Biomedical Engineering Center for Translational Research.
2007 Conference and Expo.
Friday, May 4, 2007.
University of Wisconsin-Madison, College of Engineering.
Engineering Centers Building.

Morning Agenda: Focus on Translational Research.

Prof. William L. Murphy, Departments of Biomedical Engineering, Pharmacology, and Materials Science; Ben Graf, Department of Orthopedics and Rehabilitation, and Director, Sports Medicine Division; Mark Markel, School of Veterinary Medicine, and Director, Comparative Orthopaedics Laboratory

Biologically Active Coatings on Bioresorbable Orthopedic Implants

DRAFT TRANSCRIPT, August 8, 2007

00:00
Speaker: Prof. Robert Radwin, Chair of the Department of Biomedical Engineering.

... Professor Murphy works in the creation of novel materials using bio-inspired approaches.
He uses biomaterials to define the stem cell microenvironment.
Bill is another cluster hire.
He is part of the cluster in stem cells, and he has a joint appointment in biomedical engineering and clinical pharmacology.
He also develops biomaterials for tissue regeneration and tissue engineering and novel approaches to drug delivery and gene therapy.

00:51
Speaker: William L. Murphy
OK, thank you, Rob, can everybody hear me?
OK. So, as Rob mentioned, my lab does a lot of work in materials and primarily in, kind of, new materials chemistries.
And I'll try not to go into too much detail on the detailed chemistry to avoid putting people to sleep before lunch, but I'll describe is something that's a much more applied project that we're working on in the lab. 
In collaboration with Ben Graf, who's in orthopedics and rehabilitation. 
Ben is a particularly creative orthopedic surgeon, really quite an outstanding orthopedic surgeon. 
He's really an engineer in mindset; he's quite a tinkerer. 
So, he's been a great person to work with on this. 
And Mark Markel, who's very adept at animal models for orthopedic maladies. 
So, what I'll describe is some work that we've been doing to generate biologically active codings on our resorbable bioimplants for orthopedics. 

So the clinical problem that we start with here is screw fixation during anterior cruciate ligament surgery. 
This is a very common surgery. 
Almost 240,000 ACL reconstructions are done annually. 
You probably know somebody who has had their ACL reconstructed. 
It's about a $3.5 billion market, so it's a significant market. 
A problem here is that about 5-10% of the cases require revision, and there's a very long rehabilitation time, typically about 6 months, so people lose a lot of work time in this process, and there's a lot of pain associated with it. 
One of the key limitations is that bone tendon healing and the tunnels that are formed during this process doesn't occur as you would like it to occur. 
What is done in an ACL reconstruction is that a graft, typically a tendon graft, like a hamstring tendon graph is snaked in between two tunnels in the bone -- so a tunnel in the femur and a tunnel in the tibia. 
And it's then fixed in place by screws, most commonly metal screws, but more recently bioreabsorbable screws -- meaning materials that go away with time, get replaced by tissue. 

But the issue is that this is what happens. 
So, after a period of time, after the implant has been placed, you end up with this rather large tunnel that forms during the surgery. 
And the tunnel actually expands quite significantly over time. 
There is a 60% increase in tunnel area at three years without fixation. 
When you fix it with a screw, you actually get an even larger increase in that tunnel area, so this is a significant problem. 
It really decreases the stability of the bone here, and you just don't get good healing. 
So there was a recent study from Scott Rodeo's group, where they've looked at actually adding a growth factor component embedded in a sponge, and they packed that sponge into the defect site. 
And they then see quite good healings, so the bone fills in, the bone-tendon interface looks quite nice as well. 
We thought if we could deliver this kind of a growth factor, which in this case is bone morphogenetic protein two -- I'll refer to it as BMP-2 throughout the presentation -- if we could deliver it locally and actually deliver from some of these fixation screws, we could get the best of both worlds -- mechanical fixation but also a biological effect over time. 
So this is what we became interested in doing at the recommendation of Ben Graf, my clinical collaborator. 

What we've done is we've taken a bioresorbable screw and grown a coating on it. 
We then insert into that coating drug molecules, which then as the coating is resorbed get released over time locally into the defect site, and then we get local healing of bone. 
This is our hypothesis, when we started the project. 
We started with bone screws that we got from a partner, Smith and Nephew. 
So Ben Graf has a really strong interaction with Smith and Nephew Endoscopy, and they were nice enough to send us whole bunch of screws to work with here. 
So these screws are composed of a bioresorbable polymer material. 
And so we set about trying to then coat those screws. 
And in order to coat them, we used a process that's been developed and published pretty widely. 
It's a process that involves actually a room temperature process. 
So here we incubate materials that are resorbable in a fluid that we call a modified simulated body fluid, or MSBF. 
As these materials are incubated in the fluid, a calcium-phosphate mineral forms, so we actually get a coating that's forming on these materials. 
And the key here is that it is essentially a room temperature process, so we can then incorporate biologics into the process without blowing them apart. 
Typically, when you make a coating, particularly a ceramic coating on an orthopedic implant, it's done at very high temperatures that would really deactivate essentially any biologic you'd be interested in incorporating. 
So, this is the process that we used. 
And this is just sort of an initial piece of data that shows how we've done the coating. 
So if you look at the surface of one of these coatings in an electron microscope, it looks like essentially a polymer surface -- so this is just polymer. 
If we then coat it, we get this sort of a surface, where you can see that there's something that's happened to the surface. 
If we zoom in more closely, you can see that there's this kind of plate-like mineral that's formed, and this is similar in structure and composition to bone mineral. 
I won't go into too much detail on the physical-chemical properties of this coating, but it essentially looks a lot like the mineral that's in your bone tissue. 
And we can also control the properties of these coatings. 
So this again is what the materials look. 
We can form these also on films. 
So this is a thin film of a resorbable polymer material. 
We then form a coating on it. 
And if you zoom in, you again see this plate-like structure.

So, the next step in the process here was actually to incorporate a drug and release it from these coatings, and the drug we chose based on the literature I described earlier was bone morphogenetic protein 2, or BMP-2. 
So, what we did initially was incorporate full-length BMP-2 protein into these coatings.
We can incorporate them into the coatings quite efficiently, and if we then release them over time -- here, we're looking at about a month -- they release in an almost linear fashion over time. 
So, quite a nice release profile and sort of what we were hoping for.
So now we're getting BMP-2 released locally into the defect site over an extended time frame to promote healing.

So it's important for us to check whether or not this protein remains bioactive, and if we look at the biological activity of the protein, particularly by culturing stem cells, and looking at the effect of the protein on their differentiation, we see here that when stem cells are exposed to the BMP that we've incorporated into these coatings, they show enhanced level of alkaline phosphotase activity, and this is actually a hallmark of bone differentiation. 
So this is essentially a bioactivity essay.
We're not using stem cells in our work, but this is a validation that what is being released from these coatings is active, retains its normal activity.

So we then progressed to placing these materials into a sheep model of ACL reconstruction. 
The sheep model is a nice one, because the size scale is similar, so we can actually use human implants in the knee. 
Here you're looking at a sheep tibia. 
You can see that this is a bone tunnel that's been drilled and a metal interference screw that's incorporated into it. 
In this case, you're looking at the same kind of bone tunnel, but with our resorbable coated screws in the defect site, and they're pinning a hamstring tendon in here. 
So, you're actually looking at exactly what we do during the surgery, pinning the tendon in place just as it's done in the clinical scenario. 

So these animal studies are underway, looking at the effects of these coatings on activity and on healing. 
And we're looking at both mechanics, the ability of the tendon to stay in place, and the histology -- essentially the healing process -- adjacent to the material. 

So the commercialization outcomes:
The first direct outcome has been a research contract with Smith & Nephew that'll begin shortly. 
Smith and Nephew is one of the key players in the resorbable fixation products market, and they've got about a 12 1/2% market share. 
There's a variety of other devices that this sort of coating can be applied to.
The other indirect route for these technologies to commercialize is a startup company that I co-founded a couple years back, called Tissue Regeneration Systems, and we're using similar kinds of technologies in spine applications, so there are a couple of different commercialization routes that are progressing, I think, quite well thanks to the Coulter Foundation. 

Briefly, before I finish, I want to talk about another direction we've been heading with the Coulter funding that actually generalizes this well beyond Bone Morphogenetic Protein-2. 
What we'd like to be able to do is essentially incorporate anything we want into a coating like this. 
In order to do that, we need a tag, something that's actually going to localize a molecule into a coating. 
And the way we've engineering these tags is we've looked at a natural protein that binds very strongly to the minerals that we grow -- hydroxyapatite minerals. 
The protein is called osteocalcin, and it binds with extremely high affinity to hydroxyapatite. 
You're looking at here osteocalcin bound to a mineral matrix here, and there's actually a registration between the side chains on the amino acids in osteocalcin, which is what you're looking at here, and the calcium atoms that are incorporated into the mineral. 
So this is a very high affinity interaction. 
We also have an engineered growth factor that we can derive from BMP-2, so there's a peptide sequence and a knuckle epitope of BMP-2 that retains much of the activity of BMP-2. 
So these two components, this tag that incorporates the molecule into a mineral and this active portion, we essentially pasted together, synthesized a new molecule that we can incorporate into these coatings as a proof of concept for this very broad-based technology. 

And in the interest of time, I think I'll actually skip just one slide here and go straight to our coatings.
So here what you're looking at are fluorescent micrographs of molecules that are bound to our coatings. 
And we engineered actually a series of peptide sequences that could either interact very strongly or much more weakly with our hydroxyapatite materials. 
And what you're seeing here is a molecule initially that has no interaction at all with the coating, so you see no fluorescence on the surface of the coating here. 
If we then increase the affinity of the molecule for the coating, you see some fluorescence, more fluorescence, and then in this case a very high level of fluorescence in the case where we get very high levels of binding. 
What's perhaps most interesting here is that if you look at release of these molecules from the surface of the coating, we've actually engineered a molecule here -- shown in the red line -- that doesn't come off the surface of the coating over time in solution. 
So the affinity is so strong that it's essentially linked to the surface non-covalently. 
So this allows us to do a variety of really exciting things in the orthopedics area, because hydroxyapatite is included in so many different devices. 
One of the examples of what we can do is essentially paint growth factor onto the surface of a material. 
Here, you're looking at a coating that we literally took a paint brush, dipped it into a solution of our peptide, and wrote the letter "W" onto the surface of it, washed it, and it didn't come off.  
So you can consider dip-coating or paint-coating essentially any orthopedic implant that has hydroxyapatite incorporated into it, which is a very large number of implants. 

So this is a pretty broad-based technology that we're very excited about and that Coulter has played a large part in initially developing. 
Briefly, the molecule that we engineered to bind to the surface remains active, so it remains able to push stem cells to differentiate into bone cells, so we maintain the activity of the molecule. 
And we can quantify that as well, when in comparison to control the hallmark of bone differentiation, which in this case is mineralization, increases in each engineered molecule that we've generated.

So, I want to briefly acknowledge the members of my group that have been involved in this, particularly Darilis Suarez, Jim Molenda, Jae Sam Lee, and
Leena-Jongpaiboonkit.
And Jae was particulary involved in most of this work,
so Jae has driven a lot of the work in the area of implant modification.

And I also want to particularly thank the Coulter Foundation.
Without the Coulter Foundation, we probably wouldn't be exploring a lot of these new areas of research.
Thank you.

Copyright 2007 The Board of Regents of the University of Wisconsin System

Last modified: 21 May 2009.
Created: 26 July 2007.