Jennifer H. Elisseeff

Photoencapsulation of chondrocytes in poly(ethylene oxide)‐based semi‐interpenetrating networks

A photopolymerizing hydrogel system provides an efficient method to encapsulate cells. The present work describes the in vitro analysis of bovine and ovine chondrocytes encapsulated in a poly(ethylene oxide)-dimethacrylate and poly(ethylene glycol) semi-interpenetrating network using a photopolymerization process. One day after encapsulation, (3-[4,5-dimethylthiazol-2-y1]-2, 5-diphenyl-2H-tetrazolium bromide) (MTT) and light microscopy showed chondrocyte survival and a dispersed cell population composed of ovoid and elongated cells. Biochemical analysis demonstrated proteoglycan and collagen contents that increased over 2 weeks of static incubation. Cell content of the gels initially decreased and stabilized. Biomechanical analysis demonstrated the presence of a functional extracellular matrix with equilibrium moduli, dynamic stiffness, and streaming potentials that increased with time. These findings suggest the feasibility of photoencapsulation for tissue engineering and drug delivery purposes.

In situ forming degradable networks and their application in tissue engineering and drug delivery

Multifunctional macromers based on poly(ethylene glycol) and poly(vinyl alcohol) were photopolymerized to form degradable hydrogel networks. The degradation behavior of the highly swollen gels was characterized by monitoring changes in their mass loss, degree of swelling, and compressive modulus. Experimental results show that the modulus decreases exponentially with time, while the volumetric swelling ratio increases exponentially. A degradation mechanism assuming pseudo first-order hydrolysis kinetics and accounting for the structure of the crosslinked networks successfully predicted the experimentally observed trends in these properties with degradation. Once verified, the proposed degradation mechanism was extended to correlate network degradation kinetics, and subsequent changes in network structure, with release behavior of bioactive molecules from these dynamic systems. A theoretical model utilizing a statistical approach to predict the cleavage of crosslinks within the network was used to predict the complex erosion profiles produced by these hydrogels. Finally, the application of these macromers as in situ forming hydrogel constructs for cartilage tissue engineering is demonstrated.

IN VITRO CHONDROGENESIS OF BONE MARROW-DERIVED MESENCHYMAL STEM CELLS IN A PHOTOPOLYMERIZING HYDROGEL

Mesenchymal stem cells (MSCs) from skeletally mature goats were encapsulated in a photopolymerizing poly(ethylene glycol)-based hydrogel and cultured with or without transforming growth factor beta1 (TGF) to study the potential for chondrogenesis in a hydrogel scaffold system amenable to minimally invasive implantation. Chondrogenic differentiation was evaluated by histological, biochemical, and RNA analyses for the expression of cartilage extracellular matrix components. The two control groups studied were MSCs cultured in monolayer and MSCs encapsulated in the hydrogel and cultured for 6 weeks in chondrogenic medium without TGF-beta1 (6wk-TGF). The three experimental time points for encapsulated cells studied were 0 days (0d), 3 weeks, and 6 weeks in chondrogenic medium with TGF-beta1 at 10 ng/ml (3wk+TGF and 6wk+TGF). MSCs proliferated in the hydrogels with TGF-beta1. Glycosaminoglycan (GAG) and total collagen content of the hydrogels increased to 3.5% dry weight and 5.0% dry weight, respectively, in 6wk+TGF constructs. Immunohistochemistry revealed the presence of aggrecan, link protein, and type II collagen. Upregulation of aggrecan and type II collagen gene expression compared with monolayer MSCs was demonstrated. Type I collagen gene expression decreased from 3 to 6 weeks in the presence of TGF-beta1. 6wk-TGF hydrogels produced no GAG and only moderate amounts of collagen. However, immunohistochemistry and RT-PCR demonstrated a small amount of spontaneous differentiation in this control group. This study demonstrates the ability to encapsulate MSCs to form cartilage-like tissue in vitro in a photopolymerizing hydrogel. This system may be useful for minimally invasive implantation, MSC differentiation, and engineering of composite tissue structures with multiple cellular phenotypes.

Engineering structurally organized cartilage and bone tissues

The field of tissue engineering promises to deliver biological substitutes to repair or replace tissues in the body that have been injured or diseased. The clinical demand for musculoskeletal tissues is particularly high, especially for cartilage and bone defects. Although they are generally considered biologically simple structures, musculoskeletal tissues consist of highly organized three-dimensional networks of cells and matrix, giving rise to tissue structures with remarkable mechanical properties. Although the field of cartilage and bone tissue engineering has progressed significantly in recent years, the development of structurally ordered tissues has not been accomplished. More strategies are needed to ensure that the appropriate cell and matrix organization is being achieved in the engineered tissues. This review emphasizes how different cell types and scaffold designs can be used to modulate tissue properties and engineer more complex tissue structures, with emphasis on cartilage and bone tissues.

Controlled-release of IGF-I and TGF-β1 in a photopolymerizing hydrogel for cartilage tissue engineering

Photopolymerizing hydrogel systems provide a method to encapsulate cells and implant materials in a minimally invasive manner. Controlled release of growth factors in the hydrogels may enhance the ability to engineer tissues. IGF‐I and TGF‐β were loaded in PLGA microspheres using a double emulsion technique. 125 ng and 200 pg of active IGF‐I and TGF‐β, respectively, as measured by ELISA, were released over 15 days. The growth factor containing microspheres were photoencapsulated with bovine articular chondrocytes in PEO‐based hydrogels and incubated in vitro for two weeks. Statistically significant changes in glycosaminoglycan (GAG) production compared to control gels either without microspheres or with blank spheres were observed after a 14 day incubation with IGF‐I and IGF‐I/TGF‐β microspheres combined, with a maximum density of 8.41 ± 2.5% wet weight GAG. Total collagen density was low and decreased with the IGF‐I/TGF‐β microspheres after two weeks incubation, but otherwise remained unchanged in all other experimental groups. Cell content increased 10‐fold to 0.18 ± 0.056 × 106 cells/mg wet weight and extracellular matrix (ECM) staining by H&E increased in hydrogels with IGF‐I/TGF‐β microspheres. In conclusion, photoencapsulation of microspheres in PEO‐based hydrogels provides a method to deliver molecules such as growth factors in porous hydrogel systems.

In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells

Development of clinically relevant regenerative medicine therapies using human embryonic stem cells (hESCs) requires production of a simple and readily expandable cell population that can be directed to form functional 3D tissue in an in vivo environment. We describe an efficient derivation method and characterization of mesenchymal stem cells (MSCs) from hESCs (hESCd-MSCs) that have multilineage differentiation potential and are capable of producing fat, cartilage, and bone in vitro. Furthermore, we highlight their in vivo survival and commitment to the chondrogenic lineage in a microenvironment comprising chondrocyte-secreted morphogenetic factors and hydrogels. Normal cartilage architecture was established in rat osteochondral defects after treatment with chondrogenically-committed hESCd-MSCs. In view of the limited available cell sources for tissue engineering applications, these embryonic-derived cells show significant potential in musculoskeletal tissue regeneration applications.

Effects of three-dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells.

Embryonic stem (ES) cells have the ability to self‐replicate and differentiate into cells from all three germ layers, holding great promise for tissue regeneration applications. However, controlling the differentiation of ES cells and obtaining homogenous cell populations still remains a challenge. We hypothesize that a supportive three‐dimensional (3D) environment provides ES cell‐derived cells an environment that more closely mimics chondrogenesis in vivo. In the present study, the chondrogenic differentiation capability of ES cell‐derived embryoid bodies (EBs) encapsulated in poly(ethylene glycol)‐based (PEG) hy‐drogels was examined and compared with the chondrogenic potential of EBs in conventional monolayer culture. PEG hydrogel‐encapsulated EBs and EBs in monolayer were cultured in vitro for up to 17 days in chondrogenic differentiation medium in the presence of transforming growth factor (TGF)‐β1 or bone morphogenic protein‐2. Gene expression and protein analyses indicated that EB‐PEG hydrogel culture upregulated cartilage‐relevant markers compared with a monolayer environment and induction of chondrocytic phenotype was stimulated with TGF‐β1. Histology of EBs in PEG hydrogel culture with TGF‐β1 demonstrated basophilic extracellular matrix deposition characteristic of neocartilage. These findings suggest that EB‐PEG hydrogel culture, with an appropriate growth factor, may provide a suitable environment for chondrogenic differentiation of intact ES cell‐derived EBs.

Experimental Model for Cartilage Tissue Engineering to Regenerate the Zonal Organization of Articular Cartilage

OBJECTIVE Regeneration of the zonal organization of articular cartilage may be an important advancement for cartilage tissue engineering. The first goal of this study was to validate our surgical technique as a method to selectively isolate chondrocytes from different zones of bovine articular cartilage. The second goal was to confirm that chondrocytes from different zones would have different proliferative and metabolic activities in two-dimensional (2-D) and 3-D cultures. Finally, to regenerate the zonal organization, we sought to make multi-layered constructs by encapsulating chondrocytes from different zones of articular cartilage. DESIGN Cartilage slices were removed from three (upper, middle, and lower) zones of articular cartilage of young bovine legs. Histology and biochemical composition of the cartilage slices were analyzed to confirm that they had been obtained from the proper zone. Growth kinetics and gene expression in monolayer culture and matrix formation in photopolymerizing hydrogels were evaluated. Multi-layered photopolymerizing hydrogels were constructed with chondrocytes from each zone of native cartilage encapsulated. Cell viability and maintenance of the cells in the respective layer were evaluated using the Live/Dead Viability kit and cell tracking protocols, respectively. After 3 weeks, the multi-layered constructs were harvested for histologic examination including immunohistochemistry for type II collagen. RESULTS Analysis of histology and biochemical composition confirmed that the cartilage slices had been obtained from the specific zone. Chondrocytes from different zones differed in growth kinetics and gene expression in monolayer and in matrix synthesis in 3-D culture. Cells encapsulated in each of the three layers of the hydrogel remained viable and remained in the respective layer in which they were encapsulated. After 3-week culture, each zone of multi-layered constructs had similar histologic findings to that of native articular cartilage. CONCLUSION We present this as an experimental model to regenerate zonal organization of articular cartilage by encapsulating chondrocytes from different layers in multi-layered photopolymerizing gels.

Differential Response of Adult and Embryonic Mesenchymal Progenitor Cells to Mechanical Compression in Hydrogels

Cells in the musculoskeletal system can respond to mechanical stimuli, supporting tissue homeostasis and remodeling. Recent studies have suggested that mechanical stimulation also influences the differentiation of MSCs, whereas the effect on embryonic cells is still largely unknown. In this study, we evaluated the influence of dynamic mechanical compression on chondrogenesis of bone marrow‐derived MSCs and embryonic stem cell‐derived (human embryoid body‐derived [hEBd]) cells encapsulated in hydrogels and cultured with or without transforming growth factor β‐1 (TGF‐β1). Cells were cultured in hydrogels for up to 3 weeks and exposed daily to compression for 1, 2, 2.5, and 4 hours in a bioreactor. When MSCs were cultured, mechanical stimulation quantitatively increased gene expression of cartilage‐related markers, Sox‐9, type II collagen, and aggrecan independently from the presence of TGF‐β1. Extracellular matrix secretion into the hydrogels was also enhanced. When hEBd cells were cultured without TGF‐β1, mechanical compression inhibited their differentiation as determined by significant downregulation of cartilage‐specific genes. However, after initiation of chondrogenic differentiation by administration of TGF‐β1, the hEBd cells quantitatively increased expression of cartilage‐specific genes when exposed to mechanical compression, similar to the bone marrow‐derived MSCs. Therefore, when appropriately directed into the chondrogenic lineage, mechanical stimulation is beneficial for further differentiation of stem cell tissue engineered constructs.

Adult stem cell driven genesis of human-shaped articular condyle.

Uniform design of synovial articulations across mammalian species is challenged by their common susceptibility to joint degeneration. The present study was designed to investigate the possibility of creating human-shaped articular condyles by rat bone marrow-derived mesenchymal stem cells (MSCs) encapsulated in a biocompatible poly(ethylene glycol)-based hydrogel. Rat MSCs were harvested, expanded in culture, and treated with either chondrogenic or osteogenic supplements. Rat MSC-derived chondrogenic and osteogenic cells were loaded in hydrogel suspensions in two stratified and yet integrated hydrogel layers that were sequentially photopolymerized in a human condylar mold. Harvested articular condyles from 4-week in vivo implantation demonstrated stratified layers of chondrogenesis and osteogenesis. Parallel in vitro experiments using goat and rat MSCs corroborated in vivo data by demonstrating the expression of chondrogenic and osteogenic markers by biochemical and mRNA analyses. Ex vivo incubated goat MSC-derived chondral constructs contained cartilage-related glycosaminoglycans and collagen. By contrast, goat MSC-derived osteogenic constructs expressed alkaline phosphatase and osteonectin genes, and showed escalating calcium content over time. Rat MSC-derived osteogenic constructs were stiffer than rat MSC-derived chondrogenic constructs upon nanoindentation with atomic force microscopy. These findings may serve as a primitive proof of concept for ultimate tissue-engineered replacement of degenerated articular condyles via a single population of adult mesenchymal stem cells.