Craniofacial

05/04/2015 Azran
Periodontal regeneration
Dr Craig


Slide1: So last week we started a discussion on periodontal ligament, its kind of a long lecture so I
think Im going to spill over into this session which kinds of works out because we are supposed to be
talking about alveolar bones and it is not a really large presentation so well finish up first with
periodontal ligaments and well talk about alveolar bones and in the second work well start talking
about gingiva. Tuesday, tomorrow, we will finish up gingiva then well have a CCP on periodontal
regeneration. And the object of that is to get you at your first year of dental school to be able to
predict what the outcome of wound healing is going to be if I give you a surgical set up or a wound
healing environment. That is a big objective because even my perio residents have problems with
this but we will persist and you will be successful. So anyway we are talking about periodontal
regeneration at the end of our last session. This whole area used to be exposed to the periodontal
pocket and by putting in a barrier so that cells from the periodontal ligaments had access to that
firing scaffold in the blood clot which occurs immediately after the beginning of the wound healing.
If you allow that to happen and you exclude cells from the gingival epithelium and gingival
connective tissue, you get this regeneration of a cementum periodontal ligaments and alveolar
bones and I left you with some questions last week and you are going to try to use some of the more
current literature to address those.
When you look at this environment, these cells that are kind of migrating in from the periodontal
ligaments, how do they know that they have to lay down cementum up against the tooth surface.
And how do they know then you have to lay down alveolar bones so this is not a tumor here, and its
not repair, its not a scar, its regeneration of three relatively evolved and distinct tissues. So what
recognizes it, what tells them that you know this is the root surface you have to lay down cementum.
Everyone on this side of the ocean thought it was something in the dentin that they were some
organizing influence in the dentin and well see one experiment that shows that that is not the case.
The second major question that we want to address is there a separate stem cell population that
exist within the periodontal ligaments that is specific for dental cementum, that specific for alveolar
bone ?
Or does a single pluripotential stem cell population exist in the periodontal ligament ?
So Ill show you two experiments there kind of graphic, there kind of classic that address these
questions. They totally answer them in science it is really hard to have a total complete answer.

Slide2: So this is a paper that came out a while ago now and its from Buser, Warrer and Karring and
they in a university in Switzerland and they came out with this paper. This is an old implant design, a
press-fit implant by the ITI associations. This 3 fox were testing this implants in dogs.

Slide3: The model was first you go in, and after you get the area thats healed then you go in with the
conventional implant protocol, you make your osteology, and in this case its not a screwed implant
but a press fit. The implant is made of titanium which reacts very easily with air, with oxygen.
Titanium oxide is relatively immunological privileged, the body doesnt react to it. Bone fuses with
the implant surface, its an ankylosis. Its because of the implant thats is made of titanium oxide.
Anyway, the story goes, that Karring has done a bunch of research associates. He edentate a dog
teeth, which is very hard to get out, so what happen is this, they had a whole bunch of root tip that
they left. So, T. Karring wanted to make lemonade out of lemon, His dogs were very expensive, so he
said ok what dont we go ahead and do the osteo-anatomy anyway and what do we place the
implants, so the implants are up against the root. because when you get this ankylosis to an
implant called osteo-integration, you get it because all you get is bone cells there but what if you
supply a periodontal regeneration competent cell population onto this titanium surface. If you
change the source of the cells, you will get a different kind of interface. So this is from the paper, so

here is the press fit implant and this is the retained dog roots and were gonna go focusing right
around here, so here is a little chip of tooth. So we are gonna go higher power magnification.

Slide4: Here is a little root chip and you kind of see this periodontal ligament and its compliment with
this tissue, in between the bone and implant. And this greenish stuff here is cementum and there is
some greenish stuff on the surface of the implant, and the purplish is the new bone forming.

Slide5: Still here there is the root tip and here there is this greenish material thats confluent with the
cementum from the root tip, and it has been deposited on titanium surface and here is the
periodontal ligament and you start to see the cellular nature of the periodontal ligament and its in
between the alveolar bone and the cementum

Slide6:Now if you jump to higher level of microscopy, there is something called bifringence light
microscopy. You can start to see the fibular nature of this soft tissue. And by definition, its a
periodontal ligament except this is not dentin its titanium. So here is the calcified material into
which the fiber are inserting, cementum and this is the alveolar bone. So the take away lesson, which
blew people away, is that you dont need a toot to get periodontal connective tissue attachment.
People have been trying to get periodontal ligament cells to lay down dental cementum, but so far
no one has been totally successful. So what cementum form on ? Well it can form on cementum,
dentin in the case of regeneration, and it has been shown experimentally on titanium.

Slide7: So, the last paper and this are all on your optional list. So this is a group at the national
institute of dental research, you know the NIDCR has a program that funds research. They publish
this in a journal called lancet which the english equivalence of our journal of american medical
association. They took non-erupted impacted human 3rd molars and they are able to dissect out the
periodontal ligament and they go through a protease digestion and they get a cell suspension they
put into culture and some of the clones that had grown very quickly and they came in with
monoclonal antibodies that were attached to iron bids and one of them was STRO-1 which is a
marqueur for ligaments cells and other parts of the body and they also have a molecular antibody
against this cell determinant which is associated with stem cells and other parts of the body. So what
they did is that they used those little bids to pull out cells that were STRO-1 and CD146 positive and
thats what they are showing here. they are going to be playing with this guys that have very high
levels of STRO-1 and CD146 expression and they show you where this cells are located and it still
hard to see but the cells that are STRO-1 positive seem to be expressed para-vascularly around the
vasculature of the periodontal ligament and since this is a human tissue they have monoclonal
antibodies against proteins that were specific for human and we are going to use that as a marqueur
and then they compare it agains 2 other lineages that have been previously described so one was
from dental pulp stem cells and the other was from bone mesenchymal stem cells. And there is a
relatively increased expression of transcription factors that are associated with ligament, something
called scleraxis, so they are basically showing that they got a cell population here that has very high
rate of cell division in the right environment and it similar but not identical to stem cell population
from dental pulp and from bone.

Slide8: And now they want to show is that it is truly pluripotential, so they compared the PDL stem
cell population with the dental pulp stem cell population and they induced mineralization and the
dental pulp mineralizes a lot, there is a little mineralization going on on the PDL but not very much,
and then they look at the expression, they compared the expression of certain marqueur, proteins,
gens and the PDL stem cells population vs the dental pulp and their are quiet similar with the
exception of the mineralization.

Slide9: They are indeed pluripotential. So they put them into an adipogenic media that favorises
adipocytes differentiation and they are staining here with a stain that is specific for lipids droplets.

this PDL cells can be induced to differentiate into adipocytes if you place them into this media and
they also express gene that are associated with lipogenesis. So you can induce them to become fat
cells.

Slide10: Now the interesting stuff, you put the cultures on hydroxyapatite particle and you put this
into nude mice, so they cant reject transplants. So up here, this is the PDL, this the hydroxyapatite
and this the bid that has the human PDL stem cells and whats happening, there is some calcified
materials being layer down, and up here this is PDL, this fibers are attaching into this matrix, as
opposed to this the hydroxyapatite carrier bid, this is where the bone cell population ,right ,so you
have something that looks like bone or osteocytes that are embedded in it. What you start to see is
a calcified matrix that looks very much like dental tubules.

Slide11:What they did next is that they manufactured periodontal defects. and then they supply the
human PDLSC to this defects and what they are showing you in this panels is the following they can
differentiate what is mouse and what is human, by staining with antibodies against anti human
mitochondrial proteins. And what you see here is that youve immuno-reactive human material that
is in the PDL making PDL fiber, you have material here that are right up against the previously
exposed dentin surface laying down a layer of cementum and you have immuno positive material
here that is right up against the bone surface to direct the bone formation. So at this point of time to
manage this paper, probably it is our strongest evidence that they appear to be a single PDLSC
population in the adult PDL and under the correct influences that cell population can climb out to
that fibrin scaffold and lay down cementum PDL and alveolar bone. we dont know what controls
this. Questions ?

Slide12: So, PDLSC. PDL perivascularly contains a pluripotential postnatal stem cell population. This
cells cant do everything but they can do somethings. They can become adipocytes, cementoblast,
alveolar bone cells PDL fibroblast The dentin surface appear not to be essential for cementum
formation which is kind of a surprise and if thats the case, a potential exists to bioengineer a
periodontal CT attachment that will support PDL regeneration such as dental implant.

Slide13: The PDL is very metabolically active it include several cell types including the cell rest of
malassez we dont know the function of this, but I think that they are there to support PDL
generation. It has 5 fibers group that you got to know, principal fiber groups that supports the tooth
in the alveolus and gingival fibers group that attaches the tooth the gingiva. In health the PDL
ligament fibroblast are responsible for fiber synthesis and remodeling. Finally somewhere in this
section exist a PDL stem cell population. Ok so let me switch over.

Slide1: Lets start our discussion on the alveolar bone. this is fast because you fox had a lot of
conversation on bone.

Slide2:There are several types of bone. There is the basal bone derived from mesenchyme. and there
is something called the alveolar process that is derived from ectomesenchyme which is dependent
on epithelial and mesenchymal interactions that characterize tooth formation. I f you don't have
odontogenesis you dont have alveolar process. And you can further dissect the alveolar process in
cortical plate on the bucal and lingual of the process, trabecular bone in between the cortical plate
and the bundle of the alveolus proper, and bundle bone of the alveolus proper which is where the
PDL fiber insert.

Slide3:So Im gonna show you a serie of radiograph. This is a human skull that was section sagittally,
very thin. and radiograph. down her is the bone of the maxilla, and here is the cortical plate in the
lingual and in the buccal, and we actually kind of see the shadow of the PDL and there is a little thin
bone which is the bundle of the alveolus proper. You see a little bit of trabecular bone. So if I was

doing this, if I was going to place a dental implant ill put it right in the center and we used to do that,
but nature doesnt do that. people usually put at the buccal at the expand of the buccal plate. So
here is some incisors.

Slide4: This is the same in the molar/premolar region. Here is the buccal, you can actually the bundle
bon and the alveolar bon better. Buccal plate is the thinnest in that area. also if you get to extract
this teeth, sometimes the plate gets out with the teeth. But if you come back to this site to place an
implant you will find resorption.

Slide5: The bundle bone, when you take your radiology course a lot of those structures shows up as
radiologic landmarks. And one of those landmarks is this area of radiodensity right around the tooth
that we call the lamina dura, it is the image of the bundle bone of the alveolus proper.

Slide6:Ok, stuff you got to know. This is a terrible skull, but it illustrates something really neat.
Anyways, if you look up in this area, this is the alveolar bone crest and this is the MB root and there
is actually a window or a fenestration. There is no bone, you flap that, if this little lip of bone is not
there then this become a dehiscence. So if you have a window it is called a fenestration and if you
just have a defect in the plate itself usually in the buccal it is called a dehiscence.

Slide7:If you take a skull and you make a cut throughout the bundle bone of the alveolus proper and
you are looking in to where the root was what you see is all those little fenestration, thats where 1/3
of the blood supply comes in this PDL, highly metabolic tissue needs lot of blood supply. So this little
fenestration are supplying neurovascular bundles from the underlying bone. Sometimes it is referred
to as the cribiform plate which is a terrible term.

Slide8: What determines the structure of the alveolar crest. it is height. here is a little cartoon. it has
3 panels. we already know from our PDL discussion that you need cementum, root surface exposed
so that you can have insertion of the gingival fiber groups. So, the alveolar crest is always going to be
below the CEJ. This distance varies depending on who you read. it changes form 1mm to half mm or
so, for the attachment of those gingival fibers. Sometimes they include the junctional epithelium. The
junctional epithelium is very promiscuous. We are going to focus in the space required for this
gingival fibers and thats called biologic width. What happens if you have a tooth that is erupting into
the oral cavity ? If you draw a line between the 2 CEJ, the shape of the alveolar crest is going to
mimic that because you have to have biologic width. What if you have the 3rd possibility, teeth are
consequently erupting in the oral cavity? you end up with this shape of the alveolar crest. So what
determines the shape and the heigh of the alveolar crest? It is where the CEJ are. where the bone is,
is going to determine where the soft tissue is. The tissue is the issue but the bone sets the tone.

Slide9: Composistion of the alveolar bone is the same as bone anywhere. Mineral phase, is 60%
mineral and 25% organic and 15% water by ash analysis. Mineral phase is mostly calcium and
phosphate, which are in the form of crystalline hydroxyapatite. By now you should realize that there
is several form of crystal in which calcium and phosphate participate. Protein phase, is mostly type I
collagen and about 90% is type I collagen, and non-collagenous proteins confer phenotypic
characteristics of bone.

Slide10: So years ago we made a study, I was very interested in getting a marqueur for dental
cementum. We got a whole bunch of dentin, PDL, alveolar bone, cementum and we will throw in
some gingival CT , we will run this out on SDS gel and we will try to pick out a protein that is specific
for dental cementum and we know that it is not gonna be in collagen because all the collagen are the
same for those tissue for but it is going to be in those 10% non-collagen protein matrix. So what you
get and we are going to get a real quick look at those non-collagenous protein extract 1, and at the
collagenous component, all of the collagen are the same.


Slide11: Is there a tissue specific protein present? And the answer is yes, but anyways, So this is
alveolar bone. Some of them are very enriched, this is osteocalcin right there. Some of those protein
are in all of those extract and some appear to be enriched in one. And I used to say there is PHD in
every band, but weigh our best attempt in many years we were never able to show that they were
one protein specific for dental cementum. For the purpose of our class, there appears to be proteins
that are enriched in various matrices and gives those matrices their phenotype specificity, like the
dentin proteins, and perhaps there is some in cementum but no one were able to show that.

Slide12: And this is to show you that all the tissue have all the same collagens.

Slide13: This is a section through human PDL and this is the alveolar bone and here are those PDL
fibers attaching in to the bone, this are called Sharpeys fiber. I know its alveolar bone because I
look at the matrix, the and you see how the cell processes radiate all around from this area.

Slide14: May be you will see it better in this slide. So it has to be alveolar bone, if it was cementum all
those cell processes would be pointing toward the PDL thats the only place where you can get
nutrients. Look at this fiber how they embedded in the alveolar bone and there is a layer of
osteoblast and through which those fibers are going to this layer of osteoblast periosteum, and if we
do some special staining.

Slide15: here is the PDL here is the alveolar osteoblast and those fibers are becoming incorporating
the alveolar bone. This is a zone of mineralization and notice how it is so precisely regulated.

Slide16: Now we are jumping to the electron microscopic level, so here is PDL coming in, here is
some cytoplasm from an osteoblast and those pepper like granules, these are crystal of
hydroxyapatite that are precipitating in to that type 1 collagen molecule embedding the PDL into the
alveolar bone. Pretty neat.

Slide17: Here is another electron micrograph, here is an osteoblast, here is a pre-osteblast ready to
jump in if he had to, this is part of the periosteum. So the periosteum is sequestering the
mineralization compartment from the rest of the body and this cells are laying down non-minerelized
osteoid and right here is the zona of mineralization. So here is previous osteoblast that has been
entrapped in its own matrix.

Slide18: Alveolar bon turn over in response to physiologic condition and in response to tooth
movement and the way it turns over is through the formation of osteoclast. So before, the whole
idea of bone immunoly into knowledge we knew that this guys which are big osteclast, they are
chewing up this matrix. In situation where you need calcium from the skeleton, you are going to
secrete PTH and 1,25 dihydroxy vitamin D3 which will increase the osteoclast activity. There are no
receptors for calcitropic hormones on osteoclasts, the receptor are on the osteoblasts, but they form
bone.

Slide19: So osteoclasts formation occurs from monocytes and requires M-CSF and RANKL and is
inhibited by interferon and OPG. whether you get bone resorption or formation in some respect it is
dependent on the ration of RANKL/OPG. As it turns out, in many sites of resorption osteoblast and
fibroblastic cells are the source of RANKL and OPG but in inflammatory sites like in periodontal we
never figured out why there is so many B and T cells in lesion. Ag-specific T and B cells secrete
RANKL, so its the cells of the immune system that are coming into this inflammatory lesion thats
telling osteoclast to become active and begin to resorb this matrix.

Slide20: Just some pictures, here are tow big osteoclast, here the hawship lacunae and this guys have

been caught into this enormous

Slide21: Comparison of bone and cementum. There is a lot of proteins in bone, because bone is a
vital membrane of the skeleton system we also use it as an ion reservoir like calcium phosphate and
it forms an intimate association with vasculature, you are also gonna have proteins that are associate
with vascular tissue. As opposed to cementum it is principally coll I and III and all of the non-cool
protein are also contained in bone matrix. This make us believe since cementum has one function
which is to attach the PDL to the toot, we think that it is derived from bone.

Slide22: So if we take a look at alveolar bone cells, cementum cells, cells that have form acellular
cementum and odontoblast. As it turns out, the cells that make cellular cementum are very close on
many of the gens, cell surface express to alveolar bone cells than they are to acellular cementum
cells. So cellular and acellular cementum are distinct tissues.

Slide23: So formation, youve already known the formation of alveolar bone. it forms as woven bone,
the spicules enlarges. This woven bone is remodeled by cutting cones to form the basic unit of
mature lamellar bone, the osteon. Alveolar bone is remodeled in response to forces we will have a
whole lecture on that. this is in contrast with cementum formation which is not remodeled.

Slide24: So, we will just finish up with this. So here is one tooth, here is a 2nd tooth, here is
cementum, you can see the appositional line of dental cementum. And if you look at the alveolar
bone, the 2 sides look different. this side here is being resorbed and this side is being formed. Notice
how the PDL maintain a relatively constant width. So if this are 2 teeth, and teeth always erupt
toward the midline, which way is the midline in this section? over here or over here? how many
believe that the midline is over here ? this is called in periodontal speak the resorption site and the
formation site. In ortho speak this is called the pressure side and the tension side.

Slide25: So here is the tooth, dentin and here is the cementum and you see the appositional line and
this is the PDL. And you see the osteoclast resorbing the lamellar bone.

Slide26: And if we look at the other side, here is the tooth, and the dental cementum with the
incremental lines, the PDL and this is bone that is formed.

Slide27: cutting cones, made up a group of osteoclast and they are resorbing the woven bone and
right behind them endothelial cells, bone forms an intimate association with the vasculature and
behind that, the osteoblast that into this tunnel are laying concentric rings of new bone. Their are
some papers that suggest that some of this endothelial cells can undergo transformation and
become osteoblasts.

Slide28: Summary. So, bone forms as a consequence of odontogenic epithelial/mesenchymal
interactions that characterize tooth formation in general, if you dont have tooth formation you dont
have alveolar bone. The osteon with its central vascular core. Bone deposition reflects the stresses
placed on it, called wolfs law . Cementum can be viewed as a specialized matrix derived from bone.

Gingival epithelium and connective tissue.

Slide1:

Slide2: 4 components of the Gingiva. So the oral or synonymous gingival epithelium. Why is it there?
for resistance to abrasion and penetration of pathogens and their products. It is keratinized or
parakeratinized stratified squamous epithelium. The sulcular epithelium, which is nonkeratinized or
parakeratinized stratified squamous depending on the state of inflammation the underline CT. The

junctional epithelium, which is nonkeratinized stratified squamous epithelium and finally the gingival
CT, which can be divided into superficial and deep CT we are not going to divide it for this course.

Slide3: here is the oral or gingival epithelium , here is the gingival margin, the sulcular epithelium.
Here is the junctional epithelium. Here is t he Gingival CT. So if the gingival Ct is inflamed, it has a lot
of T-cells, a lot macrophages it would probably be nonkeratinized and you will have more
pathogens.

Slide4: Here is a terrible cartoon again. Its probably not human, because the alveolar crest is actually
coronal to the CEJ, the cartoon got it correct. Clinician like to defines this depending on where the
sulcus is. They like to characterize gingiva, whether it is attached to the tooth or to the periosteum it
is called attached gingiva or of you draw an horizontal line across to the base of the sulcus everything
that is coronal is called free gingiva.

Slide5; Weve been through this several times so you guys are expect. Here is an erupting human
molar. Here is the reduced enamel epithelium on the distal cusp. There is leakage of bacterial
androgens in this area. Inflammation tends to make epithelium proliferate. As the tooth continue to
erupt you get an epithelial cuff.

Slide6: Here is the overlying oral epithelium, this tooth has already erupt in the oral cavity. Down into
the sulcular area, oral epithelium has proliferate. down here this is still a reduced epithelium, but
now this is called the junctional epithelium or 1 epithelial attachment with time these cells gets turn
over and they got replace with other epithelial cells and then it is called the secondary epithelial
attachment.

Slide7: This is attaching to the tooth surface by hemidesmosomes.

Slide8:Ok. So lets start on the oral epithelium. we are going through the 4 layers. that there is
stratum basal this is where we find the stem cells and then as they go through mitosis, the daughter
cells may decide to become differentiate and first stage of differentiation is stratum spinosum layer
this cells starts to synthesize large amount of keratin which are in those granules that are called
stratum granulosum. And finally the squamous cell layer.

Slide9: The basal cell layer. I f you look at the various types of epithelium that are present in the oral
environment. The cytoskeleton has a number of protein that are specific for epithelium and are
called cytokeratins. We have well characterized biochemistry associated with cytokeratin. The one to
remember is cytokeratin 19 which is associate with simple epithelium. junctional epithelium also
express cytokeratin, so does the REE. This cell rest of Malassez come back here again, very simple
epithelium.

Slide10:Here is the basal layer, here is a tiny capillary with a single RBC, here is the basement
membrane.notice how the basal cell membrane of this basal layer are kind of convoluted, kind of
neat.

Slide11: As it turns out every times you have an epithelium lying down against a CT you have to have
a basal membrane. You can differentiate 3 types of epithelial cells. the stem cells and under the
appropriate inductive factors can undergo mitosis and the daughter cells, one would remain a sten
cell and one would differentiate into a transit-ampligying cell. If you are looking at the basement
membrane you cant really differentiate this, but you can if you start looking at what integrins what
cell adhesion molecules are being expressed. you dont h ave to remember this interns, you just have
to remember that each one of this binds stem cells to the underline basement membrane. So
alpha2beta1 binds to collagen and fibronectin, alpha3beta1 binds laminin and other component,

alpha5beta1 binds to kalinin If a daughter cell from mitosis is induced to become a transit-
amplifying cell all of this will be down regulated. Transit-amplifying cell can still undergo limited
mitosis but the progeny come into the last compartment of keratinocytes that are present in the
basal layer so called committed cells. So this guys are committed to the keratinocytes differentiation
pathway. they will express any of this integrins. So stem cells are firmly attached, Transit-amplifying
cells are a little less attached and committed cells are not at all attached.

Slide12: Lets talk a little bit more about this basement membrane because there are some
interesting diseases that you would have to treat and may even diagnose that is associated with this
membrane. So if you look at it, here is a basal cell, keratinocytes in the gingival epithelium, you can
see all this collagen fibers. There is a specialized junction between epithelium and CT, called a
basement membrane, if we look a little closer, there is this clear area and a darker area called the
lamina densa and the clearer the lamina lucida. There is those fibers that come out, called the
anchoring fibrils, cause it seems like there are anchoring to this underlined meshwork of type I
collagen. on the keratinocytes sides, there are those hemidesmosomes, and tonofilaments. Lets go
through biolomolecular biology.

Slide13: here is a diagram. the tonofilaments converge to the hemidesmosomes.

Slide14: Whats going on here? There is a disease, called Bullous membrane pemphigoid. And a bulli
is only a latin word for blister. If I rub this all the epithelium comes off, and there is this ulceration, it
is called a positive nakolski sign. it is a relatively common disease.

Slide15: If you take a biopsy, here is the epithelium and here is the underlined CT. and right it
separates.right at the basement membrane. This a section right through this bulli.

Slide16: if you come in with an antibody against human IgD, and look under a fluorescent microscope
so that the antibody is fluorescently tagged, the area that is getting highly labeled is right at the
junction of the basal cells and the basement membrane.

Slide17: So you have a patient who is making antibody against a component of the basement
membrane. what is the auto-antigen ? This is a classic paper. She has anti-sirop, from people that
have Bullous pemphigoid, she doesn't know what the anti-sirop labels, So what she does is that she
screens cDNA library from a mouse, all the mRNA that this mouse is making. She puts that cDNA
library into an expression vector, so you can make the E-coli, which has now a library, make mRNA,
make the protein and youve got antibodies against the bullous pemphigoid antigen. Now, youve got
the cDNA, and you put in into another vector that has antibiotic resistance. You get in to the CDNA
and you delete part of the gene, now you have a recombinant that lacks part of the bullous
pempphigoid antigen gene, you inject it into an embryonic stem cells and that DNA gets
incorporated and you can implant it into a pseudopregenant mouse. An all you have to do is to take
a little piece of the tail of those mouse to find out who has the gene who got deleted. Then you
breed heterozygous offspring and hopefully youll end up with homozygous pups.

Slide18: Now, she has no idea what it encodes for. Panel B is the southern blot, so this is the DNA of
this animals. So this is the embryonic stem cells and you probe it with a probe and the you
incorporate the defective gene so this is the heterozygote with both the defective and normal gene,
and then you made them together and you hope for the best. And you end up with the homozygous,
both genes are knocked out. So this a northern blot here, so here is the wild type and here is the
bullous pemphigoid intact and here is RNA from the heterozygote, so one gene is transcribing the
good protein and one is transcribing the defective protein and now you look at the homozygous and
the gene is not being expressed at all. If you look at the western blot the same thing happen. So
what happens to the phenotype?


Slide19:So here is en electron micrograph. So this is the wild type, so here is the tonofilament and
you kind of see the hemidesmosomes that are kind of attached. so this is the wild type and in the
knock out, the bulous pemphigoid antigen knock out you dont have that attachment plaque at all, so
if you dont have the attachment plaque the cell wont be well attached to that area and it would lift
away in the lesion of a bullous pemphigoid.

Slide20: So what does it look like in a cartoon, here is a cartoon that have all the ultrastructure
names. So tonofilament is really an intermediate filament from the cytoskeleton and here is an
hemidesmosome and attaching to it into across the cell membrane into the lamina densa is this
structure, it turns this is collagen type 17. So she shown that the auto-antigen in bullous pemphigoid
is auto-antibodies against collagen 17.

Slide21: What else is present in the basement membrane ? As it turns out the basal cell layer has
more than just keratinocytes it has non-keratinocytes, it can have melanocytes that synthesize
melanin, langerhans cells that are this are dendtritic cells that will pick up pieces of antigen and
become mobile and cross the basement membrane and go to the lymph node and would display
this antigen to the immune cells. Merekel cells which are tactile sensory cells. And depending on the
degree of pathology that is present you or may not have a lymphocyte infiltration.

Slide22: What is the melanocytes look like ? Sometimes melanin can be very well presented in some
patient. So this is an african american which has a lot of melanin. the problem present for us is that
we us saddle sign for inflammation and it is very difficult for us to use those sign in patient like
this.

Slide23; Here is a little melanocyte and processes with those melanin granules that are being
presented.

Slide24: The next layer up is the prickle cell layer. You only really see it in light microscopy, using
dehydration agent. As you dehydrate this tissue, the cell begin to contract, so those areas start to
show up and this are really the desmosome that are holding the cells together.

Slide25: At a higher level, here is a EM of a spinous cell and here is another keratinocyte and the
junction here is a desmosome. A hemidesmosome is not half of a desmosme, it is 2 completely
different structures.

Slide26: the granular cell layer where keratin is being expressed.

Slide27: And then finally the squamous layer

Slide28: Im just going to finish with the EM of the squamous layer. So this cells are being removed
with time, any idea of what this stuff is ? bacterial colonies. The nice thing about the GI tract is that
you carry around about 2 pound of bacteria. And one of the strategies our successful ancestors had is
that they removed the outer layer of epithelium. the problem is you cant removed your teeth. So
teeth is the only think in the GI tract that you cant remove. We kind of have an interesting
environment as far as dentistry is interested.

Slide29: OK, so lets stop here.