158 research outputs found
Engineering the mode of morphogenetic signal presentation to promote branching from salivary gland spheroids in 3D hydrogels
No full textM.S.Xerostomia and Sjӧgren’s Syndrome are conditions associated with loss in salivary volume that is needed to regulate the health of the oral cavity. Current therapies are limited to the introduction of artificial saliva and muscarinic receptor agonists, pilocarpine and cevimeline that induce saliva secretion from residual acinar cells. Regenerative tissue engineering provides a promising platform to solve this problem in the long term by helping rebuild the gland. The salivary tissue is a highly branched network of cells, which enables an increase in surface area without a major increase in glandular volume for high fluid output. Previously we developed a fibrin hydrogel (FH) decorated with laminin-111 peptides (L1p-FH) and supports three-dimensional (3D) gland microstructures containing polarized acinar cells. Here we expand on these results and show that co-culture of gland cells with mesenchymal stem cells produces migrating branches of gland cells into the L1p-FH and we identify FGF7 as the principal morphogenetic signal responsible for branching. On the other hand, another FGF family member and know gland morphogen, FGF10 increased proliferation but did not promote migration and therefore, limited the number and length of branched structures grown into the gel. By controlling the mode of growth factor presentation and delivery, we can control the length and cellularity of branches as well as formation of new nodes/clusters within the hydrogel. Such spatial delivery of two or more morphogens may facilitate engineering of anatomically complex tissues/mini organs such as glands that can be used to address developmental questions or as platforms for drug discovery
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Engineered Vascular Tissue Generated by Cellular Self-Assembly
No full textSmall diameter vascular grafts comprised entirely from cells and cell-derived extracellular matrix (ECM) have shown promise in clinical trials and may have potential advantages as in vitro vascular tissue models. A challenge with current cell-derived tissue engineering approaches is the length of time required to generate strong, robust tissue. There is a lack of alternative methods to rapidly assemble cells into a 3D format without the support of a scaffold. Toward the goal of engineering a new approach to rapidly synthesizing vascular tissue constructs entirely from cells, we have developed and characterized a strategy for creating cell-derived tissue rings by cellular self-assembly. The focus of this thesis was to develop the system to rapidly generate engineered tissue rings, and to evaluate their structural and functional properties.
To generate tissue rings, rat smooth muscle cells (SMCs) were seeded into round-bottomed, ring-shaped agarose wells with varying inner post diameters (2, 4, and 6 mm). Within 24 hours of seeding, cells aggregated, contracted, and formed robust tissue that could be removed from their wells and handled. If kept in culture, the thickness of these tissue rings increased with time. Mechanical analysis of the tissue showed that it was stronger after only 8 days in culture than engineered tissues generated by other approaches (such as seeding cells in biopolymer gels) cultured and tested at similar time points. Histological staining of the tissue rings revealed high cell densities throughout, along with the presence of glycosaminoglycans and some collagen. We also found that we could use the tissue rings as building blocks to generate larger tubular structures. Briefly, tissue rings were removed from the agarose wells and transferred onto silicone tubing mandrels. Once the rings were placed in contact with each other on the mandrel, they were cultured to allow the rings to fuse together. We found that the ability of tissue rings to fuse decreased with increasing ring “pre-culture” duration, and that we were able to generate fully fused tissue tubes in as little as 8 days (with only one day of ring pre-culture and seven days of fusion).
In the last section of this thesis, we established the feasibility of using primary human SMCs to generate self-assembled tissue rings, similar to the self-assembled rings generated with rat SMCs. Compared to the rat SMC rings, human SMC rings were stronger, stiffer and appeared to contain increased levels of collagen. These data showed that human SMCs are capable of self-assembling into tissue rings similar to rat SMCs, and may therefore be used to create engineered human vascular tissue.
Overall, we have developed a platform technology that can be used to screen the effects of culture parameters on the structure, mechanics, and function of vascular tissue. We anticipate that through the use of this technology, we can further improve vascular grafts by better understanding factors which promote ECM synthesis and SMC contraction. We can use these results directly toward the generation of vascular grafts by fusing self-assembled cell rings together to form tissue tubes. These novel bioengineered vascular tissues may also serve as a method to produce in vitro models to help further our understanding of vascular diseases, as well as facilitate pre-clinical screening of vascular tissue responses to pharmacologic therapies
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Engineering the Keratinocyte Microenvironment: Harnessing Topography to Direct Cellular Function
No full textSkin wound healing presents a challenging and expensive clinical problem with nearly 20 million wounds requiring intervention leading to an annual cost of more than $8 million. Tissue engineered skin substitutes are valuable not only as a clinical therapy for chronic wounds and severe traumas, but also as in vitro 3D model systems to investigate wound healing and skin pathogenesis. However, these substitutes are limited by a lack of topography at the dermal-epidermal junction (DEJ). In contrast, the native DEJ is characterized by a series of dermal papillae which project upward into the epidermal layer and create physical topographic microniches that support keratinocyte stem cell clustering. In this thesis, we created novel 3D skin model systems to investigate the role of microtopography in regulating keratinocyte function and cell fate using scaffolds containing precisely engineered topographic features. We hypothesized that the microtopography of the DEJ creates distinct keratinocyte microniches that promote epidermal morphogenesis and modulate keratinocyte stem cell clustering which can be harnessed to create a more robust skin substitute that expedites wound closure. Using photolithographic techniques, we created micropatterned DEJ analogs and micropatterned dermal-epidermal regeneration matrices (µDERM) which couple a dermal support matrix to a micropatterned DEJ analog. We found that the incorporation of microtopography into our in vitro skin model resulted in a thicker, more robust epidermal layer. Additionally, we identified three distinct functional keratinocyte niches: the proliferative niche in narrow channels, the synthetic niche in wide channels and the keratinocyte stem cell niche in narrow channels and corner topographies. Ultimately, incorporation of both narrow and wide channels on a single construct allowed us to recreate native keratinocyte stem cell patterning in vitro. These model systems will allow us to investigate the role of cellular microniches in regulating cellular function and epidermal disease pathogenesis as well as to identify topographic cues that enhance the rate of wound healing
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Designing Bioengineered Skin Substitutes Containing Microfabricated Basal Lamina Analogs to Enhance Skin Regeneration
No full textBioengineered skin substitutes have been developed to treat burn and non-healing wounds; however limitations still hinder their clinical success rates. Optimizing these current design strategies requires an understanding of how biochemical and topographical features of the native tissue modulate keratinocyte processes involved in tissue functionality. In this thesis, a novel bioengineered skin substitute was developed that contains a microfabricated basal lamina analog that recapitulates the native microenvironment found at the dermal-epidermal junction (DEJ). In native skin, this microenvironment consists of both biochemical and topographical cues which play critical roles in maintaining tissue architecture and overall homeostasis with the external environment.
Therefore, we hypothesize that microfabricated basal lamina analogs with extracellular matrix cues and three-dimensional features that mimics the cellular microenvironment of the DEJ will promote enhanced epithelialization and increase epidermal stem cell clustering on the surface of bioengineered skin substitutes.
We determined that the extracellular matrix protein fibronectin (FN) found in the cellular microenvironment of the DEJ enhanced keratinocyte attachment, proliferation, and epithelialization of a collagen based basal lamina analog. It was also found that the collagen material used to create the basal lamina analog as well as the FN conjugation strategy to this material significantly influenced the bioactivity of FN and its ability to modulate keratinocyte functions through integrin based mechanism. To investigate spatial tissue organization and the role it plays in the cellular microenvironment of the DEJ on epithelialization and epidermal stem cell localization, we used photolithography coupled with materials processing techniques to create microfabricated basal lamina analogs. It was determined that epidermal thicknesses found in narrow channels of microfabricated basal lamina analogs (50 µm and 100 µm widths with 200 µm depths) were similar to cultures on de-epithelialized acellular dermis and native foreskin tissues after 7 days of in vitro culture. We also determined that the microfabricated basal lamina analogs created an epidermal stem cell niche that promoted epidermal stem cell clustering in the channels which is critical for longevity of the tissue.
Overall, we developed a platform technology that was specifically used to produce a highly functional bioengineered skin substitute with regenerative capacity that mimics native skin. We anticipate through the use of this technology, we can further improve bioengineered skin substitutes by incorporating epidermal structures of native skin including hair follicles and sweat glands as well as improve overall cosmetic appearance. Additionally, this novel bioengineered skin substitute can serve as a model system to further our understanding of pathological conditions and diseases of the skin as well as facilitate robust preclinical screenings of epidermal responses to new therapeutic agents as well as to cosmetic and chemical products
CDH2 and CDH11 act as regulators of stem cell fate decisions
No full textAbstractAccumulating evidence suggests that the mechanical and biochemical signals originating from cell–cell adhesion are critical for stem cell lineage specification. In this review, we focus on the role of cadherin mediated signaling in development and stem cell differentiation, with emphasis on two well-known cadherins, cadherin-2 (CDH2) (N-cadherin) and cadherin-11 (CDH11) (OB-cadherin). We summarize the existing knowledge regarding the role of CDH2 and CDH11 during development and differentiation in vivo and in vitro. We also discuss engineering strategies to control stem cell fate decisions by fine-tuning the extent of cell–cell adhesion through surface chemistry and microtopology. These studies may be greatly facilitated by novel strategies that enable monitoring of stem cell specification in real time. We expect that better understanding of how intercellular adhesion signaling affects lineage specification may impact biomaterial and scaffold design to control stem cell fate decisions in three-dimensional context with potential implications for tissue engineering and regenerative medicine
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