So who is an assistant professor of

chemistry and
I'm not going to this is very boring stuff

bring in all the boring stuff which
I'm going to skip as well but

he'll receive I mean or see

that they're after fifteen playing and

very few you want to Iron Man
a word had a great music

scenes and he always gave me
a hard time that I would be made

in your sent me that you don't take it
back to you on the part of my family.

I mean you carry with me the but OK.

You didn't really mean that you have
no more looking forward to that.

Thanks much appreciate
the invitation to come visit.

So I was sure that it was a perfect right.

So everything coordinated here so

I'm not going to spend a lot of
time on this introductory slide but

it's basically just making the point that
we've been thinking a lot about function.

Structure function issues for in tissues
and how we can engineer materials

to promote repair processes and so
I used the lining of the intestine.

Here is just an example to talk about this
triumvirate that we have talked about in

tissue engineering of souls matrix and
molecules.

So if we look at the sort of if we look at
the actual matrix and how it's been used

as a sort of inspiration for developing
materials in by medical applications.

Sort of on the on the bottom here.

I've got I've got shown more sort of
mechanical or structural variables as far

as like osmotic pressure models etc And
then on the top.

I have sort of more biochemical variables
and I understand a lot of nice work and

then there's a been a lot of obviously
considerable interest in delivering

exoticness growth factors to sites where
you are trying to crank up the process.

So the idea here is that we've got a
sizable segment proteins that pack a punch

and we want we want to add more of
them to drive some some process.

So there are two main strategies that
are used one is the sort of delivery

process where you have a degrading polymer
that releases therapeutic protein and

then the other option is actually just
ventilate hashing it to a material and

presenting it that way.

And so
in both these cases we're adding more.

So what we wanted to do in my
group is actually ask the question

what if we had a material that could
take things away rather than add them.

So right so matrix is the prime
regulator post transcription

regulator of the activities of sayable
signaling proteins and the way that we

think about it is so we've got our sort of
schematic of like a college and fiber and

here's a cell and here's a subtle sibling
protein so there's a competition for

this I will say when protein between
the soul receptors and the Matrix and so

the idea is that if the matrix wins out
this can get sequestered in here sometimes

inactive form sometimes an inactive form
and if it doesn't interact very much.

Then it's free to target receptors in
the cell and so we wanted to do was

just engineer matrices materials that
can perform the same function but

the question is how do we do.

So we round up so

we wanted to study ways the materials
could promote tissue repair and so

we were thinking a lot about growth
factors like everyone else but

after working on it for a while.

What we realized is that
we actually need to

start thinking a lot
more about information.

The front end of the repair process.

So this is a diagram from a few that came
out in New England Journal of Medicine and

so this is this is a skin where we've
had an injury and so in a healthy

adult you form a blood clot within twenty
seconds it's composed of fibrin and

activated Plato comes with activated
platelets and then for a few days.

Well that injury is red and
inflamed and you've got cells

crawling around the exercise matrix that
are trying to scavenge the trying to eat.

Bacteria that got in the trying
to eat that tissue etc.

So later on what happens
is this is the system

is going to thwart a repair process and
you will actually be forming the matrix.

But early on what you're basically
setting the stage for what happens later.

And so what we wanted to be
able to do is basically so

the Souls that are present
at this injury site.

Are there activities are determined by
the host of soluble signalling proteins.

But basically giving them instructions and
so well all these growth factors and

while in the review they talk mostly about
growth factors the reality is that day

three the molecules that are doing
most of the work are cytokines and

the cells that we are a particular
interest in are macrophages because

they're involved in the front end
of the process needing stuff but

then they're also the ones that later
on are actually setting the stage for

revascularization new
Matrix Matrix depositing cells.

It looks good but stuff.

Yes Yes And

I've gotten to the point now
where my students in my class.

I think are passing a hat around
to buy me a new computer and

that's exactly what I need.

That was the final bullet for that.

So the idea is what we want to do is we
want to be able to add a material here

that would indirectly control
the activities of the cells by controlling

what sayable say going proteins they would
see any idea is that if we can change

what happens at the beginning of the
inflammatory response we're hoping that we

can actually shift the final
outcome in the repair process.

That's the hope at least there's
actually a lot of there's been a lot

of studies matrix patches have studied
the interactions of growth factors.

Matrix and it also sort of
happened by any domains etc

that cause all sorts of interactions and
so like in the case of killing

when you have activated platelets at
an injury site they're dumping out large

quantities of Plato drive growth factor
which sequestered in the matrix and

then used much later on you know five
days later when you're actually ready to

start doing the repair those get released
when the fibrin matrix gets broken down

the thing with most cytokines
is that there are most of them

their interactions with the actual
matrix tend to be fairly weak and

this was actually what we were
originally studying in my lab and

from a certain perspective it's not very
interesting because you just don't see.

Many of the planetary cytokines
just a few through the matrix like.

It's just buffer and so from
a functional perspective this probably

makes sense because the idea is that

what the cytokines are doing is
they're basically like a messenger and

they're produced in response
to certain stimuli and

your tissue infections etc and

there's no reason for that message to
be delayed you when you send it out.

You want it to make it to
the other cells in the area and so

are most of what we think is that
a lot of our cytokines actually have

actually evolved to how weak interaction
is with The Matrix because of this.

And so what that means is that
it's not it's not something

that's regulated strongly
by a native matrix.

If you have an artificial matrix that
would do that would play this role and

in interacting with.

You could in principle see it.

Pick a fact if you target the right
mediators and design your materials

the right way and so that's eventually
what I'm going to tell you about.

Thank you for your patience.

So we've been doing a lot of work
looking at the macrophage was actually

going to be the saw that we're using as
a marker for how active are materials.

And so McAfee just have a sort
of check on personality where

really in the response they adopt a
phenotype which is referred to as an one.

And so this is the sort of angry
Terminator type of phenotype

where they're releasing pro-inflammatory
signals that breaking down Matrix and

are contributing to
pathogen resistance and so

this is made up this phenotype
under the influence cycle

mediators I can of tuna Quest factor and
members of the interfering family.

Late in the inflammatory response
what happens is they shift gears and

they adopt and
what's called an M two phenotype which is

under these under these
mediators members of the.

In family group.

And at this point they're releasing like
trying to generate factors to recruiting

fiberglass and they're leading
to a teach to type response and

so the community

that's probably started macrophage biology
the most is actually the cancer community.

So they are tune in to Murs we have
a lot of resident macrophages and

they are the tumor somehow convince
them to adopt this and to phenotype and

what they're doing is
they're actually releasing

molecules these all these molecules
here fuel the growth of the tumour and

so what cancer biologists would love to
do is turn these macrophages back in and

one macrophages But what we've been doing
in thinking about controlling inflammatory

responses is for conditions
where you have an over exuberant

inflammatory response it's actually
leading to continue to shoot damage.

We'd actually like to put
the brakes on this process and

ideally guide them over towards
this a little bit early.

The basic thinking is that like a hundred
thousand years ago if I'm out in

the jungle and I get cut this could be
a life threatening event because we didn't

have so we didn't know about anesthetic
techniques etc and so our immune

systems have evolved to have a very strong
response every time the skin is broken and

that the fastest way to protect
that is to scar tissue.

So what we'd like to do is find ways
of modulating phenotype with materials.

So the kinds

of conditions that we're looking at at
this point are not the sort of classical

like tissue engineering type treatments
in terms of like bone cartilage etc we're

really we've really got much more focused
on conditions like I said that of a lot of

information and where the inflammation is
actually causing further tissue damage and

so a great example of this sort of talk.

Later it was actually burns and so
what we'd like to be doing a lot of these

conditions is would like to manage
the inflammatory response locally

to avoid the sort of systemic to
avoid systemic immune responses.

So this is the point of
the talk everyone gets

disappointed because they get you
know you're excited you think this

is an important problem this guy's going
to come up with a really slick solution.

And what you see here is a high molecular
weight hydrophilic Homer I have

prevented the attachment
anybody that target specific

mediators of information like
cytokines and that space likely.

And so the thinking here is we've got so
anybody's

been developed to specifically bind it in
some cases neutralize certain proteins

like cytokines and so we have available
ones that I'll talk about later on for

all sorts of cytokines and so
what we want to do is we wanted to

what we're doing is we're attaching it to
this this big polymer and so the idea is

that by attaching it to a big hydrophilic
polymer it will stay where I put it.

And so in a sense it is kind of
like a tissue engineering approach

in terms of thinking locally but

the idea is that we're actually just
trysted in modulating immune responses.

So we've chosen to cast it for
a couple reasons.

This is the part of
the I'll talk about for

most of the talk so
it's a naturally occurring can.

So the interesting thing
about Ha is that it's so

it has this fairly
monotonous repeat sequence.

When it in the form it's recognised
by certain receptors like C.T.

forty four and what it does is it up
regulates kill it which is important in

response because you want to get your
repair cells moving in more quickly.

However when it gets chopped down chopped
into little pieces either through

oxidative stress or
magically the degradation products the.

Reactive donors damage associated
molecular patterns and so

this is part of how your immune
system looks for problems at

the sites of injury because we have this
in practically all of our tissues and so

when this gets broken down it's
a sign that there was a problem.

And so this in some a lot
of fairly complicated ways

involves your native system
spawning to respond injuries.

So what we're doing by attaching
this anybody to it is so

ironic as it by itself has we can no
interactions with most pro-inflammatory

cytokines and so what we're doing
is we're giving this sort of

giving the material sort of a new
dimension of biological activity and

we're also localizing the effects of
the anybody so and I should mention

one reason why the look in terms
of logical applications right now.

Ironic acid has been shown to be effective
at promoting healing of diabetic foot all

searchers people with sickle cell
anemia also have non-healing and

it's actually worked well for that.

And so you know if nothing else will
hopefully make it work a little bit harder

to make these base materials is simple.

Basically we do a cover to make
one of these products one of

the you know four thousand correct said
groups on the backbone of the Hanukah So

we have developed
the standard protocol and

we've analyzed the composition
with my job so

this was a lot of this work was
my first two graduate students

a city been strafe just finished his Ph D.
in chemistry and

they He's now post-doc at Harvard with
Dave moving the money and David words in

and Steve Sun is a grad student in
primary continuing the story with me.

So so
this is a picture of the purified gel for

precipitation these are Steve's
fingertips so you see that we're not.

Exactly.
In Mass production mode yet

but we can reproduce we make compositions
around this around this range.

Every one in every seven change
has gotten anybody on it.

So this what the page look right so
these are all of our caliber.

These are collaborations and these
are some of the samples that we measured.

Steve has good hands because you
know we get pretty good good.

Pretty good linear fits for
some of the details so

we image this without as far as doing
the Eliza characterization we're

basically using anybody's to measure
anybody concentrations and so

can we get decent a decent head for
the calibration curve and

so by independently measuring
the Ha concentration and

the anybody concentration we
can come up with this ratio.

So the first cytokine that we started
targeting was in the can one beta.

It's a so in immediately following injury.

We have two cytokines that get up
regulated in a better than the other one

is too many courses factor Alpha I want
to study in lab because it's small as

a marker weight of about seventeen.

And it's very soluble so it doesn't played
out on the sides of your test tube.

So it's itself a bona fide
therapeutic target for

condition and
we had a local expert in Pittsburgh micro.

Literally wrote the book on cytokines
he's one of the one of the two editors of

the cytokine handbook.

Actually most he did most of
his his work on cytokines and

in the look into it was a cancer.

But he's a lot of the early the next
work I'll show was actually done in

collaboration with him so the first
question that we had to ask was if you

just stick in and if you can really attach
an anybody to a huge charge polymer is

the anybody still going to be capable of
recognizing the energy and so there's

a second stick instrument in his lab
which basically works like a peer core.

But it what it is is it's a fiber optic.

Where you can bind anybody's or

anybody's conjugated too high on a casket
at the end of the fiberoptic And so

what you do is you don't get into
a solution that contains the energy and

in this case in one beta and then you pass
white light through the fiber optics so

when you get your head injuries.

Prevent actually get away flamed
dependents in the reflection

efficient going back to the detector and
so you is to measure changes.

Effectively in mass refractive index at
the interface and so this is these Curtis

family of curves are all the hieratic acid
gets in these are just the bare any bodies

and so what you do is you do that
to the can to the absorption and

you take it into for solution.

And then and then you watch the piece
of your antigen into solution

to these functions and you can come
up you get from the fit you get a K.

on a K. off and you take the ratio
of those not to get to your K. D..

So what we saw was that so.

This is the one with the hieratic acid in
this is the bare anybody what we see is

that the on kinetics
are basically the same.

And if anything the author can
add a little bit slower and so

what we think is happening is that
basically the the energy in his binding

is being bound by the any body and when it
forms a long live complex the higher on

a pass a basically reaches around kind of
gives it a hug and so by itself it doesn't

really have a strong affinity for
data but if it sits there long enough it

at the chain reach around and basically
help fix for a little bit longer.

But this is basically suggesting
this data are basically

suggesting that we've got it.

We have we certainly haven't abolished
the affinity of the anybody for for

these and engines.

The catch is that so this is obviously
not a natural environment for

an antibody to find it so often and so we
wanted to do one more before we move to.

Any animal experiments and so
our cells are on the lookout all over.

Most of our basic all of our
cells on the lookout for

three things bacterial products signals
from other cells like cytokines and

then energy and and most of these go
through similar signalling pathways

that all funnel into this what's called
the nuclear factor Kappa big complex and

so usually And if Kappa B. is held in an
active state out here in the cytoplasm So

here is the cell wall the cytoplasm
in the nucleus one of the signals

come down this inhibitory put it gets
lopped off in the cap and become

the nuclear membrane and and
activate the inflammatory response.

And so when you have if you can measure
the amount of benefit cap would be in

the cytoplasm versus
what's in the nucleus.

That gives you the measure of sort of an
incipient inflammatory response and so it.

The real inflammatory
response hasn't kicked in.

But this can be quantified in
correlated with with downstream events.

The nice thing about it is that this
process takes about thirty minutes.

So it's fast so this is these are also
experiments we did in his lab.

So this is actually this is this
instrument called This is an imaging site

ometer So what it does it's basically
as I said microscope hooked up

to a very fast computer.

It was developed by a company in
Pittsburgh actually called Solo mix which

was bought by thermal Fisher And so
what it does so we take these are these

are th P one macrophages it's just a macro
and we used to Staines for the cells.

We have a nuclear stain to figure out
where the new cases and then the green

that's blue stain in the blue the green
rather than anybody stain for

an F. cap of B. And so what it does is it
will image about five thousand cells and.

Figure out how much of the N.F. cap of B.

could localize is in the nuclear in
the nuke vs vs outside of the nucleus.

So we get this kind of low
dose response curves but

if you're out here on the strong response
level it becomes role of the quantitative.

And you get expanding our
crafts like this out of so.

A positive value.

So this is a unitless
measure of Translocation so

positive value implies the cells
are having a strong response and

we should see lots of information and so
the cells go bonkers when we feed them

back to our product and so
we see a big translation value.

When we dose them up with one hundred
anagrams from a leader of Iowan beta

we get a lower but
measurable positive response.

This is actually the control value so
these are the quiescent cells and

it's lightly negative.

So what we can do though we can
show here that with I one beta and

hieratic acid we basically get
the same response level so

that indicates that the HA
is not interacting.

It's not doing anything with beta and

then we can knock this back down
a chant itself has no effect.

And then we can knock down these responses
to these troubles by the treating with

anybody alone or with anybody conjugate So
we have molecular characterization and

solid base characterization indicating
that these cars trucks are capable of

neutralizing cytokines and
then we are ready to go.

Well actually I was ready to have
huge grants on this because I thought

this was great the reviewers were not so
impressed.

So they said Show us the minimum data.

How hard can it be so
seamy does not have a central

animal people that do am aware in their
labs but they do it in their own labs and

not being in the chemistry lab is
not equipped to handle surgeries and

so as the battle.

He's been very generous in helping us
with our so he's provided access to and.

This is one of his star patterns student
center that are students time Gabbert and

his family the Stanley picture
time I could find on the right.

Put your picture trying
to represent five bed.

So this is Tom who had to say with these
experiments and this is our patient.

This is a shaved anesthetized
rat the right here is of course

what we're doing in these experiments is
we're making a different one centimeter

incisions on the back of the rats on the
back of the rat and then we're talking.

We actually scrape up
the fashion a little bit

to induce a little more damage we tuck
in our gels or we're saying control.

And and then citric shot and

then usually we'll wait about
four days two to get to kind of

the peak of the inflammatory response
right before it starts to subside.

So we're going to use a few
different markers for

looking at again macrophage phenotype
is going to be the main readout so C.T.

sixty eight is a pan macrophage marker
the M one phenotype is measured by

the expression seven and
two special expression of C.

This is not a measurement of macrophage
phenotype because a lot of macrophages

were simultaneously expressed
both of these but it gave us

a semi quantitative handle on what
the effects of our materials are so

the six permits we did you
know recently high doses

in one beta antibody conjugated
How ironic acid one of these

is just a higher in a cast of control and
then the other one is higher and acid.

Plus the plus the sandy body all
the little dark circles here

are macrophages that is that
stain positively for C.T.

sixteen and you know I know I know by.

Ologists but between me and
so we did full quantification

of all the different communities to become
markers and I couldn't tell it and so

it was no friends and
so getting to prosper.

I went back and
read more about cytokine biology and so

he said that you know show
you really didn't see

a cute information there to cytokines
to get up regulated data but

there's also too many courses factor Alpha
and there's this general principle that

any important biological process will have
redundancy built and it's a pretty well

that you have two systems that
play complimentary roles and

so the nice thing about our approach
with these trials is that if you want

just I mean we spend there with
you know come on Beta side.

A party.

So when we did that we have
a very different results.

So what happened.

So.
On the left is the site that was treated

with the gel that contain
any to get off and

then here's another controls
all these little you see in

the site and

so side is to kind of occasionally see

the markers phenotype
markers that we're looking

at the other thing that was interesting
that don't doesn't really come through in

this is that when we opened up the site
for days after which suit you did so

it basically looked exactly
like it did for days earlier

we had just completely frozen the entire
process there was a little bit of a demon.

But otherwise it just was
like nothing had happened.

Whereas this controlled
proper class on tissue.

And so the good news is that curls
had an effect which was nice.

He did a lot of quantification of
the of these inflammatory mediators and

so the blue one is that
the pen macrophage marker.

So this is of so
I know some of these overlapping but

this is right and of sex for
some of the higher and so for

example these two wind up being you
know significant at least you know and

what we're seeing here is compared
to control the sailing control.

When you train with acid
with a type you actually

increase in the number of
macrophages that have invaded and

that's actually consistent with our
understanding of the damage associated

record pattern it's going to start
recruiting one time a toy sells and so

when we incorporate these things to any to
anybody that we see is we drop back down.

So it's similar to the next page number is
similar to what we saw with sailing but

it's it's it's sort of in some ways
as far as the butt of markers or

turn it can shift down in the number
of meteors information and then we're

also seeing significant decrease in the
fraction that are expressing a marker for

the M one phenotype like model
are actually having a significant effect.

We want to do is we want to lose and

what we want to do is have a nice
robust you know biomaterials and so

what we were hoping to be able to do is
actually clip it this this biological

activity into a cross thing John matrix so
independently we've been doing some work.

From Try saying how ironic
acid with a permit.

In the back with a group and
we can vary all the conditions

vary the conditions to get changed
a fraction of the minimum.

So basically you've got these chains with
these with these essentially final group

paying off the end that you can use
a radical an initiator to plumb arise and

we can do it on this rather my
chemistry students get excited

we can go then a martyr quantify.

Out here in this book in these
final pigs who can quantify

the degree of evacuation satirizing.

Yeah yeah.