Lecture 12

Lecture 12 - BioE10: Lecture 12 Professor Irina Conboy...

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Unformatted text preview: BioE10: Lecture 12 Professor Irina Conboy Tissue engineering/Regenerative Medicine 3D polymeric biomaterials for engineering tissues and organs ex-vivo Objectives: To understand the current progress and limitations as well as future strategies in biomaterials and tissue engineering. To understand the effect of mechanical stretching and shearing on cells of interest. g g Biological fundamentals: Integrins, growth factors, RGD; Extra-cellular matrix (ECM) Cytoskeletal fillamets: actin, myosin, microtubules, microfilaments What are desirable properties of biomaterials (structural part) Lutolf and Hubbell, 2005 The native ECM is the prototypical hydrogel that regulates cell function on many length scales. A: scales Integrin-binding with ECM proteins (green ligands and tan receptors), growth factor sequestration within proteoglycans (red), and cell– cell contact via cadherins (purple) occur on the scale of tens of nanometers to microns. B: Migration, which is critical in tissue regeneration, regeneration cancer metastasis, and wound healing, metastasis healing initiates on the scale of tens to hundreds of microns. Paracrine signaling that directs differentiation (pink growth factors) and proliferation (red growth factors) is also mediated on this length scale. C: Tissue homeostasis, development, and wound healing are regulated over hundreds of microns to centimeters. Here, we illustrate neutrophils being recruited to the site of a wound in the epithelium. Tibbitt and Anseth, 2009 Biotechnology and Bioengineering Synthetic–biologic hydrogels that incorporate several well-defined and orthogonal chemistries serve as robust ECM mimics for 3D cell culture. Depending on the p g application, it may be advantageous to incorporate cell- or user-defined regulation of the material properties to emulate the native dynamic environment. However, in many cases, synthetic hydrogels that , y , y y g incorporate both cell- and userdefined chemistries will be necessary. Here, we illustrate a cell cleaving MMP degradable crosslinks (yellow circles) that allow it to access sequestered growth factors ( ) and integrinq g (red) g binding sites, such as RGD (green circles). Ultimately, this cleavage allows cell motility and the deposition of ECM proteins (orange fiber).User-defined chemistries, such as photodegradable crosslinks (blue ellipses) and p g ( p ) post-gelation attachment of RGD to the network backbone, afford facile control of the dynamic biochemical and biophysical properties of the gel, thereby directing cell attachment and motility. Further, exogenous g y g application of enzymes (brown) can allow user-defined release of sequestered growth factors. Professor Kevin Healy:sIPN hydrogels Tibbitt and Anseth, 2009 Biotechnology and Bioengineering Let’s contrast Smart Biomaterials with General types of tissue analogs Think of the advantages of smart biomaterials that are provided by genetics and molecular biology toolkit 1. Single purpose acellular transient graft BioBrane is a nylon material that contains a gelatin that interacts with clotting factors in the wound. That interaction causes the dressing to adhere better, forming a more durable protective layer. Integra is a two-layered dressing. The top layer serves as a temporary synthetic epidermis; the layer below serves as a foundation for re-growth of dermal tissue. The underlying layer is made of collagen fibers that act as a lattice thro gh which the bod through hich body can begin to align cells to recreate its own dermal tissue. 2. Multi-purpose acellular graft; Hydrogel Salubria for cartilage and blood vessel replacement; neuron guidance and drug delivery d li Electron micrograph of BD PuraMatrix Peptide P M t i P tid Hydrogel Hydrogel called S l b i (d i d f H d l ll d Salubria (derived from th L ti words f " f " and "h lth ”) G the Latin d for "safe" d "healthy”). Georgia T h R i Tech Research h Corporation; Dr. David Ku (Georgia Tech). To date, tests in rats, dogs and sheep show that Salubria is biocompatible: Platelets do not adhere to it in significant quantities, and thus the chance of blood clots is greatly reduced (the problem with Dacron, which surgeons have used for artery replacement in the abdomen and legs since its development in the 1950s). In g y p g p ) addition these studies suggest Salubria can serve as a nerve guide to create a physical bridge that could dramatically increase the speed at which severed nerves heal. As an implantable drug delivery system, Salubria may work for many drugs, such as insulin and morphine, that need to be injected. Its advantage for such is that it is hydrophilic (attracted to water), rather than hydrophobic (resisted by water) like the silicone. silicone 3. Living tissue equivalents Vascular? Innervated? Organogenesis Inc. is selling its Apilgra, a living "human skin equivalent" to treat wounds and ulcers in Canada and the company is seeking approval for sales in the U.S. Additionally, LifeCell Corporation makes implantable human tissue for use in reconstructive surgery and burn treatment. Doctors are able to take a postage stampsized piece of skin from the patient and grow the skin under special tissue culture conditions. From this small piece of skin, technicians can grow enough skin to cover nearly the entire body in just 3 weeks. Cultured skin has been available in the U.S. for a decade. Artificial skins are only a temporary fix; the patient will still need skin grafts. However, with the use of artificial skins means a thinner skin graft, which allows the donor site and the patient to heal faster with fewer surgeries. The use of artificial skins has not yet been perfected and they are not surgeries right for every burn patient. Additionally scarring still results, but it may be less severe. Q: why a sheet of skin cells does not suffice as tissue replacement? 4. Nucleic Acid and protein delivery systems=scaffolds Q: Could this approach be used for delivery of shRNA? (A) Inductive factors (red circles) may be embedded or encapsulated within hydrogels, which may, in turn, be used to form 3-D scaffolds that are capable of releasing factors at the site of implantation. The highest factor concentration exists within the scaffold, with lower concentrations found within the surrounding tissue. Delivered factors target a variety of different cell populations (e.g., y y , , ) pp ( g, , , , g) ( ) myocytes, neurons, fibroblasts or osteoblasts) for various applications (e.g., muscle, nerve, bone, wound healing). (B) Substrate immobilization is characterized by factors being bound to a biomaterial. The interaction between the factor and biomaterial can be (top): (1) K on > K off, such that the factor is effectively bound irreversibly; (2) Kon=Koff, where the factor associates and dissociates from the surface at roughly equal rates; and (3) Kon < Koff, such that the factor is loaded onto the biomaterial and dissociates to serve as a delivery vehicle. Cells interact with the immobilized factors upon interaction with the biomaterial (bottom). Mechanical forces applied to a cell will propagate via cytoskeletal filaments stretching C.elegans C.elegans Shear stress Cellsalive.com Figure 7-31 Actin filaments in mammalian cell Intermediate filaments in mammalian cell Microtubules in mammalian cell Figure 7-30-Table 7-3 http://www.sumanasinc.com/webcontent/animations/content/intermediate_filaments.html http://www.wiley.com/college/pratt/0471393878/student/animations/actin_myosin/actin_myosin.swf http://www.sinauer.com/cooper/4e/animations1203.html Why mechanical integrity is important for a cell? Why did cytoskeleton evolve? C.elegans C.elegans All the elements form one mechanical network that functions as a prestressed scaffold that can withstand mechanical stress. The main cytoskeletal components (microfilaments, microtubules and intermediate filaments) are key elements in linking the cellular surface to the nucleus. Cell adhesion molecules ( dh i l l (such as cadherins and h dh i d integrins) bind to the cytoskeleton through linker proteins, in this way forming connections to adherent cells or to the extracellular matrix. On the other side, the , cytoskeleton is linked to the nuclear membrane and the underlying nuclear lamina. Q: Why would you predict and how would you check for a non-specific non specific response to stretch (consider that there are particular stress response genes, e.g. hsp-70). Correct signal transduction, cell division and cell metabolism, including ATP production can only occur with undisturbed cytoskeleton and cell shape Integrating natural and synthetic components in structural part of biomaterial. Specific example: Am J Physiol Cell Physiol 288: C30–C38, 2005. Laminin YIGSR How this biomaterial could be optimized? Polymer chemistry, peptide density, peptide composition. Cell stretching in tissue engineering and regenerative medicine What is the main difference between embryonic cells (mesoderm) dedicated to make lung tissues (or those, making heart valves and arteries) and many adult cells (e.g. hMSCs, liver cells, etc.)? Evolutionary adaptation to use “stretching” as a relevant signal (of relatively limited modulation). Schematic map of the mouse tip gene structure. Coding (black) and noncoding (green) exons are shown as vertical boxes. The initiation codon (ATG) is also shown. Approach: 1. Get rid of common mRNAs expressed by both stretched and non-stretched cells 2. DNA microarray or similar methods 3. 3 Analyze gene structure 4. Candidate gene approach: A) Expression in correct place/time/condition B) Over-expression enhances function: Over-expression substitutes for stretching; ) p p g Tip 1 myogenesis; Tip 3 adipogenesis; Tip 2 is not involved in this phenomenon C) RNAi inhibits function: Tip1 and Tip3 RNAi targeting inhibits myogenic and adipogenic cell fates (respectively) Q: What is the significance of Figures F and G? Generally speaking, gradients of biochemical molecules (proteins mostly) and multifunctional interactive signal transduction networks control cell-fate determination of stem cells and orchestrate tissue and organ formation, maintenance and repair. These biochemical cues work best in the physiological range of the matrix rigidity and mechanical stress. Understanding and recapitulating these molecular signals is the key for creating smart biomaterials (Next biomaterials. module: Stem Cell Technologies) Q: What cells-tissues are likely to recognize stretching as a g physiological signal (rather than a trauma)? Q: Would you use stretching for in vitro manufacturing of: A. Beta cells from pancreas (lost in type I diabetes)? B. Heart valves? C. Liver cells? D. Arteries? What is the best source of cells for tissue engineering? Current approach: align cells using patterned substrate (e.g. smooth muscle cells aligned on collagen grooves; hMSC-derived cells on pattered substrates, etc). Thakar et al., Biochem Biophys Res Comm (2003) Q: What will likely happen to the engineered tissue after transplantation in vivo: A. To differentiated cells? B. To di idi B T dividing precursor cells? ll ? C. Is self-renewing required for successful tissue transplantation? What is the best source of cells for engineered tissues? Myofiber: a unit of regeneration in adult muscle Basal lamina Satellite cell Laminin/PI Myonucleus Myofiber Plasma membrane Deciphering and reconstructing the biochemical and structural components of muscle ECM Using cells produced in culture by muscle stem (satellite) cells Testing this principle for engineering regenerating tissue. Work in progress. Skeletal muscle fibers (green) that are capable of self-assembly and of continuous regeneration (dividing cells are in red, BrdU incorporation), are manufactured in biochemically defined 3D micro-environment. H. Silva and I. Conboy; unpublished Conlusions: An ideal engineered tissue has chemically defined ECM that is embedded chemically-defined with biologically active molecule enabling predictably regulate tissue regeneration and/or function. The optimal source of cells, as well as the optimal rigidity and mechanical stress conditions are determined based on the physiological ranges of signals that operate during normal organogenesis and tissue remodeling. Check your understanding: You should be able to compare the functional properties of several classes of biomaterials discussed in this lecture; You should be able to propose strategies for optimal control of cell adhesion and cell functionality in an engineered tissue You should understand the criteria for selection of cells for tissue engineering You should understand the function and structure of cytoskeletal filaments ...
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