Phyllis Wang National Institutes of Health Table of Contents Illustrations 2 Introduction... 4 Scope of the Report. 5 Evaluation Criteria. 5 Evaluation of Scaffold Materials. 8 Natural Scaffold Materials.. 8 Synthetic Scaffold Materials 11 Evaluation of Scaffold Manufacturing Techniques.. 18 Electrospinning.. 19 Molding.. 20 Particulate Leaching. 21 Phase Separation of Emulsions. 22 Recommendations 23 References 26 Evaluation of Novel Tissue Scaffolds for Implantation 1
Phyllis Wang National Institutes of Health Illustrations Figures Figure 1. A. Two units of cellulose joined by a beta glycosidic linkage.. 9 Modified from http://www.mansfield.ohio-state.edu/~sabedon/071amylo.gif B. Two units of alginate joined by a beta-glycosidic linkage. http://www.scientificpsychic.com/fitness/alginate1.gif Figure 2. Two units of hyaluronic acid joined by a beta-glycosidic linkage 9 http://upload.ecvv.com/upload/product/200801/2007621142832477566_ Hyaluronic_Acid_Heparin_Sodium.jpg Figure 3. Triple-helical structure of native collagen 10 http://www.3dchem.com/imagesofmolecules/collagen2.jpg Figure 4. Fibrin, in turquoise, holds together platelets (purple) and red blood cells (red)... 11 http://www.bss.phy.cam.ac.uk/~jrb75/fibrin.jpg Figure 5. A general ester bond. 11 http://en.wikipedia.org/wiki/file:ester-general.svg Figure 6. PGA, PLA, and PLGA monomers 12 http://www.drugdeliverytech.com/media/publicationsarticle/001446.jpg Figure 7. Cross-sectional view of compact and trabecular bone. 13 Modified from http://www.onjcenter.com/images/bone.jpg Figure 8. Crystalline hydroxyapatite 14 http://content.answcdn.com/main/content/img/elsevier/vet/gr95.jpg Figure 9. Powdered tricalcium phosphate 14 http://www.runkai.net/en/cp/cp3-1.jpg Figure 10. Water molecule showing electrical charges.. 15 http://www.amnh.org/learn/courses/images/w3e1_1.jpg Figure 11. Structure of polyethylene glycol, one monomer shown. 17 http://upload.wikimedia.org/wikipedia/commons/4/42/polyethylene_glycol.png Evaluation of Novel Tissue Scaffolds for Implantation 2
Phyllis Wang National Institutes of Health Figure 12. Structure of polyacrylamide, one monomer shown 17 http://www.tiptheplanet.com/images/c/c9/poly.png Figure 13. Diagram of general electrospinning apparatus... 19 Modified from http://www.ciam.unibo.it/polymer_science/images/ electrospinning.jpg Figure 14. Linearly oriented fibers on rotating metal collector... 20 http://www.people.vcu.edu/~glbowlin/images/electrospinning.jpg Figure 15. Phase separation apparatus. 22 Modified from A.L. Wooten, Agglomeration of cell-loaded polymeric porous microbeads as a precursor to in vitro tissue generation. PhD rotation paper, Washington University, 2010. Tables Table 1. Sources and degradation times of linear aliphatic polyester polymer 13 Table 2. Majors classes of cross-linking initiators and representative chemicals 16 All images modified from http://en.wikipedia.org Table 3. Effects of various process parameters on electrospun membranes 20 Table 4. Evaluation of Scaffold Materials... 24 Table 5. Evaluation of Scaffold Manufacturing Technique. 24 Evaluation of Novel Tissue Scaffolds for Implantation 3
Phyllis Wang National Institutes of Health Introduction Every year, approximately 9000 Americans die while waiting for an organ transplant [1]. The reason for this high mortality is that a patient who suffers serious organ damage because of disease, accident, or congenital defect has very limited treatment options. Often, receiving a graft or transplant from a human or animal donor are the patient s only choices. These procedures carry substantial risks: In addition to undergoing invasive and traumatizing surgery, patients who survive a transplant operation must take immune-suppressing drugs for the remainder of their lives to prevent organ rejections. These drugs increase patients susceptibility to other diseases such as bacterial infections and do not always work. Even if the drugs successfully prevent rejection, the transplanted organ itself still wears out within five to ten years [2], sending the patient back to the operating room to undergo yet another dangerous surgical procedure. A severe and prolonged shortage of donor organs exists in the United States and around the world. Because of this shortage and the risks associated with transplant operations, the medical industry needs to develop better alternatives to organ grafts and transplants, and the field of tissue engineering has emerged in the past 20 years to fill this gap. Tissue engineering seeks to create replacements for biological tissues that can repair or enhance existing tissue. Tissue engineering often attempts to integrate living cells with manmade materials to create structures that mimic the target tissue s original biological functions. To encourage research in this field, in 2009, the U.S. Congress has passed the Organ Transplant Shortage Act, an $80 million bill funding tissue engineering research. Within the field of tissue engineering, implantable tissue scaffolds are manmade structures that assist the body in regenerating damaged or missing tissue. To be effective, a scaffold must be physically and chemically compatible with a wound site, degradable by the human body s natural enzymatic processes, structurally stable for the time required to regenerate a damaged tissue, and bioactive to encourage cellular growth and differentiation. Evaluation of Novel Tissue Scaffolds for Implantation 4
Phyllis Wang National Institutes of Health Scope of the Report At the request of the U.S. Senate s Health, Education, and Labor Committee, the Center for Scientific Review (a division of the National Institutes of Health), has prepared this recommendation report evaluating the medical feasibility of new tissue scaffolds and presenting funding recommendations. This report addresses the following topics: Significant natural and synthetic scaffold materials scientists are currently studying. Benefits and shortcomings of each material s physical properties. Important manufacturing techniques scientists are using to fabricate novel scaffolds. Advantages, disadvantages, and potential applications of each technique. Based on these areas, the report rates each scaffold material and manufacturing method according to several key criteria and issues recommendations regarding which scaffolds show the most promise for medical use and thus should receive the most funding. Evaluation Criteria In the context of clinical implantation in patients, the NIH will utilize the following six criteria in this report, listed in order of importance, to evaluate tissue scaffolds and their manufacturing techniques: 1. Compatibility capability of two materials to coexist and function in the same environment. In the context of tissue engineering, compatibility requires that an implanted scaffold be both nontoxic and immune neutral. Nontoxic means that the scaffold material does not harm or interfere with the human body s internal functions. Immune neutral means that the scaffold material does not trigger an inflammatory response from the patient s immune system. The inflammatory response is the immune system s defense against foreign biological invaders such as viruses or bacteria. Our immune system tends to recognize and destroy any foreign organic material that enters the body, whether harmful or not. This hostile response is the basis of organ transplant rejection [2], and our immune system s sensitivity makes compatibility a particular challenge when implementing natural scaffold materials. Evaluation of Novel Tissue Scaffolds for Implantation 5
Phyllis Wang National Institutes of Health 2. Biodegradability methods and time over which natural processes break down a material. As patients participate in daily activities such as walking, sitting and bending over, implanted materials undergo structural wear and tear that alter an implant s shape and location within the body. Damaged implants eventually cause pain and require surgical removal. Therefore it is dangerous to leave a tissue scaffold implanted indefinitely in situ. On the other hand, removing and replacing a scaffold poses its own dangers. After a scaffold has been implanted and native tissues, organs, and blood vessels have grown around the scaffold, it is impossible to remove the scaffold without causing severe trauma to the implant site. Biodegradability requires that the human body s own enzymatic processes be able to digest an implanted scaffold material. Ideally, a particular scaffold will degrade at the same rate its surrounding tissue grows [3]. Furthermore, the residues of this breakdown process must be nontoxic, immune compatible, and excretable by the liver and kidneys. 3. Structural Stability a material s ability to maintain its original form over time. An implanted scaffold must be stable over its intended lifetime because a localized defect in a scaffold, such as shrinkage, can become a defect in the regenerated tissue such as lower bone density. 4. Bioactivity ability to actively promote cellular growth, differentiation, and repair. In the past, scientists pursued scaffolds that were as biologically inert as possible to avoid the pitfalls of toxicity and immune incompatibility. Today, the field of tissue engineering has undergone a reversal and is now seeking to develop implants that are not only fully compatible with the human body, but that also dramatically improve the rate of tissue regrowth. This quest for higher bioactivity is the driving force behind development of new scaffold materials and manufacturing methods. Bioactivity is also driving research toward smaller and smaller physical scales. Because oxygen dissolved in blood can travel only about 200 µm [4], or the width of several human hairs, cells growing in the center of an implanted scaffold are likely to become Evaluation of Novel Tissue Scaffolds for Implantation 6
Phyllis Wang National Institutes of Health hypoxic, or oxygen-starved, and die within several days. To overcome this problem, scientists are increasingly pursuing micro-sized scaffolds and experimenting with different physical structures and chemical signaling compounds to encourage new blood vessels to grow into and around a porous scaffold. 5. Applications potential uses in various parts of the body for different types and sizes of defects. Different physical properties make each scaffold material suitable for different tissue repair sites. For example, fibrous scaffolds are best for muscle repair because they are structurally similar to muscle fibers, while minerals are best for bone repair because bones contain high mineral content [3]. The most promising scaffold materials and manufacturing techniques will be those that have either a broad range of potential applications or demonstrated high applicability to a specific tissue type. 6. Cost expenses incurred or saved during materials processing, manufacturing, and clinical implementation. Three key factors influence the cost of an implant. The first is customization. Medical treatments are becoming increasingly personalized, and tissue engineering is no exception to this trend. We anticipate that future implantable scaffolds will be tailormade for each individual patient in two ways: (1) custom molded to fit specific wound sites and (2) seeded with a patient s own stem cells, tissue grafts, or signaling molecules to maximize bioactivity and minimize rejection. The more customized an implant is, the higher its cost. A second factor influencing cost is manufacturing. Mass production is the most effective way of manufacturing any product, so scaffolds that are easy to mass-produce will be the most cost effective in the long run. In issuing funding recommendations, we will discuss potential challenges in scaling up current scaffold fabrication processes. Evaluation of Novel Tissue Scaffolds for Implantation 7
Phyllis Wang National Institutes of Health The third factor that influences implant cost is clinical implementation. The more invasive the implantation procedure, the higher the hospitalization, surgery, and recovery costs. Scaffolds that require minimal surgery will be both cost effective and safer for patients. Evaluation of Scaffold Materials The NIH broadly classifies scaffold materials into two categories natural materials, which come from existing biological matter, and synthetic, or manmade materials. The following two sections of this report describe the physical properties of the most significant natural and synthetic scaffold materials scientists are currently studying and evaluate the benefits and drawbacks of each material. Natural Scaffold Materials In general, the advantage of using natural scaffold materials in tissue scaffolds is that natural materials tend to have high bioactivity. The disadvantage of natural materials is that they are more likely than synthetic materials to exhibit immune incompatibility. Immune incompatibility occurs because organic matter contains molecule-sized chemical markers known as antigens. Antigens are typically small proteins or sugars attached to the surface of cells. The human immune system recognizes these antigens as foreign invaders and attacks them [2], causing implant rejection. Therefore, the most important area of natural scaffold research is to mitigate immune incompatibility. Three of the most promising natural scaffolds are polysaccharides, collagen, and clotting factors. Polysaccharides The prefix poly- means many, and -saccharide refers to sugars. Polysaccharides are multiunit sugars. The most common polysaccharides consist of carbon rings joined to one another by chemical bonds, called glycosidic linkages. A single polysaccharide molecule may contain thousands of rings, giving the molecule fibrous properties. Glucose, the main component of cane Evaluation of Novel Tissue Scaffolds for Implantation 8
Phyllis Wang National Institutes of Health sugar used in everyday cooking, is an example of a polysaccharide. In tissue engineering, the most popular polysaccharides are alginate and hyaluronic acid. Alginate is a component of the cell walls of brown algae, a plant that lives in shallow ocean areas [5]. Its structure is similar to that of cellulose, or common plant fiber. Like cellulose, alginate s main function is to provide structural stability. And like cellulose, alginate s physical strength comes from the bond connecting sugar units to one another A B Figure 1. A. Two units of cellulose joined by a beta glycosidic linkage. B. Two units of alginate joined by a betaglycosidic linkage. (compare Figure 1A and 1B). This bond is called a beta-glycosidic linkage, and it is strong because it resists chemical breakdown. In fact, doctors recommend eating vegetables while dieting because cellulose fibers are difficult for humans to digest and thus do not provide as many calories as other foods. In the context of tissue engineering, alginate s structural stability makes it an excellent candidate for building scaffolds. Because tissue repair and regeneration take months to years to complete, a scaffold must be structurally stable for such an extended period of time. Furthermore, alginate promotes wound healing. In fact, the medical industry already incorporates alginate into bandages for wrapping burn wounds. Hyaluronic acid is a component of human connective tissue. Connective tissue includes ligaments, joints, and tendons. The two roles of connective tissue in the body are to hold bones, muscles, and organs in place and to facilitate movements such as walking and running [2]. Figure 2. Two units of hyaluronic acid joined by a beta-glycosidic linkage. Evaluation of Novel Tissue Scaffolds for Implantation 9
Phyllis Wang National Institutes of Health Hyaluronic acid has a similar structure to alginate and cellulose, including betaglycosidic linkages that render it structurally stable (see Figure 2). Research has found that hyaluronic acid contributes to cartilage growth and sunburn repair, making it an excellent candidate for improving the rate of tissue regeneration [3]. The cosmetics industry already incorporates small concentrations of dissolved hyaluronic acid into facial lotions, toners, and masks as a wrinkle-reducer. Hyaluronic acid also demonstrates a favorable biodegradability profile, meaning that scientists understand how the human body breaks it down. For example, we know that the average person contains about 15 grams of hyaluronic acid, five of which break down and regenerate every day [6]. Collagen Figure 3. Triple-helical structure of native collagen. Collagen, the second most promising natural scaffold material, is the major component of the extra-cellular matrix, or ECM, found in all animal cells including humans. The extra-cellular matrix is a fluid-filled bath that surrounds animal tissues. Its purpose is to provide structural support, assist cell-to-cell communication, and direct cell growth and repair [2]. This last role, directing growth and repair, makes collagen highly bioactive and therefore an attractive candidate for tissue scaffold engineering. Collagen s structure is a fibrous coil consisting of three protein strands wrapped around each other (see Figure 3). However, collagen is not useful in its native form and must undergo processing before implementation as a scaffold. This processing denatures, or unravels, the coil so that engineers can reshape it into a desired structure. Processing requires dissolving powdered collagen in boiling water, pouring the liquid mixture into a mold, and letting the mixture resolidify by cooling. The first step, boiling in water, unravels the triple coil, thus reducing collagen s structural stability and changing its biodegradability profile. The most abundant source of denatured collagen gelatin usually comes from the bones and hooves of pigs, and thus poses immune compatibility challenges [2]. Major directions in collagen research include characterizing the biodegradability of gelatin and improving its structural stability by hybridizing gelatin with stronger materials. Evaluation of Novel Tissue Scaffolds for Implantation 10
Phyllis Wang National Institutes of Health Clotting factors Clotting factors are a class of molecules involved in the first step of wound healing, namely forming a clot, or scab, to prevent further blood loss. Clotting factors include numerous substances, but tissue scaffold engineers are primarily interested in two factors: fibrinogen and thrombin [3]. When combined, these two factors react to form a solid fibrous mesh called fibrin, shown in turquoise in Figure 4. Fibrin s role is to bind together blood cells and other tissues [2]. Its binding abilities give it potential as a scaffold material. However, fibrin biodegrades extremely rapidly; it begins to break down within several days after forming, and completes its degradation process within several weeks [3]. Fibrin s short life span makes it more applicable for short-term or emergency tissue regeneration treatments and less applicable for long-term therapy. An alternative direction of clotting factor research is to combine fibrin with more durable substances such as polysaccharides or various synthetic materials to slow down its biodegradability. Figure 4. Fibrin, in turquoise, holds together platelets (purple) and red blood cells (red). Synthetic Scaffold Materials Polysaccharides, collagen, and clotting factors demonstrate that the major weaknesses of using natural implant materials are immune incompatibility and structural stability. To mitigate these problems, scientists have increasingly turned to synthetic scaffold materials. This section describes the properties of three of the most promising synthetic scaffolds: polyester polymers, calcium phosphates, and hydrogels. Polyester Polymers Polyesters are chemical compounds that contain multiple ester bonds. An ester bond joins two chemical groups and contains one carbon atom bonded to two oxygen atoms (see Figure 5). R and R denote generic chemical groups, Figure 5. A general ester bond. which usually include additional carbon atoms. Polyesters already have many applications, particularly in the clothing and furniture industries. Evaluation of Novel Tissue Scaffolds for Implantation 11
Phyllis Wang National Institutes of Health In the context of tissue engineering, the most popular synthetic scaffold materials are linear aliphatic polyester polymers [2]. Polymers are large molecules made up of smaller repeating units connected by chemical bonds, similar to polysaccharides. Aliphatic means fatty, which describes polyesters composed primarily of the elements carbon and hydrogen. The primary reason for the popularity of these polyester polymers is that the FDA has already approved three polymers for limited clinical use in humans [3]: Poly-glycolic acid (PGA) Poly-lactic acid (PLA) Poly-lactic-co-glycolic acid (PLGA) Figure 6 shows the repeating unit, or monomer, of each polyester. The n and m denote varying numbers of monomers. Chemists synthesize these Figure 6. PGA, PLA, and PLGA monomers. compounds using byproducts from oil purification, and they can control the length of the polymers (the values of n and m) by adjusting reaction conditions including temperature, pressure, presence of a catalyst, and concentrations of starting materials. The human body contains enzymes such as butyrylcholinesterase and acetylcholinesterase that degrade esters by hydrolysis, a reaction that adds water to compounds containing ester bonds [2]. After implantation in the body, poly-glycolic acid (PGA) breaks down within several months via this ester hydrolysis mechanism [4]. On the other hand, poly-lactic acid (PLA) takes several years to break down [4]. The reason for their differing biodegradability is the differing strength of the ester bonds: Each unit of poly-lactic acid contains an additional -CH 3 group compared to poly-glycolic acid. This -CH 3 group repels water, slowing down the hydrolysis process. Differing biodegradability of PLA and PGA allows chemists to carefully tune the degradation time frame of a scaffold by using PLGA, poly-lactic-co-glycolic acid. PLGA contains both PLA and PGA units. By varying the ratio of PLA to PGA, chemists can synthesize polymers that take from several months to several years to degrade. This flexibility renders polyester polymers attractive for a wide range of short term to long term tissue engineering applications. Evaluation of Novel Tissue Scaffolds for Implantation 12
Phyllis Wang National Institutes of Health Polyester polymers also offer versatility in manufacturing. Newly-synthesized polymers come in powder or granulated form, and they can be dissolved, molded, or even woven into many different structures with different physical properties. Other non-fda-approved polyester polymers include poly-caprolactone, poly-hydroxyl butarate, and poly-propylene fumarate. Properties of these polyesters are summarized in Table 1. Table 1. Sources and degradation times of linear aliphatic polyester polymers. [2] Compound Abbreviation Degradation Time Source Poly-glycolic acid PGA 2-3 months petroleum Poly-lactic acid PLA 1-2 years petroleum Poly-lactic-co-glycolic acid PLGA 2 months-2 years petroleum Poly-caprolactone PCL > 2 years petroleum Poly-hydroxyl butyrate PHB > 2 years fermentation Poly-propylene fumarate PPF unknown petroleum Calcium Phosphates Calcium phosphates are a group of inorganic minerals that make up about half of the total bone mass in humans [2]. While each bone has a unique structure, all bones generally consist of two layers: a solid outer layer called compact bone and a porous, soft core called trabecular bone, which includes the bone marrow (see Figure 7). Calcium phosphate forms porous networks that provide structural rigidity in both layers, but is denser and more abundant in the compact outer layer. Within the trabecular layer, soft tissues including blood vessels, collagen, and growth factors contribute to a spongy, flexible texture. The bone marrow is the site of greatest cell growth and cell turnover. Although both layers are porous, trabecular bone is much more so, with a porosity of over 30% [2]. The most popular calcium phosphates for tissue engineering are calcium hydroxyapatite and tricalcium phosphate. Figure 7. Cross-sectional view of compact and trabecular bone. Evaluation of Novel Tissue Scaffolds for Implantation 13
Phyllis Wang National Institutes of Health Calcium hydroxyapatite is the natural form of calcium phosphate found in human bone. Its molecular formula is Ca 10 (PO 4 ) 6 OH 2, and in its pure form, appears as sharp, solid crystals (see Figure 8). Commerical hydroxyapatite comes from two sources: The first and most common source is by direct synthesis, where chemists allow calcium and phosphorous precursors calcium Figure 8. Crystalline hydroxyapatite. (http://content. answcdn.com/main/content/img/els evier/vet/gr95.jpg) carbonate CaCO 3 and ammonium hydrogen phosphate (NH 4 ) 2 HPO 4 to react in solution, forming hydroxyapatite as a solid precipitate [7]. The second method involves heating coral skeletons, which contain calcium carbonate, to initiate their transformation into hydroxyapatite [7]. The heating process burns away protein residues, preventing immune incompatibility. However, hydroxyapatite derived from coral tends to be structurally weaker and less uniform than hydroxyapatite synthesized in the laboratory. Tricalcium phosphate, molecular formula Ca (PO ) is 3 4 2, called bone dust because it forms when burning calcium hydroxyapatite. In its pure state, tricalcium phosphate appears as a fine white powder (see Figure 9). It already has several medical applications as an antacid and calcium dietary supplement. Tricalcium phosphate exists in nature as a rock mixed with sandstone and phosphorous oxides [8]. Chemists can also synthesize tricalcium phosphate directly by reacting calcium and phosphoric acid. Figure 9. Powdered tricalcium phosphate. (http://www.runkai.net/ en/cp/cp3-1.jpg) The advantage of calcium phosphates is that both hydroxyapatite and tricalcium phosphate are osteoconductive and osteoinductive. Osteoconductive refers to their ability to promote both bone cell adhesion and differentiation, while osteoinductive refers to their ability to induce new bone Evaluation of Novel Tissue Scaffolds for Implantation 14
Phyllis Wang National Institutes of Health cell growth. Studies have shown that osteoconductivity arises from the attachment of bone morphogenetic proteins that direct the formation of new bone cells [3]. On the other hand, the major challenge of working with calcium phosphates is mimicking the porous structure of natural bone. Pure calcium phosphates are usually solid crystals with geometry dictated by atomic size and spacing. These crystals are brittle, meaning they fracture easily and are thus difficult to mold into alternate forms. Promising calcium phosphate research seeks to combine calcium phosphates with more flexible materials such as collagen in order to improve elasticity. Hydrogels Hydrogels are cross-linked polymers that can absorb a great deal of water, up to 99%, without dissolving into liquid form [3]. Cross-linking is a chemical process that transforms linear polymer chains into an interconnected, three-dimensional network by forming bonds between polymer chains. Cross-linking typically enhances the mechanical and thermal stability of materials. This enhanced stability allows hydrogel polymers to hold water while still maintaining solid form, making hydrogels capable of filling tissue defects of various sizes and shapes. The degree of cross-linking within a hydrogel correlates directly with its mechanical strength and inversely with the amount of water it can hold: the more extensively linked a gel, the stronger the gel is but the less water it can absorb. Hydrogels are advantageous in scaffold engineering because the human body contains about 90% water [2], allowing hydrogels to achieve high compatibility and bioactivity by providing a favorable environment for regenerative agents including stem cells, signalling molecules, and growth factors. Designing hydrogels requires a tradeoff between bioactivity from water content and structural stability from mechanical strength. Figure 10. Water molecule showing electrical charges. Most hydrogel polymers are anionic, meaning they contain negative electric charge. These negative charges are key to hydrogels ability to absorb water: Water molecules of molecular formula H 2 O are highly electrically polar, with oxygen atoms carrying partial negative charges and hydrogen atoms carrying partial positive charges (see Figure 10). Because opposite electrical charges attract, negatively charged polymers can bind to the Evaluation of Novel Tissue Scaffolds for Implantation 15
Phyllis Wang National Institutes of Health positively charged hydrogen atoms of water. Since polymers contain many subunits and therefore many sites of negative charge, a hydrogel can bind many water molecules, resulting in a high water-to-polymer ratio. Chemists can control the water content of a hydrogel by adjusting the concentration of cations, or positively charged species, in the hydrogel mixture. Metal cations such as sodium, Na +, and calcium, Ca 2+, carry stronger positive charge than the hydrogen atoms of water. In competition with hydrogen, these cations bind preferentially to negatively charged polymers, neutralizing some of the polymers negative charges and preventing those sites from binding water. The more cations in a hydrogel mixture, the less water it can absorb. Chemists also control the water content of hydrogels by increasing or decreasing the amount of cross-linking present. Cross-linking requires the use of a chemical initiator, a highly reactive compound that breaks previously stable bonds between polymer units, allowing the formation of new bonds. Because these cross-linking agents are very reactive, a major safety concern about hydrogels is the potential toxicity of cross-linking initiators. Table 2 shows the most common initiators and briefly describes their toxicity concerns. Table 2. Majors classes of cross-linking initiators and representative chemicals. [9] Initiator Class Representative Chemical Toxicity Concerns Acetic Corrosive, flammable, Acid anhydrides anhydride respiratory irritant Aldehydes Azo-compounds Glutaraldehyde Azobenzene Corrosive, respiratory irritant Mutagen (causes DNA mutations) Peroxides Photoinitiators Hydrogen peroxide Nitrogen dioxide + UV radiation Corrosive, explosive, respiratory irritant Carcinogen (causes cancer) X-ray radiation N/A Carcinogen (causes cancer) Evaluation of Novel Tissue Scaffolds for Implantation 16
Phyllis Wang National Institutes of Health The two major classes of hydrogels used in tissue scaffolds are polyethylene glycol (PEG) and polyacrylamide derivatives: Polyethylene glycol ( PEG) polymers are available in a wide range of polymer lengths, from tens to several hundreds of subunits long. Solutions containing longer polymers typically have higher viscosity, or fluid thickness, than solutions containing shorter polymers. Figure 11 shows the chemical structure of one subunit of polyethylene glycol. Note the presence of oxygen atoms, which bind hydrogen in water. In addition Figure 11. Structure of polyethylene glycol, one monomer shown. to toxicity of cross-linking agents, PEG has the disadvantage of being non-biodegradable. Current studies seek to improve its degradability by combining PEG with degradable polyesters or by adding degradable bonds to the carbon backbone of the polymer [3]. Polyacrylamide (Figure 12) and its derivatives currently have applications as cosmetic wrinkle-fillers and contact lens materials. Like polyethylene glycol, polyacrylamide is not biodegradable in its pure form [3]. An additional toxicity concern unique to polyacrylamide is toxicity Figure 12. Structure of polyacrylamide, one monomer associated with the precursor monomer, acrylamide. In its shown. unpolymerized form, free acrylamide is a potent neurotoxin a dangerous chemical that damages nerve cells [10]. Once polymerized however, polyacrylamide is non-toxic and chemically unreactive. While polymerization reactions typically go to completion, trace amounts of unreacted acrylamide may remain, posing health risks when implanted. Scientists also harbor concerns regarding the potential release of free acrylamide into the body if further technological development succeeds in making polyacrylamide biodegradable [3]. Evaluation of Novel Tissue Scaffolds for Implantation 17
Phyllis Wang National Institutes of Health Evaluation of Scaffold Manufacturing Techniques In addition to containing compatible, degradable, stable, and bioactive materials, a tissue scaffold must also be the correct size and shape for a particular wound site. The physical property all scaffolds must have, regardless of their final implant location, is porosity. Porosity is important for two reasons: 1. A porous structure contains higher surface area than a solid structure of the same volume, allowing more cells to attach, grow, and replicate. Providing sufficient space for cell growth is critical because cells secrete signaling molecules that cause neighboring cells to die if grown in overcrowded conditions [2]. 2. A porous structure allows the diffusion of nutrients, wastes, oxygen, and carbon dioxide in and out of the scaffold. Diffusion is the movement of a chemical, usually a liquid or gas, from an area of high concentration to an area of low concentration. In the human body, diffusion is the principle mechanism by which substances move between the bloodstream and the tissues. The diffusion limit, or range, of oxygen in blood is about 200 µm, or the width of several human hairs [4]. Therefore in order to be viable, a segment of tissue must be in close proximity to a blood vessel. A porous scaffold provides enough room for blood vessels to grow inward and transport substances to cells growing at the center of a scaffold. To achieve a porous structure, researchers have developed the following four general synthetic strategies: Electrospinning Molding Particulate leaching Phase separation of emulsions The following sections describe each synthetic protocol, which materials can undergo each protocol, and what physical properties the final products exhibit. Evaluation of Novel Tissue Scaffolds for Implantation 18
Phyllis Wang National Institutes of Health Electrospinning Many tissues in the body including nerve cells, muscle, and connective tissue have fibrous or threadlike structures. Electrospinning is a synthetic technique that produces networks of fibrous mesh with higher surface area than conventional textile manufacturing techniques. The key to the higher surface area and higher Figure 13. Diagram of general electrospinning apparatus. bioactivity of electrospun membranes is the small diameter of electrospun thr eads. Conventional textile manufacturing, such as silk spinning, typically produces threads at least several hundred micrometers wide. On the other hand, electrospinning achieves nano- or micrometer thin fibers by using high voltage to transform liquid solutions of dissolved polymer into threads. First, droplets of dissolved polymer solution exude slowly through a syringe and needle. The needle connects to a high voltage source, which imparts electrical charge to the polymer droplet. An oppositely charged metal collector, situated about five to ten inches away from the needle, attracts the charged droplet, forcing the droplet to elongate into a thin thread and accelerate toward the collector (see Figure 13). As the thread travels toward the collector, the liquid solvent in the thread evaporates, leaving behind a solid polymer thread. Using a syringe pump a machine that ejects fluid through a syringe at a constant low rate provides a steady flow of polymer solution that becomes a long, continuous thread. Electrospinning is highly sensitive to process conditions such as polymer solution viscosity, voltage, collector-needle distance, syringe pump rate, temperature, and humidity. Adjusting these process conditions produces membranes with different properties. Table 3 summarizes the effects of these parameters on the final membrane. Evaluation of Novel Tissue Scaffolds for Implantation 19
Phyllis Wang National Institutes of Health Table 3. Effects of various process parameters on electrospun membranes. Parameter Effect on Membrane Polymer solution viscosity Higher viscosity = thicker threads Voltage (5 to 30 kilovolts) Collector-needle distance Syringe pump rate Temperature Humidity Higher voltage = thinner threads Shorter distance = denser mesh Faster pump rate = thicker threads and denser mesh Higher temperature = thinner threads Higher humidity = thicker threads Both natural and synthetic polymers can undergo electrospinning. Popular electrospinning candidates include poly-glycolic acid, poly-lactic-co-glycolic acid, poly-caprolactone, collagen, and fibrinogen [3]. The structure of the membrane changes depending on the shape of the collector. The stationary metal plate shown in Figure 13 collects Figure 14. Linearly oriented fibers on rotating metal collector. randomly oriented fibers. A rotating collector can collect linearly oriented fibers, seen in Figure 14. The major drawback of electrospinning is that thus far, it produces only flat two-dimensional membranes and cannot yet produce three-dimensional scaffolds. Attempts to produce three- on top of one another have dimensional scaffolds by layering multiple membranes yielded mechanically weak structures [3]. Molding Unlike electrospinning, molding can produce three-dimensional scaffolds. In general, molding works by pouring a dissolved scaffold material into a mold, letting the material harden, and removing the mold to leave behind a scaffold with a specific shape and internal pore structure. Evaluation of Novel Tissue Scaffolds for Implantation 20
Phyllis Wang National Institutes of Health One of the most promising molding techniques described by Ma [11] uses small spheres of paraffin wax as a template. When poured into a mold such as a small beaker or funnel, paraffin spheres tend to sink to the bottom of the mold and pack together in an orderly fashion. When heated briefly, these aggregated spheres melt slightly and stick together, producing a continuous spherical network. After removal of the paraffin template, the gaps in the scaffold left behind form an orderly and interconnected porous network. Changing the size of the template paraffin spheres produces different sizes of pores in the final scaffold, while changing the scaffold material produces different mechanical properties. To undergo paraffin molding, a scaffold material must be soluble in a liquid that does not dissolve paraffin wax. Varying solubility makes it possible to remove the paraffin template after the scaffold hardens without dissolving the scaffold itself. Paraffin wax dissolves in specific hydrocarbon-based solvents such as paint thinner, making it incompatible with some organic polymers. Finding alternatives to paraffin for use as templates is a promising direction of scaffold engineering. Particulate Leaching Particulate leaching is similar to molding in that it uses a template material to produce a porous structure. In particulate leaching, developed by Mikos and Temenoff [12], salt crystals occupy spaces that become pores upon removal of the salt. Salt crystals have the distinct advantage of being soluble in water. Since most scaffold materials are insoluble or only slightly soluble in water (to remain structurally stable when implanted in the body), soaking a dried scaffold in water successfully removes the salt particles without affecting the scaffold. Thus, particulate leaching is applicable to a wide range of scaffold materials. Particulate leaching can produce a range of pore sizes depending on the size of the salt crystals used and the ratio between salt and scaffold material. A major drawback of particulate leaching is that current technology cannot yet finely control pore shape and inter-pore connections. Evaluation of Novel Tissue Scaffolds for Implantation 21
Phyllis Wang National Institutes of Health Phase Separation of Emulsions Many scaffold materials form emulsions when mixed with aqueous solutions. An emulsion is a mixture of two liquids that do not dissolve well in one another. In an emulsion, the less abundant liquid typically forms spherical droplets within the more abundant liquid. Everyday examples of emulsions include mayonnaise (oil droplets in lemon juice) and vinaigrette salad dressing (oil droplets in vinegar). Forming an emulsion requires vigorous agitation such as shaking to force two incompatible liquids to mix. A common example is shaking salad dressing containers to resuspend the oily top layer with the heavier bottom layer containing spices. Aqueous solutions are liquids that contain mostly water. Because scaffold materials are insoluble in water, chemists usually dissolve scaffold materials in hydrocarbon-based organic solvents such as dichloromethane or toluene. These solutions are immiscible, or insoluble, with water. Emulsions of organic and aqueous solutions, like vinaigrette sauces, are unstable and settle out over time into separate layers. The principle of unstable emulsions is useful for fabricating tissue scaffolds because when the aqueous layer is less abundant than the organic layer, the aqueous layer forms droplets that become pores upon removal. The following synthetic protocol from Choi [13] uses poly-lactic-co-glycolic acid and dissolved gelatin as an illustrative example of the phase separation technique: 1. Poly-lactic-co-glycolic acid dissolves in an organic solution of dichloromethane, forming a 2% polymer solution which is insoluble in water. 2. Powdered gelatin (denatured collagen) dissolves in water, forming a 15% aqueous gelatin solution, the less abundant aqueous layer. 3. A handheld emulsifier mixes the two solutions vigorously, forming a uniform mixture containing spherical aqueous droplets suspended in a continuous organic solution. Figure 15. Phase separation apparatus. Evaluation of Novel Tissue Scaffolds for Implantation 22
Phyllis Wang National Institutes of Health 4. After pouring the emulsion into a syringe and letting it sit, the emulsion begins to separate into two layers. The top layer grows richer in aqueous gelatin, while the bottom layer grows poorer in gelatin. 5. A syringe pump exudes droplets from the top layer through a narrow needle into a collection bath containing cold water (see Figure 15). 6. After stirring overnight in the cold water bath, the dichloromethane solvent evaporates, allowing the droplets to harden into PLGA beads containing gelatin. 7. Soaking the beads in hot water dissolves the gelatin droplets, which gradually seep out of the PLGA beads, leaving behind interconnected pores. The overall size of these beads depends on the inner diameter of the needle through which the precursor droplets pass. The principle behind PLGA bead synthesis is that each bead acts as a miniature scaffold that can be injected through medical needles directly into the wound site of a patient without the need for invasive surgery. Producing beads of the correct size merely requires using the correct needle when collecting bead droplets. The standard needle size is 18 gauge, which produces beads several hundred micrometers in diameter, about the width of several human hairs [4]. In vivo (live animal or human) studies have shown that cells seeded into these porous PLGA microbeads can grow and survive when implanted into the shoulders of mice [14]. The next step of microbead scaffold research is to study the behavior of stem cells, signaling molecules, and growth factors in interaction with PLGA beads. A toxicity concern is potential poisoning from residual solvent, dichloromethane, which is a corrosive carcinogen [15]. Recommendations Based on the previous evaluations of scaffold materials and scaffold manufacturing techniques, the following tables tally up the benefits and drawbacks of each scaffold using a numeric system. Each scaffold earns of 1 to 5 for each of the six categories from Scope of the Report, with 1 indicating low potential for success and 5 indicating very high potential for success. Regarding cost, 1 indicates high cost and 5 indicates low cost. Evaluation of Novel Tissue Scaffolds for Implantation 23
Phyllis Wang National Institutes of Health Table 4. Evaluation of Scaffold Materials. Criteria Polysaccharides Collagen Clotting Polyester Calcium Hydrogels Factors Polymers Phosphates Compatibility 3 (immune 3 (immune 3 (immune incompatibility) incompatibility) incompatibility) 5 5 4 Biodegradability 5 3 2 5 5 2 Structural Stability 4 3 2 5 2 3 Bioactivity 5 5 5 4 5 4 Applications 4 4 2 5 3 (Bone only) 4 Cost 3 5 1 2 4 5 Sum 24 23 15 26 21 22 Table 5. Evaluation of Scaffold Manufacturing Techniques. Criteria Electrospinning Molding Particulate Phase Separation Leaching of Emulsions Structural Stability 2 (membranes are 5 5 5 typically millimeters thin) Bioactivity 4 4 2 (pores not 5 connected) Applications 2 (two- dimensional only) 5 5 5 Cost 4 3 (mold destroyed) 5 2 (difficult to scale up) Sum 12 13 12 17 Based on these tabulated values, the most promising scaffold materials are natural polysaccharides and synthetic polyester polymers, while the most promising manufacturing process is phase separation of emulsions. In terms of research funding, the NIH recommends preferentially funding projects with the greatest potential of achieving rapid clinical implementation. To this end, the polyester polymers are particularly attractive because several polymers have already garnered FDA approval for human use, dramatically speeding up the process of clinical implementation. Evaluation of Novel Tissue Scaffolds for Implantation 24
Phyllis Wang National Institutes of Health Funding should also go to projects that develop protocols for efficiently scaling up existing manufacturing processes, which thus far have been carried out almost exclusively in small-scale laboratories. A major roadblock to implementing scaffolds is guaranteeing the viability of cells within large scaffolds. Because the diffusion limit of oxygen in blood is so small, only microscale scaffolds such as polymer beads are currently useful. Larger implants will need to promote the growth of blood vessels in order to become useful for medical applications. The NIH recommends funding fundamental research that studies the growth mechanism of blood vessels in an effort to induce new capillary growth in scaffolds. Finally, because every scaffold material and synthetic technique exhibit unique advantages and disadvantages, the NIH concludes that the most promising scaffolds will be hybrid structures that combine multiple materials, thus taking advantage of favorable properties while mitigating unfavorable properties. The Senate should give special funding priority to daring research proposals that seek to study hybrid scaffold technologies. While tissue engineering is still a long way from replacing organ transplants entirely, scientists around the world are aggressively pursuing new tissue scaffolds, and the NIH is confident that the field of tissue engineering will greatly improve the quality of medical care in the future. Evaluation of Novel Tissue Scaffolds for Implantation 25
Phyllis Wang National Institutes of Health References [1] S. Satel. (2006, May 15). Death s waiting list. New York Times [Online]. Available: http://www.nytimes.com/2006/05/15/opinion/15satel.html [2] K. McCance and S. Huether, Pathophysiology: The Biological Basis for Disease in Adults and Children. St. Louis, Missouri: Mosby, Inc., 2002. [3] P.X. Ma, Scaffolds for tissue fabrication, Materials Today, vol. 7, no. 5, pp. 30-40, May 2004. [4] A.L. Wooten, Agglomeration of cell-loaded polymeric porous microbeads as a precursor to in vitro tissue generation. PhD rotation paper, Washington University, 2010. [5] D.J. McHugh, Production and utilization of products from commercial seaweeds, Food and Agricultural Organization of the United Nations, Rome, FAO 288, 1987. [6] R. Stern, Hyaluronan catabolism: a new metabolic pathway, European Journal of Cell Biology, vol. 83, no. 7, pp. 317-325, Aug. 2004. [7] K. Gross, Synthesis of hydroxyapatite powders, Azomaterials, Warriewood, Australia, July 2004. [8] U.S. Department of Agriculture: Bureau of Plant Industry, Occurrence of beta tricalcium phosphate in northern Mexico. Beltsville, Maryland, 1951. [9] Sigma Aldrich Product Catalog. (2010). Sigma Aldrich [Online]. Available: http:// www.sigmaldrich.com [10] Materials safety data sheets: acrylamide, J.T. Baker, Phillipsburg, New Jersey, MSDS no. A1550, Feb. 2009. [11] P.X. Ma and J.W. Choi, Biodegradable polymer scaffolds with well-defined interconnected spherical pore network, Tissue Engineering, vol. 7, no. 1, pp. 23-33, 2001. [12] A.G. Mikos and J.S. Temenoff. (2000). Formation of highly porous biodegradable scaffolds for tissue engineering. Electronic Journal of Biotechnology [Online]. Available: http://www.scielo.cl/scielo.php?pid=s0717-34582000000200003&script=sci_arttext&tlng =en [13] S.W. Choi, I.W. Cheong, J.H. Kim, and Y. Xia, Preparation of uniform microspheres using a simple fluidic device and their crystallization into close-packed lattices, Small, vol. 5, no. 4, pp. 454-459, 2009. [14] S.W. Choi, Y.C. Yeh, Y. Zhang, H.W. Sung, and Y. Xia, Uniform beads with controllable pore sizes for biomedical applications, Small, vol. 6, no. 14, pp. 1492-1498, 2010. [15] Materials safety data sheets: methylene chloride. J.T. Baker, Phillipsburg, New Jersey, MSDS no. M4420, Aug. 2008. Evaluation of Novel Tissue Scaffolds for Implantation 26