Garret D. Nicodemus

Cell Encapsulation in Biodegradable Hydrogels for Tissue Engineering Applications

Encapsulating cells in biodegradable hydrogels offers numerous attractive features for tissue engineering, including ease of handling, a highly hydrated tissue-like environment for cell and tissue growth, and the ability to form in vivo. Many properties important to the design of a hydrogel scaffold, such as swelling, mechanical properties, degradation, and diffusion, are closely linked to the crosslinked structure of the hydrogel, which is controlled through a variety of different processing conditions. Degradation may be tuned by incorporating hydrolytically or enzymatically labile segments into the hydrogel or by using natural biopolymers that are susceptible to enzymatic degradation. Because cells are present during the gelation process, the number of suitable chemistries and formulations are limited. In this review, we describe important considerations for designing biodegradable hydrogels for cell encapsulation and highlight recent advances in material design and their applications in tissue engineering.

The role of hydrogel structure and dynamic loading on chondrocyte gene expression and matrix formation

Crosslinked poly(ethylene glycol) (PEG) hydrogels are attractive scaffolds for cartilage tissue engineering because of their ability to mimic the aqueous environment and mechanical properties of native cartilage. In this study, hydrogel crosslinking density was varied to study the influence of gel structure and the application of dynamic loading (continuous, 1 Hz, 15% amplitude strain) on chondrocyte gene expression over approximately 1 week culture. Gene expression was quantified using real-time RT-PCR for collagen II and aggrecan, the major cartilage extracellular matrix (ECM) components, and collagen I, an indicator of chondrocyte de-differentiation. When chondrocytes were encapsulated in PEG gels with low or high crosslinking, a high collagen II expression compared to collagen I expression (1000 or 100,000:1, respectively) indicated the native chondrocyte phenotype was retained. In the absence of loading, relative gene expression for collagen II and aggrecan was significantly higher (e.g., 2-fold and 4-fold, respectively, day 7) in the low crosslinked gels compared to gels with higher crosslinking. Dynamic loading, however, showed little effect on ECM gene expression in both crosslinked systems. To better understand the cellular environment, ECM production was qualitatively assessed using an in situ immunofluorescent technique and standard histology. A pericellular matrix (PCM) was observed as early as day 3 post-encapsulation and the degree of formation was dependent on gel crosslinking. These results suggest the PCM may protect the cells from sensing the applied loads. This study demonstrates that gel structure has a profound effect on chondrocyte gene expression, while dynamic loading has much less of an effect at early culture times.

Designing 3D photopolymer hydrogels to regulate biomechanical cues and tissue growth for cartilage tissue engineering.

PurposeSynthetic hydrogels fabricated from photopolymerization are attractive for tissue engineering for their controlled macroscopic properties, the ability to incorporate biological functionalities, and cell encapsulation. The goal of the present study was to exploit the attractive features of synthetic hydrogels to elucidate the role of gel structure and chemistry in regulating biomechanical cues.MethodsCartilage cells were encapsulated in poly(ethylene glycol) (PEG) hydrogels with different crosslinking densities. Cellular deformation was examined as a function of gel crosslinking. The effects of continuous versus intermittent dynamic loading regimens were examined. RGD, a cell adhesion peptide, was incorporated into PEG gels and subjected to mechanical loading. Chondrocyte morphology and activity was assessed by anabolic and catabolic ECM gene expression and matrix production by collagen and glycosaminoglycan production.ResultsCell deformation was mediated by gel crosslinking. In the absence of loading, anabolic activity was moderately upregulated while catabolic activity was significantly inhibited regardless of gel crosslinking. Dynamic loading enhanced anabolic activities, but continuous loading inhibited catabolic activity, while intermittent loading stimulated catabolic activity. RGD acted as a mechanoreceptor to influence tissue deposition.ConclusionsWe demonstrate the ability to regulate biomechanical cues through manipulations in the gel structure and chemistry and cartilage tissue engineering.

Mechanical loading regimes affect the anabolic and catabolic activities by chondrocytes encapsulated in PEG hydrogels

OBJECTIVE Mechanical loading of cell-laden synthetic hydrogels is one strategy for regenerating functional cartilage. This work tests the hypothesis that type of loading (continuous vs intermittent) and timing when loading is applied (immediate vs delayed) influence anabolic and catabolic activities of chondrocytes when encapsulated in poly(ethylene glycol) (PEG) hydrogels. METHODS Primary bovine chondrocytes encapsulated in PEG hydrogels were subjected to unconfined dynamic compressive strains applied continuously or intermittently for 1 week (i.e., immediate) or intermittently for 1 week but after a 1 week free-swelling (FS) period (i.e., delayed). Anabolic activities were assessed by gene expression for collagen II and aggrecan (AGC) and extracellular matrix (ECM) deposition by (immuno)histochemistry. Catabolic activities were assessed by gene expression for matrix metalloproteinases, MMP-1, 3, and 13. RESULTS Intermittent loading (IL) upregulated ECM and MMP expressions, e.g., 2-fold, 16-fold and 8-fold for collagen II, MMP-1, MMP-3, respectively. Continuous loading upregulated AGC expression 1.5-fold but down-regulated MMP-1 (3-fold) and -3 (2-fold) expressions. For delayed loading, chondrocytes responded to FS conditions by down-regulating MMP expressions (P<0.01), but were less sensitive to loading when applied during week 2. Spatially, deposition of ECM molecules was dependent on the timing of loading, where immediate loading favored enhanced collagen II deposition. CONCLUSIONS The type and timing of dynamic loading dramatically influenced ECM and MMP gene expression and to a lesser degree matrix deposition. Our findings suggest that early applications of IL is necessary to stimulate both anabolic and catabolic activities, which may be important in regenerating and restructuring the engineered tissue long-term.

Gel structure has an impact on pericellular and extracellular matrix deposition, which subsequently alters metabolic activities in chondrocyte-laden PEG hydrogels

While designing poly(ethylene glycol) hydrogels with high moduli suitable for in situ placement is attractive for cartilage regeneration, the impact of a tighter crosslinked structure on the organization and deposition of the matrix is not fully understood. The objectives of this study were to characterize the composition and spatial organization of new matrix as a function of gel crosslinking and study its impact on chondrocytes in terms of anabolic and catabolic gene expression and catabolic activity. Bovine articular chondrocytes were encapsulated in hydrogels with three crosslinking densities (compressive moduli 60, 320 and 590 kPa) and cultured for 25 days. Glycosaminoglycan production increased with culture time and was greatest in the gels with lowest crosslinking. Collagens II and VI, aggrecan, link protein and decorin were localized to pericellular regions in all gels, but their presence decreased with increasing gel crosslinking. Collagen II and aggrecan expression were initially up-regulated in gels with higher crosslinking, but increased similarly up to day 15. Matrix metalloproteinase (MMP)-1 and MMP-13 expression were elevated (∼25-fold) in gels with higher crosslinking throughout the study, while MMP-3 was unaffected by gel crosslinking. The presence of aggrecan and collagen degradation products confirmed MMP activity. These findings indicate that chondrocytes synthesized the major cartilage components within PEG hydrogels, however, gel structure had a significant impact on the composition and spatial organization of the new tissue and on how chondrocytes responded to their environment, particularly with respect to their catabolic expression.

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.

Dynamic compressive loading influences degradation behavior of PEG-PLA hydrogels

Biodegradable hydrogels are attractive 3D environments for cell and tissue growth. In cartilage tissue engineering, mechanical stimulation has been shown to be an important regulator in promoting cartilage development. However, the impact of mechanical loading on the gel degradation kinetics has not been studied. In this study, we examined hydrolytically labile gels synthesized from poly(lactic acid)‐b‐poly(ethylene glycol)‐b‐poly‐(lactic acid) dimethacrylate macromers, which have been used for cartilage tissue engineering. The gels were subject to physiological loading conditions in order to examine the effects of loading on hydrogel degradation. Initially, hydrogels were formed with two different cross‐linking densities and subject to a dynamic compressive strain of 15% at 0.3, 1, or 3 Hz. Degradation behavior was assessed by mass loss, equilibrium swelling and compressive modulus as a function of degradation time. From equilibrium swelling, the pseudo‐first‐order reaction rate constants were determined as an indication of degradation kinetics. The application of dynamic loading significantly enhanced the degradation time for the low cross‐linked gels (P < 0.01) while frequency showed no statistical differences in degradation rates or bulk erosion profiles. In the higher cross‐linked gels, a 3 Hz dynamic strain significantly increased the degradation kinetics resulting in an overall faster degradation time by 6 days compared to gels subject to the 0.3 and 1 Hz loads (P < 0.0001). The bioreactor set‐up also influenced overall degradation behavior where the use of impermeable versus permeable platens resulted in significantly lower degradation rate constants for both cross‐linked gels (P < 0.001). The compressive modulus exponentially decreased with degradation time under dynamic loading. Together, our findings indicate that both loading regime and the bioreactor setup influence degradation and should be considered when designing and tuning a biodegradable hydrogel where mechanical stimulation is employed. Biotechnol. Bioeng. 2009; 102: 948–959.

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