Justine J. Roberts

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.

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.

On the role of hydrogel structure and degradation in controlling the transport of cell-secreted matrix molecules for engineered cartilage

Damage to cartilage caused by injury or disease can lead to pain and loss of mobility, diminishing ones quality of life. Because cartilage has a limited capacity for self-repair, tissue engineering strategies, such as cells encapsulated in synthetic hydrogels, are being investigated as a means to restore the damaged cartilage. However, strategies to date are suboptimal in part because designing degradable hydrogels is complicated by structural and temporal complexities of the gel and evolving tissue along multiple length scales. To address this problem, this study proposes a multi-scale mechanical model using a triphasic formulation (solid, fluid, unbound matrix molecules) based on a single chondrocyte releasing extracellular matrix molecules within a degrading hydrogel. This model describes the key players (cells, proteoglycans, collagen) of the biological system within the hydrogel encompassing different length scales. Two mechanisms are included: temporal changes of bulk properties due to hydrogel degradation, and matrix transport. Numerical results demonstrate that the temporal change of bulk properties is a decisive factor in the diffusion of unbound matrix molecules through the hydrogel. Transport of matrix molecules in the hydrogel contributes both to the development of the pericellular matrix and the extracellular matrix and is dependent on the relative size of matrix molecules and the hydrogel mesh. The numerical results also demonstrate that osmotic pressure, which leads to changes in mesh size, is a key parameter for achieving a larger diffusivity for matrix molecules in the hydrogel. The numerical model is confirmed with experimental results of matrix synthesis by chondrocytes in biodegradable poly(ethylene glycol)-based hydrogels. This model may ultimately be used to predict key hydrogel design parameters towards achieving optimal cartilage growth.

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.

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.

An Instrumented Bioreactor for Mechanical Stimulation and Real-Time, Nondestructive Evaluation of Engineered Cartilage Tissue

Functional tissue engineering involves the application of physical loads to promote the development of tissue constructs that can withstand the mechanical demands encountered in vivo [1]. Specifically, the goal of functional tissue engineering of articular cartilage is to develop an engineered cartilage construct that exhibits structure and function sufficient to replace or repair damaged articular cartilage. To accomplish this goal, bioreactors have been developed to apply mechanical stimulation to cell-laden constructs. Design strategies may impart various types of load including hydrostatic pressure, compression, or shear [2–5]. However, few bioreactors include instrumentation that allow for continuous monitoring of tissue development. The successful in vitro development of functional tissue-engineered constructs could benefit from a method of assessment that allows for continuous evaluation of tissue while not compromising construct integrity, preserving the construct for continuous development and eventual implantation. Current methods for evaluating extracellular matrix (ECM) development and mechanical properties are time consuming and destructive to the construct, and require numerous replicates to obtain a comprehensive overview of construct quality. Nondestructive, continuous evaluation of a tissue construct during development can be useful not only for final clinical use, but also for determining appropriate bioreactor parameters to achieve sufficient structure and function. Nondestructive measurement systems have been developed to assess construct mechanical properties as well as bulk-tissue development [6,7]. Preiss-Bloom et al. developed a bioreactor to mechanically stimulate tissue-engineered cartilage and measure real-time force response [6]. The bioreactor was outfitted with load sensors to continuously log construct resistance to deformation by the bioreactor. Such measurements give insight into the change in construct stiffness during stimulation and development in the bioreactor. Hagenmuller et al. developed a bioreactor that combines mechanical loading and online microcomputed tomography (μCT) for monitoring the development of engineered bone tissue [7]. Cartridge-like culture chambers were designed to allow for sterile mechanical stimulation and μCT monitoring of mineral deposition without removing the constructs. Another potential method for nondestructive assessment of tissue formation is ultrasound. Ultrasonic techniques are sensitive to mechanical and biochemical properties of cartilage [8–10] and have the potential to nondestructively assess the quality of tissue-engineered cartilage during development. Ultrasonic waves are utilized to acquire acoustic images and make localized quantitative measurements of tissue properties. Propagation and scattering of ultrasonic waves depend on tissue composition and structure [11]. Specifically, the reflection coefficient, the fraction of ultrasound reflected from an interface with different acoustic impedances, is one parameter commonly used to evaluate tissue characteristics [12–16]. A number of studies have been conducted to examine the feasibility of ultrasound as a tool for diagnosis of osteoarthritis by measuring changes in ultrasonic parameters after spontaneous and selective enzymatic degradation of cartilage tissue [17–20]. Ultrasound has also been used as a tool for monitoring in vivo cartilage tissue development and repair [21–23]. However, ultrasound has only recently been used as a measurement tool for the evaluation of tissue-engineered cartilage [8,24] and has yet to be implemented for real-time evaluation of tissue development. The objective of this work was to develop an instrumented bioreactor that could be utilized to stimulate and nondestructively evaluate tissue-engineered cartilage. Our dynamic compression bioreactor is instrumented with an ultrasonic transducer, load cells, and a video microscope for assessing ECM development and mechanical properties of tissue-engineered cartilage. Chondrocyte-laden hydrogel constructs were placed in the bioreactor and subjected to a three-part loading regime including: (1) a ramp, (2) sinusoidal compression, and (3) no load. This regime was repeated twice per day for 7 days. Constructs were nondestructively evaluated with ultrasound on days 0 and 7. Constructs were also evaluated on days 0 and 7 for cell viability, cell number, sulfated glycosaminoglycan (sGAG), and collagen content. Histological sections were stained for sGAG and collagen with safranin O and Massons trichrome, respectively.

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.

Inclusion of a collagen-GAG sponge core improves tangent modulus of multi-phase PEGDM hydrogel constructs

Nearly 27 million people in the United States suffer from osteoarthritis (OA).[1] While surgical options are available for patients suffering from OA, focal treatments, such as resection and mosaicplasty, rarely succeed in regenerating fully functional cartilage. Tissue engineering holds potential for developing more effective repair strategies.Copyright

Optical trapping for tissue scaffold fabrication

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 because 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 are encapsulated by photopolymerization of monomer via projection of a 512x512 spatial light modulator (SLM) illuminated at 405 nm. High 405 nm sensitivity and complete insensitivity to 1064 nm is enabled by a lithium acylphosphinate (LAP) salt photoinitiator. Use of a polyethylene glycol dimethacrylate (PEGDMA) based monomer is compared to that of polyethylene glycol (PEG) hydrogels formed by thiol-ene photo-click chemistry for patterning structures with cellular resolution, and for maintaining cell viability. Cells patterned in thiol-ene with RGD are shown to retain viability up to 4 days after the trapping and encapsulation procedure. Further, cells patterned in thiol-ene with RGD and a degradable ester link, are shown to fuse, indicating the initial stages of development of multi-nucleated cells.

The Mechanical Behavior of Engineered Hydrogels

Agarose and poly(ethylene-glycol) (PEG) are commonly used as scaffolds for cell and tissue engineering applications [1]. Agarose is a natural biomaterial that is thought to be inert [2] and permits growing cells and tissues in a three-dimensional suspension [3]. Gels synthesized from photoreactive poly(ethylene glycol) (PEG) macromonomers are well suited as cell carriers because they can be rapidly photopolymerized in vivo by a chain radical polymerization that is not toxic to cells, including chondrocytes. This paper explores the differences in mechanical behavior between agarose, a physically cross-linked hydrogel, and PEG, a chemically cross-linked hydrogel, to set the foundation for choosing hydrogel properties and chemistries for a desired tissue engineering application.Copyright