Charles F.

Cox, DMD, PhD

Director, Research & Developm ent
Phoenix Dental Inc. Fenton, Michigan
BIOCOMPATIBILITY OF DENTAL AGENTS:
A SERIES OF VI FACTOIDS
PART-I: DENTAL BIOCOMPATIBILITY – A HISTORICAL PERSPECTIVE
PART-II: WHICH CAME FIRST? – IN VITRO OR IN VIVO BIOCOMPATIBILITY ANALYSIS
PART-III: IN VIVO BIOCOMPATIBILITY ANALYSIS
PART-IV: IN VITRO BIOCOMPATIBILITY ANALYSIS
PART-V: WHICH BIOCOMPATIBILITY TEST IS MOST IMPORTANT ?
PART-VI: A BACTERIOMETIC SEAL IS THE REAL DEAL

PART-V: WHICH BIOCOMPATIBILITY TEST IS MOST IMPORTANT ?

Now that you’ve read FACTOIDS #I thru #IV, you may think that the answer is
simple. But, if you’ve followed the chronological comparison of in vitro versus in vivo
testing, you may realize that no simple answer has yet presented itself—a proper
scientific answer for biocompatibility testing is simply not easily reported by a simple yes
or no response.
A number of in vivo animal & human usage studies (Kakahashi 1968, Brännström
1968, Bergenholtz 1978, Cox et al 1982,1984,1987, 1992) each demonstrated—
beyond a doubt—that certain bacteria & their toxins can easily infiltrate into enamel
lamella & restorative channels & rapidly penetrate down to the EDJ. From this point,
the bacteria & toxins can easily spread along the EDJ interface, where they penetrate
into & through the dentine tubules. From this point, they may initiate a low-grade
irritation response to the odontoblast cells, whereby these cells may respond by
depositing a thin layer of reactionary dentine. However, if the insult persists as a
chronic effect, the primary odontoblasts may die—whereby certain undifferentiated cells
of the pulp stroma may respond to form new odontoblastoid cells that then deposit a
new layer of reparative dentine directly adjacent to the secondary dentine. However, if
the bacterial insult is persistent, it may easily cause pulp inflammation & regional pulp
necrosis (Van Hassell). Such dynamic factors of microleakage & bacteria are extremely
difficult to control & even more so, they are difficult to simulate within in vitro bench top
tests—the ISO committee is still waiting for such well controlled & in vitro tests to be
repeated by ―certified research laboratories‖.
Lets now consider some additional research publications to allow you to make the
decision for yourself: 1) In vivo research data in dog teeth were first published in the
1936 Brit Dent Jour by Dr. Manley of Birmingham, England. He reported that the

1
 Charles F. Cox, DMD, PhD
Director, Research & Developm ent
Phoenix Dental Inc. Fenton, Michigan
H3PO4 component of silicate cement was the toxic factor to vital canine dental pulps. 2)
It was 2-decades later that in vitro tissue culture tests were first published on silver-tin
dental amalgam by Dr. Kawahara & colleagues from Osaka University in 1955. 3) In
1968, Dr. Brännström demonstrated in controlled human studies, that silicate cement
was non-toxic to vital pulp cells—bacteria were the causative agents for inflammation
through microleakage along the restorative interface. 4) In 1977—after years of
personal efforts with colleagues—Dr. William Cotton organized the 1st IADR PBG
Symposium in Copenhagen, which strongly advocated for the creation of harmonized
st
biocompatibility standards—he was 1 to move towards harmonization of
biocompatibility testing. 5) After years of meetings the ISO committee published their
1st Book of Harmonized Biocompatibility testing standards in 1999. 6) In 2009, the ISO
committee published revised testing standards that serve as suggested standards,
which may be chosen for evaluation by the ―developing group‖ for biocompatibility
validation.
ARE TOXIC AGENTS PRESENT IN TODAYS DENTAL RESTORATIVE AGENTS?
Today, the fact remains. There are certain agents that are minor & major
components of restorative dental systems that range from irritational, mutagenic,
carcinogenic & toxic to vital human tissues. For instance, research publications report
that the toxic chemistry of formalin (OSHA Tech guide 2006), aldehydes (O’Brien et al.
2005), glutaraldehyde (Sano et al. 2005), formocresol (Block 2010), phenol (Conning
1970), hydroxy ethyl methacrylates (HEMA), (Mathias et al. 2006), bis-phenyl-A (BPA)
(FDA 2010), & ketones (Breeze 1984); are known irritants that range from mild-to-
toxic within the International Medical & Dental profession (Autian 1972, 1974).
Isn’t, it somewhat curious that due to that rather infamous grandfather clause that
still lingers in the dental industry since the 1950’s era, that the dental profession is still
able to continue to employ many of these irritational & toxic agents as a minor
component of ―newly developed‖ dental products. And, in addition to the various
chemicals, certain metals (nickel, cadmium, zinc, copper) that are commonly found in
many cosmetics also are known to cause initial sensitizing reactions to humans. In
1999, Yamanaka reported more intense irritational reactions to human dentine & vital
pulp tissues! Again, even with the current continued ISO acceptance of these
grandfathered, yet known irritating dental products, many worldwide dental agencies
continue to permit the clinical use of these agents—even though some are classified as

2
 Charles F. Cox, DMD, PhD
Director, Research & Developm ent
Phoenix Dental Inc. Fenton, Michigan
germicidal (glutaraldehyde, Sano 2005). In addition, studies have documented that
high concentrations of alcohols, aldehydes & acetone may rapidly dehydrate &
denature vital cells, collagen fibers & other proteins especially when improperly placed
by the clinician. Today, there still remains on the commercial dental marketplace,
endodontic & pædiatric restorative agents that contain lead, formaldehyde &
glutaraldehyde, which are known irritants & are potentially toxic, not only to the patient
but more importantly to all office personnel who work in the clinic environment on a
daily basis.
WE NOW POSE AN APPARENT QUESTION: CAN IN VITRO RESEARCHERS WHO
OFTEN REPRESENT VARIOUS COMPANIES INTERESTS—HAVE IT BOTH WAYS ?
SHOULD THEY BE ABLE TO CHOOSE THEIR OWN FAVORITE TEST ?
As we’ve reached this point, we can now understand that the ISO–10993-5-2009
committee in vitro tests recommendations are only suggestions. They only serve as the
first biocompatibility ―test gate‖, especially when a new dental restorative product or
device is developed for use in the oral cavity. Once the developing group has passed
their own personally chosen in vitro biocompatibility test—they can then move to the
next in vivo biocompatibility-testing gate—again an animal system of their choice.
Animal tests involve the surgical placement of the agent into the connective tissues of
the tissues of rats or rabbits for certain periods of time along with positive & negative
control agents such as ZnOE, silicate or ZnPh cements for evaluation of their
histological responses.
Still, we are now confronted by the fact that initial in vitro tests are not completely
reliable to define a possible false positive or a false-negative test outcome. For
instance, if you challenge those same in vitro research laboratories who adhere to their
―own suggested‖ in vitro test & then ask them if they have routinely employed a known
toxic control agent e.g. glutaraldehyde in their system, they often respond that they do
not concern themselves with such a control test—especially since those agent
(glutaraldehyde, phenol, acetone, HEMA) have long been grandfathered as legally
acceptable restorative dental agents. If you ask those same in vitro researchers who sit
on today’s Pulp Biology committees that discuss & define testing models & then
challenge them with the critical issue of bacterial infection use in their own in vitro
system, they quickly respond they ―are only required to follow the suggested BS EN

3
 Charles F. Cox, DMD, PhD
Director, Research & Developm ent
Phoenix Dental Inc. Fenton, Michigan
ISO–10993-5 standards‖—some of these same people sit on committees to define
standards. Isn’t it ironic that these researchers can continue to have it both ways?
IS THERE AN IDEAL IN VITRO BIOCOMPATIBILITY TEST ALTERNATIVE TO USING
LIVE ANIMALS?
Well, I’ve presented—what I consider to be important concerns about
biocompatibility testing. So let me present a possible solution to what I see as a testing
conundrum. As we think about an ideal ISO in vitro biocompatibility test, the research
person/group needs to demonstrate a proficiency of understanding of enamel, dentine,
cementum & pulp substrates of the tooth. More importantly, they need to consider the
dynamic dimension of the oral bacterial biofilm & their toxins, which easily penetrate
from the tooth surface & into the vital pulp.
Today, there appears to be greater research focus on mechanical bond strength
testing of new restorative agents when submitted for the intended clinical use.
Nakabayashi (1992, 1997) determined that the general breaking (cohesive) capacity of
―normal‖ human dentine is approximately 21-megapascals (MPa), whereas the general
breaking capacity of normal human enamel is approximately 52-MPa (Shimada 1992).
In addition, (Craig & Peyton 1956) verified that mineralized substrates have regional
morphological extremes of density—demonstrated by their indentation tests. Ten
Cate’s text (2002) has eloquently demonstrated that enamel, dentine & pulp tissues are
variable substrates in their developmental, aging, physiological & morphological
dimensions—today’s researchers should be responsible to be knowledgeable about all
tooth substrates before they begin their research studies.
WHERE MIGHT THE RESEARCH ISSUES NOW GO ? As one searches the www to
identify published in vivo & in vitro studies of new dental materials—it is obvious that in
vitro publications far exceed all other tests. Personal experience reflects that a well
defined & controlled ISO in vivo usage primate study takes several years from inception
to completion as well as requiring a strong funding base. In contrast, an in vitro study is
much less time consuming & much less expensive. As future ISO committees move
towards harmonization of biocompatibility testing, they should consider to move beyond
bond strength testing & to explore the development of tests that will to merge the more
important biological issue of a bacteriometic restorative seal—from both a biological as
well as mechanical ―sealed‖ perspective. ESSENTIALLY-THE SEAL IS THE DEAL !