparin to bind and stabilize growth factors. Basic fibroblast growth factor and vascular endothelial growth factor are heparin-binding growth factors that synergistically enhance angiogenesis. Mild, in situ encapsulation of both basic fibroblast growth factor and vascular endothelial growth factor and subsequent bioactive dual release has not been demonstrated from heparin-crosslinked hydrogels, and the combined long-term delivery of both growth factors from biomaterials is still a major challenge. Both basic fibroblast growth factor and vascular endothelial growth factor were encapsulated in poly(vinyl alcohol)-heparin hydrogels and demonstrated controlled release. A model cell line, BaF32, was used to show bioactivity of heparin and basic fibroblast growth factor released from the gels over multiple days. Released basic fibroblast growth factor promoted higher human umbilical vein endothelial cell outgrowth over 24 h and proliferation for 3 days than the poly(vinyl alcohol)-heparin hydrogels alone. The release of vascular endothelial growth factor from poly(vinyl alcohol)-heparin hydrogels promoted human umbilical vein endothelial cell outgrowth but not significant proliferation. Dual-growth factor release of basic fibroblast growth factor and vascular endothelial growth factor from poly(vinyl alcohol)-heparin hydrogels resulted in a synergistic effect with significantly higher human umbilical vein endothelial cell outgrowth compared to basic fibroblast growth factor or vascular endothelial growth factor alone. Poly(vinyl alcohol)-heparin hydrogels allowed bioactive growth factor encapsulation and provided controlled release of multiple growth factors which is beneficial toward tissue regeneration applications.

A comparative study of enzyme initiators for crosslinking phenol-functionalized hydrogels for cell encapsulation.

BACKGROUND: Dityrosine crosslinking in proteins is a bioinspired method of forming hydrogels. This study compares oxidative enzyme initiators for their relative crosslinking efficiency and cytocompatibility using the same phenol group and the same material platform. Four common enzyme and enzyme-like oxidative initiators were probed for resulting material properties and cell viability post-encapsulation. RESULTS: All four initiators can be used to form phenol-crosslinked hydrogels, however gelation rates are dependent on enzyme type, concentration, and the oxidant. Horseradish peroxidase (HRP) or hematin with hydrogen peroxide led to a more rapid poly (vinyl alcohol)-tyramine (PVA-Tyr) polymerization (10-60 min) because a high oxidant concentration was dissolved within the macromer solution at the onset of crosslinking, whereas laccase and tyrosinase require oxygen diffusion to crosslink phenol residues and therefore took longer to gel (2.5+ hours). The use of hydrogen peroxide as an oxidant reduced cell viability immediately post-encapsulation. Laccase- and tyrosinase-mediated encapsulation of cells resulted in higher cell viability immediately post-encapsulation and significantly higher cell proliferation after one week of culture. CONCLUSIONS: Overall this study demonstrates that HRP/H2O2, hematin/H2O2, laccase, and tyrosinase can create injectable, in situ phenol-crosslinked hydrogels, however oxidant type and concentration are critical parameters to assess when phenol crosslinking hydrogels for cell-based applications.

Promoting Cell Survival and Proliferation in Degradable Poly(vinyl alcohol)-Tyramine Hydrogels.

A photopolymerizable-tyraminated poly(vinyl alcohol) (PVA-Tyr) system that has the ability to covalently bind proteins in their native state was evaluated as a platform for cell encapsulation. However, a key hurdle to this system is the radicals generated during the cross-linking that can cause oxidative stress to the cells. This research hypothesized that incorporation of anti-oxidative proteins (sericin and gelatin) into PVA-Tyr gels would mitigate any toxicity caused by the radicals. The results showed that although incorporation of 1 wt% sericin promoted survival of the fibroblasts, both sericin and gelatin acted synergistically to facilitate long-term 3D cell function. The encapsulated cells formed clusters with deposition of laminin and collagen, as well as remaining metabolically active after 21 d.

Copolymerization of an indazole ligand into the self-polymerization of dopamine for enhanced binding with metal ions.

5,6-Dihydroxy-1H-indazole (DHI) is able to self-polymerize through the same mussel-inspired chemistry responsible for generating poly(dopamine) (PDA), demonstrating the potential to expand this class of catecholamine-exclusive chemistry onto heterocyclic catechol derivatives for the preparation of functional materials. Although DHI exhibits slower polymerization kinetics compared to dopamine, the two chemical species are compatibly polymerizable under the same reaction conditions and allow the preparation of copolymer coatings in different molar ratios. Of these copolymers, the 1 : 3-copolymer (DHI-to-dopamine ratio) has demonstrated adequate structural stability as a polymer coating. While PDA performs as an intact framework, the incorporated DHI enhances the colloidal stability and provides additional coordinating functionality through the pyrazole moieties. The 1 : 3-copolymer was fabricated into polymer capsules which exhibit negligible cytotoxicity towards murine dermal fibroblasts (L929) and enhanced binding behaviour towards copper(ii). This represents a new channel for fabricating cargo carriers for biomedical applications that involve the use of transition metal-based species.

Tailoring Stimuli Responsiveness using Dynamic Covalent Cross-Linking of Poly(vinyl alcohol)-Heparin Hydrogels for Controlled Cell and Growth Factor Delivery.

Heparin-based hydrogels are attractive for cell encapsulation and drug delivery because of the ability of heparin to bind native proteins. However, heparin-based hydrogels have received little attention for their potential as stimuli-sensitive materials. Biosynthetic, poly(vinyl alcohol) (PVA)-heparin hydrogels were formed using dynamic, covalent cross-linking. Hydrogel stimuli-sensitivity was tailored by tuning the concentration of heparin to PVA. Relatively thermally and pH stable hydrogels were produced when formed from only the synthetic, nonionic PVA polymer cross-linked via hydrazone bonds. Cross-linking in the ionic biopolymer heparin, to form PVA-heparin gels, has a profound impact on thermal stability, with degradation ranging from over 6 months to only 4 days across 25-50 °C. PVA-heparin hydrogels degrade within 18 days at basic pH (10), while not fully degrading over 6 months at lower pH (4, 7.4). This finding is attributed to the anionic repulsion of carboxyls and sulfates in heparin. PVA-heparin macromers were cytocompatible and enabled mild cell encapsulation, in addition to providing pH-controlled growth factor release. Overall, it is demonstrated that the biopolymer heparin can be used to create pH and temperature-responsive hydrogel biomaterials for cell and drug delivery.

Interaction of hyaluronan binding peptides with glycosaminoglycans in poly(ethylene glycol) hydrogels.

This study investigates the incorporation of hyaluronan (HA) binding peptides into poly(ethylene glycol) (PEG) hydrogels as a mechanism to bind and retain hyaluronan for applications in tissue engineering. The specificity of the peptide sequence (native RYPISRPRKRC vs non-native RPSRPRIRYKC), the role of basic amino acids, and specificity to hyaluronan over other GAGs in contributing to the peptide-hyaluronan interaction were probed through experiments and simulations. Hydrogels containing the native or non-native peptide retained hyaluronan in a dose-dependent manner. Ionic interactions were the dominating mechanism. In diH2O the peptides interacted strongly with HA and chondroitin sulfate, but in phosphate buffered saline the peptides interacted more strongly with HA. For cartilage tissue engineering, chondrocyte-laden PEG hydrogels containing increasing amounts of HA binding peptide and exogenous HA had increased retention and decreased loss of cell-secreted proteoglycans in and from the hydrogel at 28 days. This new matrix-interactive hydrogel platform holds promise for tissue regeneration.

Comparison of photopolymerizable thiol-ene PEG and acrylate-based PEG hydrogels for cartilage development.

When designing hydrogels for tissue regeneration, differences in polymerization mechanism and network structure have the potential to impact cellular behavior. Poly(ethylene glycol) hydrogels were formed by free-radical photopolymerization of acrylates (chain-growth) or thiol-norbornenes (step-growth) to investigate the impact of hydrogel system (polymerization mechanism and network structure) on the development of engineered tissue. Bovine chondrocytes were encapsulated in hydrogels and cultured under free swelling or dynamic compressive loading. In the acrylate system immediately after encapsulation chondrocytes exhibited high levels of intracellular ROS concomitant with a reduction in hydrogel compressive modulus and higher variability in cell deformation upon compressive strain; findings that were not observed in the thiol-norbornene system. Long-term the quantity of sulfated glycosaminoglycans and total collagen was greater in the acrylate system, but the quality resembled that of hypertrophic cartilage with positive staining for aggrecan, collagens I, II, and X and collagen catabolism. The thiol-norbornene system led to hyaline-like cartilage production especially under mechanical loading with positive staining for aggrecan and collagen II and minimal staining for collagens I and X and collagen catabolism. Findings from this study confirm that the polymerization mechanism and network structure have long-term effects on the quality of engineered cartilage, especially under mechanical loading.

Three dimensional live cell lithography.

We investigate holographic optical trapping combined with step-and-repeat maskless projection stereolithography for fine control of 3D position of living cells within a 3D microstructured hydrogel. C2C12 myoblast cells were chosen as a demonstration platform since their development into multinucleated myotubes requires linear arrangements of myoblasts. C2C12 cells are positioned in the monomer solution with multiple optical traps at 1064 nm and then encapsulated by photopolymerization of monomer via projection of a 512x512 spatial light modulator illuminated at 405 nm. High 405 nm sensitivity and complete insensitivity to 1064 nm was enabled by a lithium acylphosphinate (LAP) salt photoinitiator. These wavelengths, in addition to brightfield imaging with a white light LED, could be simultaneously focused by a single oil immersion objective. Large lateral dimensions of the patterned gel/cell structure are achieved by x and y step-and-repeat process. Large thickness is achieved through multi-layer stereolithography, allowing fabrication of precisely-arranged 3D live cell scaffolds with micron-scale structure and millimeter dimensions. Cells are shown to retain viability after the trapping and encapsulation procedure.

Comparative study of the viscoelastic mechanical behavior of agarose and poly(ethylene glycol) hydrogels.

This study presents a comparative investigation into differences in the mechanical properties between two hydrogels commonly used in cartilage tissue engineering [agarose vs. poly(ethylene glycol) (PEG)], but which are formed through distinctly different crosslinking mechanisms (physical vs. covalent, respectively). The effects of hydrogel chemistry, precursor concentration, platen type (nonporous vs. porous) used in compression bioreactors, and degradation (for PEG) on the swelling properties and static and dynamic mechanical properties were examined. An increase in precursor concentration resulted in decreased equilibrium mass swelling ratios but increased equilibrium moduli and storage moduli for both hydrogels (p < 0.05). Agarose displayed large stress relaxations and a frequency dependence indicating its viscoelastic properties. Contrarily, PEG hydrogels displayed largely elastic behavior with minimal stress relaxation and frequency dependence. In biodegradable PEG hydrogels, the largely elastic behavior was retained during degradation. The type of platen did not affect static mechanical properties, but porous platens led to a reduced storage modulus for both hydrogels implicating fluid flow. In summary, agarose and PEG exhibit vastly different mechanical behaviors; a finding largely attributed to differences in their chemistries and fluid movement. Taken together, these design choices (hydrogel chemistry/structure, loading conditions) will likely have a profound effect on the tissue engineering outcome.

Incorporation of biomimetic matrix molecules in PEG hydrogels enhances matrix deposition and reduces load-induced loss of chondrocyte-secreted matrix.

Poly(ethylene glycol) (PEG) hydrogels offer numerous advantages in designing controlled 3D environments for cartilage regeneration, but offer little biorecognition for the cells. Incorporating molecules that more closely mimic the native tissue may provide key signals for matrix synthesis and may also help in the retention of neotissue, particularly when mechanical stimulation is employed. Therefore, this research tested the hypothesis that exogenous hyaluronan encapsulated within PEG hydrogels improves tissue deposition by chondrocytes, while the incorporation of Link-N (DHLSDNYTLDHDRAIH), a fragment of link protein that is involved in stabilizing hyaluronan and aggrecan in cartilage, aids in the retention of the entrapped hyaluronan as well as cell-secreted glycosaminoglycans (GAGs), particularly when dynamic loading is employed. The incorporation of Link-N as covalent tethers resulted in a significant reduction, ~60%, in the loss of entrapped exogenous hyaluronan under dynamic stimulation. When chondrocytes were encapsulated in PEG hydrogels containing exogenous hyaluronan and/or Link-N, the extracellular matrix (ECM) analogs aided in the retention of cell-secreted GAGs under loading. The presence of hyaluronan led to enhanced deposition of collagen type II and aggrecan. In conclusion, our results highlight the importance of ECM analogs, specifically hyaluronan and Link-N, in matrix retention and matrix development and offer new strategies for designing scaffolds for cartilage regeneration.

Degradation improves tissue formation in (un)loaded chondrocyte-laden hydrogels.

BACKGROUND: Photopolymerizable poly(ethylene glycol) (PEG) hydrogels offer a platform to deliver cells in vivo and support three-dimensional cell culture but should be designed to degrade in sync with neotissue development and endure the physiologic environment. QUESTIONS/PURPOSES: We asked whether (1) incorporation of degradation into PEG hydrogels facilitates tissue development comprised of essential cartilage macromolecules; (2) with early loading before pericellular matrix formation, the duration of load affects matrix production; and (3) dynamic loading in general influences macroscopic tissue development. METHODS: Primary bovine chondrocytes were encapsulated in hydrogels (n = 3 for each condition). The independent variables were hydrogel degradation (nondegrading PEG and degrading oligo(lactic acid)-b-PEG-b-oligo(lactic acid) [PEG-LA]), culture condition (free swelling, unconfined dynamic compressive loading applied intermittently for 1 or 4 weeks), and time (up to 28 days). The dependent variables were neotissue deposition through biochemical contents, immunohistochemistry, and compressive modulus. RESULTS: Degradation led to 2.3- and 2.9-fold greater glycosaminoglycan and collagen contents, respectively; macroscopic cartilage-like tissue formation comprised of aggrecan, collagen II and VI, link protein, and decorin; but decreased moduli. Loading, applied early or throughout culture, did not affect neotissue content in either hydrogel but affected neotissue spatial distribution in degrading hydrogels where 4 weeks of loading appeared to enhance hydrogel degradation resulting in tissue defects. CONCLUSIONS: PEG-LA hydrogels led to macroscopic tissue development comprised of key cartilage macromolecules under loading, but hydrogel degradation requires further tuning. CLINICAL RELEVANCE: PEG-LA hydrogels have potential for delivering chondrocytes in vivo to replace damaged cartilage with a tissue-engineered native equivalent, overcoming many limitations associated with current clinical treatments.