Sujata K. Bhatia

Coagulation Facilitates Tumor Cell Spreading in the Pulmonary Vasculature during Early Metastatic Colony Formation

Coagulation has long been known to facilitate metastasis. To pinpoint the steps where coagulation might play a role in the metastasis, we used three-dimensional visualization of direct infusion of fluorescence labeled antibody to observe the interaction of tumor cells with platelets and fibrinogen in isolated lung preparations. Tumor cells arrested in the pulmonary vasculature were associated with a clot composed of both platelets and fibrin(ogen). Initially, the cells attached to the pulmonary vessels were rounded. Over the next 2 to 6 hours, they spread on the vessel surface. The associated clot was lysed coincident with tumor cell spreading. To assess the importance of clot formation, we inhibited coagulation with hirudin, a potent inhibitor of thrombin. The number of tumor cells initially arrested in the lung of hirudin-treated mice was essentially the same as in control mice. However, tumor cell spreading and subsequent retention of the tumor cells in the lung was markedly inhibited in the anticoagulated mice. These associations of the tumor cells with platelets were independent of tumor cell expression of P-selectin ligands. This work identifies tumor cell spreading onto the vascular surface as an important component of the metastatic cascade and implicates coagulation in this process.

Pathway engineering strategies for production of beneficial carotenoids in microbial hosts

Carotenoids, such as lycopene, β-carotene, zeaxanthin, canthaxanthin and astaxanthin have many benefits for human health. In addition to the functional role of carotenoids as vitamin A precursors, adequate consumption of carotenoids prevents the development of a variety of serious diseases. Biosynthesis of carotenoids is a complex process and it starts with the common isoprene precursors. Condensation of these precursors and subsequent modifications, by introducing hydroxyl- and keto-groups, leads to the generation of diversified carotenoid structures. To improve carotenoid production, metabolic engineering has been explored in bacteria, yeast, and algae. The success of the pathway engineering effort depends on the host metabolism, specific enzymes used, the enzyme expression levels, and the strategies employed. Despite the difficulty of pathway engineering for carotenoid production, great progress has been made over the past decade. We review metabolic engineering approaches used in a variety of microbial hosts for carotenoid biosynthesis. These advances will greatly expedite our efforts to bring the health benefits of carotenoids and other nutritional compounds to our diet.

Biomaterials for clinical applications

Coronary Artery Disease.- Stroke.- Pneumonia.- COPD.- Diarrheal Diseases.- HIV/AIDS.- Tuberculosis.- Lung Cancer Chapter 9 Lung cancer .- Traumatic Injuries Chapter 10 Traumatic Injuries .- Prematurity Chapter 11 Prematurity .- Conclusion Chapter 12 Conclusion .

Tissue engineering for clinical applications

Tissue engineering is increasingly being recognized as a beneficial means for lessening the global disease burden. One strategy of tissue engineering is to replace lost tissues or organs with polymeric scaffolds that contain specialized populations of living cells, with the goal of regenerating tissues to restore normal function. Typical constructs for tissue engineering employ biocompatible and degradable polymers, along with organ‐specific and tissue‐specific cells. Once implanted, the construct guides the growth and development of new tissues; the polymer scaffold degrades away to be replaced by healthy functioning tissue. The ideal biomaterial for tissue engineering not only defends against disease and supports weakened tissues or organs, it also provides the elements required for healing and repair, stimulates the bodys intrinsic immunological and regenerative capacities, and seamlessly interacts with the living body. Tissue engineering has been investigated for virtually every organ system in the human body. This review describes the potential of tissue engineering to alleviate disease, as well as the latest advances in tissue regeneration. The discussion focuses on three specific clinical applications of tissue engineering: cardiac tissue regeneration for treatment of heart failure; nerve regeneration for treatment of stroke; and lung regeneration for treatment of chronic obstructive pulmonary disease.

Metabolic engineering for the production of clinically important molecules: Omega-3 fatty acids, artemisinin, and taxol

Driven by requirements for sustainability as well as affordability and efficiency, metabolic engineering of plants and microorganisms is increasingly being pursued to produce compounds for clinical applications. This review discusses three such examples of the clinical relevance of metabolic engineering: the production of omega‐3 fatty acids for the prevention of cardiovascular disease; the biosynthesis of artemisinic acid, an anti‐malarial drug precursor, for the treatment of malaria; and the production of the complex natural molecule taxol, an anti‐cancer agent. In terms of omega‐3 fatty acids, bioengineering of fatty acid metabolism by expressing desaturases and elongases, both in soybeans and oleaginous yeast, has resulted in commercial‐scale production of these beneficial molecules. Equal success has been achieved with the biosynthesis of artemisinic acid at low cost for developing countries. This is accomplished through channeling the flux of the isoprenoid pathway to the specific genes involved in artemisinin biosynthesis. Efficient coupling of the isoprenoid pathway also leads to the construction of an Escherichia coli strain that produces a high titer of taxadiene‐the first committed intermediate for taxol biosynthesis. These examples of synthetic biology demonstrate the versatility of metabolic engineering to bring new solutions to our health needs.

Polysaccharide-Based Tissue Adhesives for Sealing Corneal Incisions

Purpose: To investigate the ability of a novel polysaccharide-based tissue adhesive to seal corneal incisions, and to determine the effect of the tissue adhesive on corneal endothelial cells. Methods: A polysaccharide-based tissue adhesive composed of dextran aldehyde and star PEG amines was applied to a 5-mm corneal incision on an enucleated rabbit eye, and the leak pressure of the eye was measured. The tissue adhesive was additionally incubated in direct contact with bovine corneal endothelial cells to evaluate cytotoxicity. Results: The polysaccharide-based tissue adhesive was successful in sealing corneal incisions to pressures of > 10 psi (500 mmHg). The tissue adhesive was non-cytotoxic to bovine corneal endothelial cells. Conclusions: Polysaccharide-based tissue adhesives are efficacious in sealing corneal wounds and are non-cytotoxic to corneal endothelial cells. Such adhesives represent a promising new technology for ophthalmic surgery.

Tissue scaffold surface patterning for clinical applications.

Patterned scaffold surfaces provide a platform for highly defined cellular interactions, and have recently taken precedence in tissue engineering. Despite advances in patterning techniques and improved tissue growth, no clinical studies have been conducted for implantation of patterned biomaterials. Four major clinical application fields where patterned materials hold great promise are antimicrobial surfaces, cardiac constructs, neurite outgrowth, and stem cell differentiation. Specific examples include applications of patterned materials to (i) counter infection by antibiotic resistant bacteria, (ii) establish proper alignment and contractile force of regrown cardiac cells for repairing tissue damaged by cardiac infarction, (iii) increase neurite outgrowth for central nervous system wound repair, and (iv) host differentiated stem cells while preventing reversion to a pluripotent state. Moreover, patterned materials offer unique advantages for artificial implants which other constructs cannot. For example, by inducing selective cell adhesion using topographical cues, patterned surfaces present cellular orientation signals that lead to functional tissue architectures. Mechanical stimuli such as modulus, tension, and material roughness are known to influence tissue growth, as are chemical stimuli for cell adhesion. Scaffold surface patterns allow for control of these mechanical and chemical factors. This review identifies research advances in scaffold surface patterning, in light of pressing clinical needs requiring organization of cellular interactions.

Engineering Biomaterials for Regenerative Medicine

Preface: The clinical imperative for regenerative medicine(Sujata K. Bhatia, Harvard University) Cellular Recruitment and Delivery CHAPTER 1. Biomaterial surfaces for the isolation of hematopoietic stem and progenitor cells (Srinivas D. Narasipura and Michael R. King, Cornell University) CHAPTER 2. Matrix stiffness: A regulator of cellular behavior and tissue formation (Brooke N. Mason, Joseph P. Califano, and Cynthia A. Reinhart-King, Cornell University) Oxygen Delivery CHAPTER 3. Oxygen supply for tissue engineering (Whitney L. Stoppel and Susan C. Roberts, University of Massachusetts-Amherst) Tuning of Mechanical Properties CHAPTER 4. Adhesion behavior of soft materials (Santanu Kundu, National Institute of Standards and Technology, and Edwin P. Chan, University of Massachusetts-Amherst) CHAPTER 5. PLA-PEO-PLA hydrogels and their mechanical properties(Gregory N. Tew and Surita R. Bhatia, University of Massachusetts-Amherst) Control of Inflammation and Host Response CHAPTER 6. Host response to biomaterials (Anjelica L. Gonzalez-Simon, Yale University, and Omolola Eniola-Adefeso, University of Michigan) CHAPTER 7. Modulation of the wound healing response through oxidation active materials (Paritosh P. Wattamwar and Thomas D. Dziubla, University of Kentucky) Biologically Inspired Materials for Tissue Regeneration CHAPTER 8. Gecko-inspired tape-based adhesives (Woo Kyung Cho, Maria Jose Maio Nunes Pereira, Nora Lang, Kyungheon Lee, Shwetha Mureli, Andreas Zumbuehl, Cathryn Sundback, Peter T. Masiakos, David J. D. Carter, Jeffrey Borenstein, Lino Ferreira, Robert Langer, and Jeffrey M. Karp, Harvard-MIT) CHAPTER 9. Heparin-functionalized materials in tissue engineering applications (Christopher McGann and Kristi Kiick, University of Delaware) Clinical Applications of Tissue Regeneration CHAPTER 10. Tissue engineering strategies for vocal fold repair and regeneration (Alexandra J. E. Farran, Zhixiang Tong, Robert L. Witt, and Xinqiao Jia, University of Delaware) CHAPTER 11. Non-viral gene delivery for applications in regenerative medicine (Kory Blocker and Millicent Sullivan, University of Delaware) CHAPTER 12. Chitosan-based delivery system for tissue regeneration and chemotherapy (Sungwoo Kim and Yunzhi Yang, University of Texas Health Science Center) CHAPTER 13. Conclusion: Translating tissue engineering into successful therapies (Sujata K. Bhatia, Harvard University)

Sealing and Healing of Clear Corneal Incisions with an Improved Dextran Aldehyde-PEG Amine Tissue Adhesive

Purpose: To create a non-cytotoxic, spontaneously curing tissue adhesive that is strongly bonding and persistent enough that 1–2 μL is capable of sealing a clear corneal incision throughout the first five days of healing. Methods: A novel prototype delivery device capable of delivering 1–2 μL of a two-component adhesive delivered aqueous solutions of dextran aldehyde and star PEG amine, which mixed by diffusion and crosslinked to form an adhesive hydrogel. Adhesive hydrogels were tested for rates of degradation in phosphate-buffered saline, leak pressures when used to seal clear corneal incisions in enucleated rabbit eyes, and in vitro cytotoxicity when placed in contact with NIH3T3 fibroblast cells. Two formulations were used in vivo to seal clear corneal incisions in New Zealand White rabbits. Wound integrity after 1, 3, 5 and 7 days of healing was assessed by measuring the leak pressures of enucleated eyes. Results: Tissue adhesives formed by combining aqueous solutions of dextran aldehyde (MW 10,000, 50% oxidized) and an 8-arm star poly(ethylene glycol) (MW 10,000) having two primary amine groups at the end of each arm gave mean leak pressures as high as 141 ± 35 mm Hg and exhibited no in vitro cytotoxicity. When 1–2 μL was used in vivo to seal clear corneal incisions in New Zealand White rabbits, the adhesive maintained an eye leak pressure of at least 120 mm Hg and remained visibly present at the wound site for 5 days. Conclusions: The combination of an 8-arm star poly(ethylene glycol), MW 10,000, having two primary amine groups at the end of each arm and dextran aldehyde (MW 10,000, 50% oxidized) forms a tissue adhesive that cures spontaneously, is non-cytotoxic, and is strongly bonding and persistent enough that 1–2 μL is capable of sealing a clear corneal incision through the first 5 days of healing.

Rolling Adhesion Kinematics of Yeast Engineered To Express Selectins

Selectins are cell adhesion molecules that mediate capture of leukocytes on vascular endothelium as an essential component of the inflammatory response. Here we describe a method for yeast surface display of selectins, together with a functional assay that measures rolling adhesion of selectin‐expressing yeast on a ligand‐coated surface. E‐selectin‐expressing yeast roll specifically on surfaces bearing sialyl‐Lewis‐x ligands. Observation of yeast rolling dynamics at various stages of their life cycle indicates that the kinematics of yeast motion depends on the ratio of the bud radius to the parent radius (B/P). Large‐budded yeast “walk” across the surface, alternately pivoting about bud and parent. Small‐budded yeast “wobble” across the surface, with bud pivoting about parent. Tracking the bud location of budding yeast allows measurement of the angular velocity of the yeast particle. Comparison of translational and angular velocities of budding yeast demonstrates that selectin‐expressing cells are rolling rather than slipping across ligand‐coated surfaces.