1 00:00:02,370 --> 00:00:08,809 82% of soldiers in battle suffer from traumatic limb injuries. Many of these injuries are 2 00:00:08,809 --> 00:00:15,750 large bone defects. Engineers at MIT are trying to create materials that mimic the function 3 00:00:15,750 --> 00:00:20,810 of the bone's natural healing processes. The structure and properties of these materials 4 00:00:20,810 --> 00:00:27,559 promote bone regeneration. This video is part of the Structure-Function-Properties video 5 00:00:27,559 --> 00:00:33,079 series. The structure, function, and properties of a system are related and depend on the 6 00:00:33,079 --> 00:00:39,679 processes that define or create the system. Hi, my name is Nisarg Shah, and I am a graduate 7 00:00:39,679 --> 00:00:44,820 student in Professor Paula Hammond's lab in the chemical engineering department at MIT. 8 00:00:44,820 --> 00:00:49,999 In the Hammond Lab, we design novel materials for tissue engineering, gene and drug delivery, 9 00:00:49,999 --> 00:00:56,010 and energy applications. My research in particular, focuses on developing and assembling different 10 00:00:56,010 --> 00:00:59,929 materials for bone tissue regeneration. 11 00:00:59,929 --> 00:01:05,930 Before watching this video, you should be familiar with the concepts of pH and pKa, 12 00:01:05,930 --> 00:01:11,000 and with the common chemical functional groups. After watching this video, you will be able 13 00:01:11,000 --> 00:01:17,280 to explain how the concepts of pH and pKa are useful in the design of materials via 14 00:01:17,280 --> 00:01:24,280 layer-by-layer assembly. Many of the materials our lab develops are based on a technique 15 00:01:27,390 --> 00:01:33,740 called layer-by-layer assembly. Layer- by-layer assembly utilizes electrostatic interactions 16 00:01:33,740 --> 00:01:40,530 to create layers of chemical species of alternating charge. These chemical species could be anything 17 00:01:40,530 --> 00:01:47,530 -- polymers, proteins, small molecules -- the key is that they have the appropriate positive 18 00:01:47,660 --> 00:01:52,039 or negative charge to incorporate them into the film. 19 00:01:52,039 --> 00:01:58,020 We assemble these films using a simple dip method. First, we take our substrate or surface 20 00:01:58,020 --> 00:02:03,990 that we wish to coat and bombard it with free radical oxygen in an instrument called a plasma 21 00:02:03,990 --> 00:02:10,169 cleaner. This not only cleans the surface of contaminants, but also leaves the surface 22 00:02:10,169 --> 00:02:17,170 with a negative charge. Next, we take our substrate and dip it into a solution of positively 23 00:02:17,519 --> 00:02:24,040 charged molecules. The molecules in solution bind to the substrate because of Coulombic 24 00:02:24,040 --> 00:02:25,060 interactions. 25 00:02:25,060 --> 00:02:31,219 This process is self-adsorption limited, meaning that once the newly adsorbed layer neutralizes 26 00:02:31,219 --> 00:02:37,359 the charged sites on the surface, additional molecules will not bind. This creates layers 27 00:02:37,359 --> 00:02:40,590 that are on the order of nanometers thick. 28 00:02:40,590 --> 00:02:47,590 After a rinse step to remove any unbound components, the substrate is dipped into a solution of 29 00:02:47,590 --> 00:02:53,510 negatively charged molecules. Interactions with the charges on the previous layer allow 30 00:02:53,510 --> 00:03:00,329 a new layer to be deposited. Again, we would rinse to remove any unbound components. We 31 00:03:00,329 --> 00:03:05,829 can continue this process and dip our growing film into solutions of positively and negatively 32 00:03:05,829 --> 00:03:12,829 charged molecules, with rinse steps in between, building up our coating layer-by-layer. Many 33 00:03:13,349 --> 00:03:19,980 of the films I work with have on the order of 40 layers. We use polymers in many of our 34 00:03:19,980 --> 00:03:26,620 layers. These long chain, high molecular weight molecules form more stable layers than small 35 00:03:26,620 --> 00:03:31,900 molecules. The long polymer chains from one layer weave through other layers creating 36 00:03:31,900 --> 00:03:38,819 an interlocking structure. Also, because we use negatively and positively charged polymers 37 00:03:38,819 --> 00:03:45,819 in these layers, the polymers ionically crosslink, adding to the stability of the overall film. 38 00:03:47,709 --> 00:03:53,430 Polymers whose repeating unit contains a charged group are called polyelectrolytes. Thus, many 39 00:03:53,430 --> 00:03:59,150 people refer to the films that we create as polyelectrolyte multilayers. For every polymer 40 00:03:59,150 --> 00:04:06,150 or small molecule I incorporate into my layer-by-layer assembly, I have to be mindful of it's pKa 41 00:04:06,510 --> 00:04:12,889 and select appropriate assembly conditions to ensure they have the desired charge. For 42 00:04:12,889 --> 00:04:18,660 example, one of the polymers that I use in a layer-by-layer assembly is polyacrylic acid. 43 00:04:18,660 --> 00:04:23,620 You don't need to worry about it's exact chemical structure. The important thing to note is 44 00:04:23,620 --> 00:04:29,870 the repeating carboxylic acid group. Another polymer that I use in my layer-by-layer assembly 45 00:04:29,870 --> 00:04:35,389 contains repeating amine groups. Let's say I'm creating my layer-by-layer assembly at 46 00:04:35,389 --> 00:04:42,389 a pH equal to 4. If the pKa of the carboxylic acid groups on our first polymer is approximately 47 00:04:43,470 --> 00:04:50,470 4.5, and the pKa of the amine groups on our second polymer is approximately 6.5, what 48 00:04:51,120 --> 00:04:56,330 would the charge on each of these polymers be? Pause the video and take a moment to think 49 00:04:56,330 --> 00:05:03,330 about it. At a pH of 4, some of the carboxylic acid groups on our first polymer would be 50 00:05:08,009 --> 00:05:14,539 deprotonated, resulting in a negative charge. At the same pH, some of the amine groups on 51 00:05:14,539 --> 00:05:21,539 our second polymer pick up an extra hydrogen, resulting in a positive charge. Because so 52 00:05:26,819 --> 00:05:31,970 many molecules could be viable candidates for incorporation into these films, they are 53 00:05:31,970 --> 00:05:38,039 being used in a wide variety of applications. My main interest is using these films in tissue 54 00:05:38,039 --> 00:05:39,810 engineering applications. 55 00:05:39,810 --> 00:05:46,060 So, first I have to ask myself, what problem do I want to solve? Then, I have to ask myself 56 00:05:46,060 --> 00:05:52,550 how these multilayer films could be useful. What function do they need to have? What properties 57 00:05:52,550 --> 00:05:58,000 of the film would help to achieve that function? Taking all of this into account, how should 58 00:05:58,000 --> 00:06:00,409 I structure my film? 59 00:06:00,409 --> 00:06:05,770 One of the problems I became particularly interested in is that of large bone defects. 60 00:06:05,770 --> 00:06:10,479 Large bone defects are large gaps in the bone that result from trauma. 61 00:06:10,479 --> 00:06:14,860 Although bone is capable of regeneration, when there are large defects, some sort of 62 00:06:14,860 --> 00:06:19,639 intervention therapy is needed to bridge the defect for proper healing. 63 00:06:19,639 --> 00:06:24,389 These intervention therapies typically involve taking bone tissue either from another location 64 00:06:24,389 --> 00:06:31,060 in the patient's body or from a deceased donor source and grafting it into the defect site. 65 00:06:31,060 --> 00:06:36,720 While bone taken from the patient may be immune compatible and be more viable, this method 66 00:06:36,720 --> 00:06:42,729 has limitations. Tissue injury and trauma at the site of bone removal causes patients 67 00:06:42,729 --> 00:06:48,650 pain and long healing times. Bone tissue from a deceased donor may cause an unfavorable 68 00:06:48,650 --> 00:06:55,490 immune response. The processes used to prepare these tissues for implantation may also compromise 69 00:06:55,490 --> 00:06:57,219 their mechanical properties. 70 00:06:57,219 --> 00:07:00,949 Our lab had an idea for a possible solution to this problem. We asked ourselves, can we 71 00:07:00,949 --> 00:07:01,800 design a scaffold to bridge these large defects that will stimulate the growth of new bone 72 00:07:01,800 --> 00:07:06,979 tissue? The idea was to create a rigid, porous scaffold coated with a multilayer film. The 73 00:07:06,979 --> 00:07:12,530 multilayer film would deliver biological molecules that would stimulate the growth of bone both 74 00:07:12,530 --> 00:07:18,060 on the surface and throughout the scaffold. Over time, the scaffold would slowly degrade, 75 00:07:18,060 --> 00:07:24,300 leaving the new bone tissue behind. In selecting components for both the scaffold and these 76 00:07:24,300 --> 00:07:30,610 films, we looked to the biological process of wound healing for inspiration. The idea 77 00:07:30,610 --> 00:07:35,530 was that if we could mimic the wound healing process in bone, we could potentially regenerate 78 00:07:35,530 --> 00:07:41,229 tissue that is mechanically and chemically identical to native bone tissue. 79 00:07:41,229 --> 00:07:46,770 The bulk of our scaffold consisted of a polymer that would slowly degrade when placed in the 80 00:07:46,770 --> 00:07:52,610 body. This polymer was mixed with calcium phosphate, a significant component of native 81 00:07:52,610 --> 00:07:58,610 bone. Our thought was that calcium phosphate would promote the attachment, growth, and 82 00:07:58,610 --> 00:08:02,840 migration of cells that produce bone tissue within the scaffold. 83 00:08:02,840 --> 00:08:07,289 We then used layer-by-layer assembly to create a multilayered film on the surface of the 84 00:08:07,289 --> 00:08:13,560 scaffold. The film contained layers of polymers and layers of biological molecules that we 85 00:08:13,560 --> 00:08:19,240 wanted to release into the defect site. One of the molecules was a protein called bone 86 00:08:19,240 --> 00:08:24,749 morphogenetic protein-2. This protein is a growth factor that stimulates mesenchymal 87 00:08:24,749 --> 00:08:30,479 stem cells from the bone marrow to transform into bone tissue producing cells. 88 00:08:30,479 --> 00:08:35,000 The biodegradable properties of the polymers we used in the film would help to release 89 00:08:35,000 --> 00:08:39,570 the protein into the surrounding tissue when the scaffold is implanted. 90 00:08:39,570 --> 00:08:43,510 In addition to identifying molecules that would give us the function and properties 91 00:08:43,510 --> 00:08:49,980 that we desired, we had to choose an assembly pH that would ensure they had the desired 92 00:08:49,980 --> 00:08:55,180 charge. 93 00:08:55,180 --> 00:09:00,540 To test our hypothesis that these materials would lead to bone growth, we implanted our 94 00:09:00,540 --> 00:09:04,440 coated scaffold into a rat quadriceps muscle. 95 00:09:04,440 --> 00:09:09,639 In this model, the scaffold was placed in a location where bone is not normally found, 96 00:09:09,639 --> 00:09:15,180 so that we would know that any bone created on the scaffold was due to the coating. 97 00:09:15,180 --> 00:09:20,750 Our experiments were successful in that we saw the deposition of bone minerals and collagen 98 00:09:20,750 --> 00:09:26,800 on our scaffolds within 4 weeks. Of course, there are more experiments to be done to see 99 00:09:26,800 --> 00:09:31,550 if this system will work in the same way in a large defect site. 100 00:09:31,550 --> 00:09:36,829 The main lesson I learned in these experiments was that for bone tissue regeneration to occur, 101 00:09:36,829 --> 00:09:43,450 my multilayer-coated scaffolds needed to provide two key functions: to encourage bone producing 102 00:09:43,450 --> 00:09:50,450 cells to attach, migrate through, and deposit tissue in the scaffold; and to encourage mesenchymal 103 00:09:51,760 --> 00:09:58,760 stem cells to transform into bone producing cells. 104 00:10:01,290 --> 00:10:08,290 In this video, we hope that you saw how general chemistry concepts such as pH and pKa continue 105 00:10:16,990 --> 00:10:22,060 to be useful beyond the classroom and into research settings. Here, we saw how we need 106 00:10:22,060 --> 00:10:28,440 We also saw how considering the desired function and properties of a material can help us rationally 107 00:10:28,440 --> 00:10:35,440 design its structure.