Transdermal photopolymerization of hydrogels for tissue engineering

Thesis (Ph.D.)--Harvard--Massachusetts Institute of Technology Division of Health Sciences and Technology, 1999.Includes bibliographical references.by Jennifer Hartt Elisseeff.Ph.D

Microarray Embedding/Sectioning for Parallel Analysis of 3D Cell Spheroids.

Three-dimensional cell spheroid models can be used to predict the effect of drugs and therapeutics and to model tissue development and regeneration. The utility of these models is enhanced by high throughput 3D spheroid culture technologies allowing researchers to efficiently culture numerous spheroids under varied experimental conditions. Detailed analysis of high throughput spheroid culture is much less efficient and generally limited to narrow outputs, such as metabolic viability. We describe a microarray approach that makes traditional histological embedding/sectioning/staining feasible for large 3D cell spheroid sample sets. Detailed methodology to apply this technology is provided. Analysis of the technique validates the potential for efficient histological analysis of up to 96 spheroids in parallel. By integrating high throughput 3D spheroid culture technologies with advanced immunohistochemical techniques, this approach will allow researchers to efficiently probe expression of multiple biomarkers with spatial localization within 3D structures. Quantitative comparison of staining will have improved inter- and intra-experimental reproducibility as multiple samples are collectively processed, stained, and imaged on a single slide

Derivation of Chondrogenically-Committed Cells from Human Embryonic Cells for Cartilage Tissue Regeneration

Background: Heterogeneous and uncontrolled differentiation of human embryonic stem cells (hESCs) in embryoid bodies (EBs) limits the potential use of hESCs for cell-based therapies. More efficient strategies are needed for the commitment and differentiation of hESCs to produce a homogeneous population of specific cell types for tissue regeneration applications. Methodology/Principal Findings: We report here that significant chondrocytic commitment of feeder-free cultured human embryonic stem cells (FF-hESCs), as determined by gene expression and immunostaining analysis, was induced by coculture with primary chondrocytes. Furthermore, a dynamic expression profile of chondrocyte-specific genes was observed during monolayer expansion of the chondrogenically-committed cells. Chondrogenically-committed cells synergistically responded to transforming growth factor-b1 (TGF-b1) and b1-integrin activating antibody by increasing tissue mass in pellet culture. In addition, when encapsulated in hydrogels, these cells formed cartilage tissue both in vitro and in vivo. In contrast, the absence of chondrocyte co-culture did not result in an expandable cell population from FF-hESCs. Conclusions/Significance: The direct chondrocytic commitment of FF-hESCs can be induced by morphogenetic factor

OA cartilage derived chondrocytes encapsulated in poly(ethylene glycol) diacrylate (PEGDA) for the evaluation of cartilage restoration and apoptosis in an in vitro model

Osteoarthritis (OA) is characterized by cartilage attrition, subchondral bone remodeling, osteophyte formation and synovial inflammation. Perturbed homeostasis caused by inflammation, oxidative stress, mitochondrial dysfunction and proapoptotic/antiapoptotic dysregulation is known to impair chondrocyte survival in joint microenvironments and contribute to OA pathogenesis. However, the molecular mechanisms underlying the programmed cell death (apoptosis) of chondral cells are not yet well defined. The present study was conducted to evaluate apoptosis of chondrocytes from knee articular cartilage of patients with OA. The aim of this study was to investigate and compare the apoptosis through the expression of caspase-3 in tissue explants, in cells cultured in monolayer, and in cells encapsulated in a hydrogel (PEGDA) scaffold. Chondrocytes were also studied following cell isolation and encapsulation in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Specifically, articular cartilage specimens were assessed by histology (Hematoxlyn and Eosin) and histochemistry (Safranin-O and Alcian Blue). The effector of apoptosis caspase-3 was studied through immunohistochemistry, immunocytochemistry and immunofluorescence. DNA strand breaks were evaluated in freshly isolated chondrocytes from human OA cartilage using the TUNEL assay, and changes in nuclear morphology of apoptotic cells were detected by staining with Hoechst 33258. The results showed an increased expression of caspase-3 in tissue explants, in pre-confluent cells and after four passages in culture, and a decreased expression of caspase-3 comparable to control cartilage in cells encapsulated in hydrogels (PEGDA) after 5 weeks in culture. The freshly isolated chondrocytes were TUNEL positive. The chondrocytes after 5 weeks of culture in hydrogels (PEGDA) showed the formation of new hyaline cartilage with increased cell growth, cellular aggregations and extracellular matrix (ECM) production. This is of particular relevance to the use of OA cells and tissue engineering in the therapeutic approach to patients

Evolution of Autologous Chondrocyte Repair and Comparison to Other Cartilage Repair Techniques

Articular cartilage defects have been addressed using microfracture, abrasion chondroplasty, or osteochondral grafting, but these strategies do not generate tissue that adequately recapitulates native cartilage. During the past 25 years, promising new strategies using assorted scaffolds and cell sources to induce chondrocyte expansion have emerged. We reviewed the evolution of autologous chondrocyte implantation and compared it to other cartilage repair techniques. Methods. We searched PubMed from 1949 to 2014 for the keywords “autologous chondrocyte implantation” (ACI) and “cartilage repair” in clinical trials, meta-analyses, and review articles. We analyzed these articles, their bibliographies, our experience, and cartilage regeneration textbooks. Results. Microfracture, abrasion chondroplasty, osteochondral grafting, ACI, and autologous matrix-induced chondrogenesis are distinguishable by cell source (including chondrocytes and stem cells) and associated scaffolds (natural or synthetic, hydrogels or membranes). ACI seems to be as good as, if not better than, microfracture for repairing large chondral defects in a young patient’s knee as evaluated by multiple clinical indices and the quality of regenerated tissue. Conclusion. Although there is not enough evidence to determine the best repair technique, ACI is the most established cell-based treatment for full-thickness chondral defects in young patients

Photomodulation of Cellular Gene Expression in Hydrogels

Biomaterials are designed to mimic aspects of various extracellular matrix environments, through chemical modifications to input biological or chemical signals. However, the dynamic nature and timing of gene expression during cellular events is much more difficult to mimic and control in these synthetic environments. Here, we utilized concepts of photochemistry combined with click chemistry for synthetic biology applications to modulate cellular gene expression in poly­(ethylene glycol) (PEG) hydrogels. Specifically, a genetic inducer, isopropyl β-d-1-thiogalactopyranoside (IPTG), is covalently linked to PEG via a biocompatible and easy to synthesize 2-(2-azido-6-nitrophenyl)­ethoxycarbonyl (ANPEOC) photocleavable moiety that, on a short exposure to UV light, effectively releases IPTG and activates gene expression of enhanced green fluorescence protein (EGFP). We anticipate that combining concepts of material chemistry with synthetic biology will further enable the construction of highly defined engineered niches that are capable of controlling both intrinsic and extrinsic cellular events

Tissue Extracellular Matrix Nanoparticle Presentation in Electrospun Nanofibers

Biomaterials derived from the decellularization of mature tissues retain biological and architectural features that profoundly influence cellular activity. However, the clinical utility of such materials remains limited as the shape and physical properties are difficult to control. In contrast, scaffolds based on synthetic polymers can be engineered to exhibit specific physical properties, yet often suffer from limited biological functionality. This study characterizes composite materials that present decellularized extracellular matrix (DECM) particles in combination with synthetic nanofibers and examines the ability of these materials to influence stem cell differentiation. Mechanical processing of decellularized tissues yielded particles with diameters ranging from 71 to 334 nm. Nanofiber scaffolds containing up to 10% DECM particles (wt/wt) derived from six different tissues were engineered and evaluated to confirm DECM particle incorporation and to measure bioactivity. Scaffolds containing bone, cartilage, and fat promoted osteogenesis at 1 and 3 weeks compared to controls. In contrast, spleen and lung DECM significantly reduced osteogenic outcomes compared to controls. These findings highlight the potential to incorporate appropriate source DECM nanoparticles within nanofiber composites to design a scaffold with bioactivity targeted to specific applications