WEBVTT 00:00:02.370 --> 00:00:08.809 82% of soldiers in battle suffer from traumatic limb injuries. Many of these injuries are 00:00:08.809 --> 00:00:15.750 large bone defects. Engineers at MIT are trying to create materials that mimic the function 00:00:15.750 --> 00:00:20.810 of the bone's natural healing processes. The structure and properties of these materials 00:00:20.810 --> 00:00:27.559 promote bone regeneration. This video is part of the Structure-Function-Properties video 00:00:27.559 --> 00:00:33.079 series. The structure, function, and properties of a system are related and depend on the 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 00:00:39.679 --> 00:00:44.820 student in Professor Paula Hammond's lab in the chemical engineering department at MIT. 00:00:44.820 --> 00:00:49.999 In the Hammond Lab, we design novel materials for tissue engineering, gene and drug delivery, 00:00:49.999 --> 00:00:56.010 and energy applications. My research in particular, focuses on developing and assembling different 00:00:56.010 --> 00:00:59.929 materials for bone tissue regeneration. 00:00:59.929 --> 00:01:05.930 Before watching this video, you should be familiar with the concepts of pH and pKa, 00:01:05.930 --> 00:01:11.000 and with the common chemical functional groups. After watching this video, you will be able 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 00:01:17.280 --> 00:01:24.280 layer-by-layer assembly. Many of the materials our lab develops are based on a technique 00:01:27.390 --> 00:01:33.740 called layer-by-layer assembly. Layer- by-layer assembly utilizes electrostatic interactions 00:01:33.740 --> 00:01:40.530 to create layers of chemical species of alternating charge. These chemical species could be anything 00:01:40.530 --> 00:01:47.530 -- polymers, proteins, small molecules -- the key is that they have the appropriate positive 00:01:47.660 --> 00:01:52.039 or negative charge to incorporate them into the film. 00:01:52.039 --> 00:01:58.020 We assemble these films using a simple dip method. First, we take our substrate or surface 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 00:02:03.990 --> 00:02:10.169 cleaner. This not only cleans the surface of contaminants, but also leaves the surface 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 00:02:17.519 --> 00:02:24.040 charged molecules. The molecules in solution bind to the substrate because of Coulombic 00:02:24.040 --> 00:02:25.060 interactions. 00:02:25.060 --> 00:02:31.219 This process is self-adsorption limited, meaning that once the newly adsorbed layer neutralizes 00:02:31.219 --> 00:02:37.359 the charged sites on the surface, additional molecules will not bind. This creates layers 00:02:37.359 --> 00:02:40.590 that are on the order of nanometers thick. 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 00:02:47.590 --> 00:02:53.510 negatively charged molecules. Interactions with the charges on the previous layer allow 00:02:53.510 --> 00:03:00.329 a new layer to be deposited. Again, we would rinse to remove any unbound components. We 00:03:00.329 --> 00:03:05.829 can continue this process and dip our growing film into solutions of positively and negatively 00:03:05.829 --> 00:03:12.829 charged molecules, with rinse steps in between, building up our coating layer-by-layer. Many 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 00:03:19.980 --> 00:03:26.620 layers. These long chain, high molecular weight molecules form more stable layers than small 00:03:26.620 --> 00:03:31.900 molecules. The long polymer chains from one layer weave through other layers creating 00:03:31.900 --> 00:03:38.819 an interlocking structure. Also, because we use negatively and positively charged polymers 00:03:38.819 --> 00:03:45.819 in these layers, the polymers ionically crosslink, adding to the stability of the overall film. 00:03:47.709 --> 00:03:53.430 Polymers whose repeating unit contains a charged group are called polyelectrolytes. Thus, many 00:03:53.430 --> 00:03:59.150 people refer to the films that we create as polyelectrolyte multilayers. For every polymer 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 00:04:06.510 --> 00:04:12.889 and select appropriate assembly conditions to ensure they have the desired charge. For 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. 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 00:04:23.620 --> 00:04:29.870 the repeating carboxylic acid group. Another polymer that I use in my layer-by-layer assembly 00:04:29.870 --> 00:04:35.389 contains repeating amine groups. Let's say I'm creating my layer-by-layer assembly at 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 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 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 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 00:05:08.009 --> 00:05:14.539 deprotonated, resulting in a negative charge. At the same pH, some of the amine groups on 00:05:14.539 --> 00:05:21.539 our second polymer pick up an extra hydrogen, resulting in a positive charge. Because so 00:05:26.819 --> 00:05:31.970 many molecules could be viable candidates for incorporation into these films, they are 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 00:05:38.039 --> 00:05:39.810 engineering applications. 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 00:05:46.060 --> 00:05:52.550 how these multilayer films could be useful. What function do they need to have? What properties 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 00:05:58.000 --> 00:06:00.409 I structure my film? 00:06:00.409 --> 00:06:05.770 One of the problems I became particularly interested in is that of large bone defects. 00:06:05.770 --> 00:06:10.479 Large bone defects are large gaps in the bone that result from trauma. 00:06:10.479 --> 00:06:14.860 Although bone is capable of regeneration, when there are large defects, some sort of 00:06:14.860 --> 00:06:19.639 intervention therapy is needed to bridge the defect for proper healing. 00:06:19.639 --> 00:06:24.389 These intervention therapies typically involve taking bone tissue either from another location 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. 00:06:31.060 --> 00:06:36.720 While bone taken from the patient may be immune compatible and be more viable, this method 00:06:36.720 --> 00:06:42.729 has limitations. Tissue injury and trauma at the site of bone removal causes patients 00:06:42.729 --> 00:06:48.650 pain and long healing times. Bone tissue from a deceased donor may cause an unfavorable 00:06:48.650 --> 00:06:55.490 immune response. The processes used to prepare these tissues for implantation may also compromise 00:06:55.490 --> 00:06:57.219 their mechanical properties. 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 00:07:00.949 --> 00:07:01.800 design a scaffold to bridge these large defects that will stimulate the growth of new bone 00:07:01.800 --> 00:07:06.979 tissue? The idea was to create a rigid, porous scaffold coated with a multilayer film. The 00:07:06.979 --> 00:07:12.530 multilayer film would deliver biological molecules that would stimulate the growth of bone both 00:07:12.530 --> 00:07:18.060 on the surface and throughout the scaffold. Over time, the scaffold would slowly degrade, 00:07:18.060 --> 00:07:24.300 leaving the new bone tissue behind. In selecting components for both the scaffold and these 00:07:24.300 --> 00:07:30.610 films, we looked to the biological process of wound healing for inspiration. The idea 00:07:30.610 --> 00:07:35.530 was that if we could mimic the wound healing process in bone, we could potentially regenerate 00:07:35.530 --> 00:07:41.229 tissue that is mechanically and chemically identical to native bone tissue. 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 00:07:46.770 --> 00:07:52.610 body. This polymer was mixed with calcium phosphate, a significant component of native 00:07:52.610 --> 00:07:58.610 bone. Our thought was that calcium phosphate would promote the attachment, growth, and 00:07:58.610 --> 00:08:02.840 migration of cells that produce bone tissue within the scaffold. 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 00:08:07.289 --> 00:08:13.560 scaffold. The film contained layers of polymers and layers of biological molecules that we 00:08:13.560 --> 00:08:19.240 wanted to release into the defect site. One of the molecules was a protein called bone 00:08:19.240 --> 00:08:24.749 morphogenetic protein-2. This protein is a growth factor that stimulates mesenchymal 00:08:24.749 --> 00:08:30.479 stem cells from the bone marrow to transform into bone tissue producing cells. 00:08:30.479 --> 00:08:35.000 The biodegradable properties of the polymers we used in the film would help to release 00:08:35.000 --> 00:08:39.570 the protein into the surrounding tissue when the scaffold is implanted. 00:08:39.570 --> 00:08:43.510 In addition to identifying molecules that would give us the function and properties 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 00:08:49.980 --> 00:08:55.180 charge. 00:08:55.180 --> 00:09:00.540 To test our hypothesis that these materials would lead to bone growth, we implanted our 00:09:00.540 --> 00:09:04.440 coated scaffold into a rat quadriceps muscle. 00:09:04.440 --> 00:09:09.639 In this model, the scaffold was placed in a location where bone is not normally found, 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. 00:09:15.180 --> 00:09:20.750 Our experiments were successful in that we saw the deposition of bone minerals and collagen 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 00:09:26.800 --> 00:09:31.550 if this system will work in the same way in a large defect site. 00:09:31.550 --> 00:09:36.829 The main lesson I learned in these experiments was that for bone tissue regeneration to occur, 00:09:36.829 --> 00:09:43.450 my multilayer-coated scaffolds needed to provide two key functions: to encourage bone producing 00:09:43.450 --> 00:09:50.450 cells to attach, migrate through, and deposit tissue in the scaffold; and to encourage mesenchymal 00:09:51.760 --> 00:09:58.760 stem cells to transform into bone producing cells. 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 00:10:16.990 --> 00:10:22.060 to be useful beyond the classroom and into research settings. Here, we saw how we need 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 00:10:28.440 --> 00:10:35.440 design its structure.