978

Slow Release of Tetracycline

Hydrochloride From a Cellulose
Membrane Used in Guided Tissue

Regeneration
Cristina Markman* Sergio E.L. Fracalanzza* Arthur B. Novaes, Jr.*
and Arthur B. Novaes*

The objective of this study was to evaluate if the biologic membrane utilized for
GTR can be impregnated by tetracycline- hydrochloride and if the chemotherapeutic
agent, once impregnated, can be released in minimal inhibitory concentrations for a
period compatible with clinical application. Initially, an in vitro study was done with
cellulose membranes cut in pieces measuring 9 cm2. A volume of 100 containing a
72,000 g/ml solution f tetracycline was dispensed onto each fragment, and dried for
70 minutes at 37C. Four pieces measuring 0.5 cm2 were cut from different points of
the 9 cm2 membrane (presumably, containing 400 g of tetracycline), placed in test
tubes containing 4 ml of sterile deionized water, and agitated for 2 minutes. A standard
curve was made from known concentrations of tetracycline and compared to 10 of
the test solutions obtained by the elution of the 0.5 cm2 fragments. The concentrations
were determined through the bioassay technique in 3 duplicate experiments. The samples
recovered from the membrane fragments had a mean of 101 g/ml of tetracycline
liberated, demonstrating that the membrane was impregnated homogeneously by the
chemotherapeutic agent. In a second phase, an in vivo study was carried out to determine
the length of time the drug was liberated from the membranes and at which concentrations, in the presence of an inflammatory process. Fourteen 0.5 cm2 fragments containing
400 g of tetracycline were placed in 14 polypropylene chambers containing 200 of
thioglycolate medium. The chambers were implanted in the peritoneal cavities of 14
mice, one chamber per animal, and left in from 1 to 14 days. They were then removed
and the concentrations of tetracycline determined from 20 samples using a bioassay.
The results showed that the antibiotic was released slowly from the 1st through the 12th
day in decreasing concentrations that varied from 218 to 20.8 g/ml. The impregnated
cellulose membrane can probably be used in GTR acting as a membrane and as a slowrelease device, liberating the chemotherapeutic agent in concentrations high enough to
eliminate periodontopafhic microorganisms. / Periodontol 1995;66:978-983.

Key Words: Membranes, artificial; membranes, barrier; tetracycline/therapeutic

guided tissue regeneration.

Guided tissue

regeneration (GTR) is an accepted therapeutic modality for the treatment of furcation lesions1-2
and interproximal defects.2 It can also be used for bone
regeneration following tooth extractions and reconstruction of alveolar ridges,3 and in association with osseointegrated implants.4-5
Systemic antibiotics are usually prescribed as part of
Federal University of Rio de Janeiro, Brazil.
'University of Sao Paulo at Ribeirao Preto, Brazil.

the

use;

to avoid the formation of microabthat

scesses
may occur around the third week of healing.6-7
They may also be prescribed to avoid contamination of
the membrane and consequent infection in situations
where the membrane becomes exposed due to soft tissue
recession.8 Tetracycline hydrochloride (Te-HCl), used systemically or locally, is an antibiotic commonly recommended for various reasons: it is highly efficacious
against the majority of periodontopathic bacteria,9-11 it has
an acidic pH and can be used as a root demineralizing

surgical protocol

Volume 66
Number 11

agent,12

MARKMAN, FRACALANZZA, NOVAES JR, NOVAES

it inhibits human

collagenase13

and bone rsorp-

tion,14 and it is associated with bone formation.1516 Although a generally accepted mode of administration, the

routine use of systemic antibiotics17 may cause different

levels of side-effects. In order to avoid the systemic use
of antibiotics and possible side-effects, it would be ideal
if the membrane used for GTR could deliver the antibiotic

locally.

The objective of this study was to evaluate if a membrane used in GTR* can be impregnated homogeneously
in vitro by Te-HCl. We also determined in vivo if the
drug was liberated from the membrane and, if so, at which
concentrations and for how long.

MATERIALS AND METHODS

979

Slow Release of the Te-HCl From the Cellulose

Membrane: In Vivo Analysis
Membranes measuring 0.5 cm2 impregnated with 400 g/
ml of Te-HCl were placed into polypropylene chambers
measuring 15 mm in length made from the body of 1 ml
syringes. One end of the chamber had been previously
sealed with a nitrocellulose filter with pores measuring
0.22 . All the material utilized had been previously sterilized. The membrane was introduced into the chamber
along with 200 of thioglycolate medium. The chamber
was then sealed with another nitrocellulose filter with the
same pore size. The thioglycolate medium was used as a
diluent because it also induces inflammation in the surrounding tissues as it diffuses through the pores in the
nitrocellulose filter, mimicking the inflammation present
in the periodontal tissues of patients with periodontal disease.

Membrane

Analysis

Impregnation

With Te-HCl: In Vitro

The sterile cellulose membrane was cut into pieces measuring 9 cm2 (3X3 cm). To each piece of the membrane
100 of a 72,000 g/ml solution of Te-HCl was dispensed under sterile conditions such that the whole membrane was homogeneously covered by the solution. The
membranes were placed in Petri dishes and dried at 37C.
The 9 cm2 pieces were then cut in smaller pieces measuring 0.5 cm2 and stocked in a desiccator at 10C.
Theoretically, each 0.5 cm2 piece should contain 400
g of Te-HCl. To evaluate this hypothesis, four 0.5 cm2
membranes taken from various parts of the original 9 cm2
membrane were placed in four separate test tubes containing 4 ml sterile deionized water, and then agitated for
2 minutes.
The concentration of Te-HCl in the solution was determined through the bioassay technique. The MuellerHinton agar medium containing Bacillus subtilis (ATCC
6633) as the test bacteria was perforated with sterile cylinders measuring 6 mm in diameter. To each 6 mm hole
10 of known concentrations of Te-HCl were dispensed
in order to obtain a standard curve. A 10 sample removed from each of the test tubes containing the impregnated pieces of the membranes was also dispensed into
separate empty holes. The experiment was always done
in duplicate.
The dishes were incubated for 18 hours at 37C and
the diameter of the inhibition halos measured. The concentration of the antibiotic in each of the membranes was
calculated from the mathematic curve obtained through
linear regression analysis from the experimental points on
the standard curve.19 The experiment was repeated three
times, each on a different day.

'Gengiflex,

BioFill Produtos

Biotecnologicus, Curitiba, PR,

Brazil.

The chambers were introduced into the peritoneal cavity of 14 adult Swiss mice. This experiment was conducted according Federal University protocol for animal
studies. A 15-mm incision was made in the trichotomized
abdomen of the previously anesthetized animals, gaining
access to the peritoneal cavity. One chamber was introduced into the peritoneal cavity of each of the 14 mice
so that it adapted to the internal organs without damage
to them. The incisions were sutured and the animals were
kept warm until recovery from anesthesia. One animal
was sacrificed each day for the next 14 days. A new access to the peritoneal cavity was made to recover the
chambers and to analyze the reaction of the tissues to the

implantation.
Immediately following

retrieval of the chambers, their

removed with a sterile tip adapted to a micropipette from a small hole made in one of the nitrocellulose filters and transferred to a sterile tube.
20-1
of
each
tube
diluted
in
medium
was
thioglycolate
sample
1:10 to 1:80 in serial dilution, always in duplicate.
The concentration of Te-HCl was determined through
the bioassay technique as described earlier, with the exception that the standard curve was prepared with Te-HCl
diluted in thioglycolate medium.
contents

were

RESULTS
The membranes absorbed the antibiotic well without apparent alteration in their physical characteristics.
The in vitro experiment was designed to evaluate the
uniformity of the impregnation of the membrane with TeHCl. Four 0.5 cm2 pieces obtained from the original 9.0
cm2 membrane were identified as Tl, T2, T3, and T4.
They were placed in deionized sterile water, agitated for
the elution of the antibiotic, and the resultant solution
tested through the bioassay using Bacillus subtilis as the
indicator bacteria. The experiment was repeated three
times. As can be seen in Table 1, the mean diameter of
the halos of inhibition for Tl, T2, T3, and T4 in the three

980

J Periodontol
November 1995

TETRACYCLINE-TREATED CELLULOSE MEMBRANE USE IN GTR

Table 1. Evolution of the Homogeneous Distribultion of Te-HCl in

the Membranes Analyzed Through the Bioassay
Concentrations of
TE + HCL

Diameter of the Halo of Inhibition

Og/ml)

Experiment

3.12*
6.25
12.50
25.00
50.00
100.00
200.00
400.00

<6
7
13
17
22
25
29
32

TV

26
25
26
25
25.5

T2
T,
T4

Mean T, and T,

1000

Experiment

(in mm)
Experiment 3

<6

11

7
14
18
23
25
29
33
25
26
25
25
25.0

13
20
22

Concentrations of Te-HCl used for the standard

en

3
0)

25
29

.c

32
34

TS

29

o
i

100

>>

28
28
28

>.

28.2

ta

curve.

T T2, T3, T4, represents the experimental fragments of the impregnated

membrane. Each fragment was placed in 4 ml of distilled water, and
after elution the concentrations of the antibiotic

were

10

determined.

c
a

experiments is 25.5 mm, 25.0 mm, and 28.2 mm, respectively. The concentrations of Te-HCl in the fragments
were obtained using the equation shown in Table 2. The
concentrations were 111 g/ml, 95 g/ml, and 98.0 g/
ml, which were very similar to the initial 100 g/ml con-

centration. These values also indicated that the concentration of Te-HCl in the 0.5 cm2 fragments were close to
400 g, because the membranes were placed into 4 ml
of water. The mathematical curves obtained from the
equations shown in Table 2 are represented in Figure 1.
In the second stage, the capacity of the membranes impregnated with Te-HCl to liberate the antibiotic in vivo,
in the presence of an inflammatory process similar to that
which occurs in periodontal disease, was evaluated, as
well as the concentrations obtained after periods from 1
to 14 days.
Following periods of 1 to 14 days, the animals were
sacrificed and the peritoneal cavity re-entered to retrieve
the chambers. Examination of the surrounding tissues did
not reveal exudates or any signs of generalized inflammation. In the animal sacrificed after day 1, the extrem-

Experiment 1
Experiment 2

Experiment 3

J_

1
10

20

40
Size of the halo of Inhibition
30

(mm)

Figure 1. Mathematical curve obtained through linear regression analysis of the experimental points in the standard curve.
ities of the chamber had adhered to the intestinal loops.
The tissues in that area showed hyperemia and edema,
indicating the beginning of a discrete inflammatory process. The process increased from the second to the fourth
day, when a membranous capsule surrounded the chamber
and the presence of a highly vascularized inflammatory
tissue was present, especially close to the extremities of
the chamber. The process remained stable up to the 14th

day.

Table 2. Determination of the Concentrations of Te-HCl in the Fragments of the Impregnated Membranes Through the Equation Obtained Through Linear Regression Analysis of the Experimental
Points

Concentrations of Te-HCl in the

Experiment
1
2
3

Equation
log
log
log

y
y
y

=
=
=

0.21 + 0.072.x*
0.16 + 0.072.x
-0.47 + 0.087.x

rxy*

T,

T2

T3

0.99
0.99
0.99

120.0

102.0
107.0
93.0

120,0

*Coefficient of correlation.
'Each fragment was eluted in 4 ml of water.
'Standard errors for Equations 1, 2 and 3 are
level (P < 0,01).
\

'In g/ml.

91.2
112.0

respectively 0.06,

Fragment
T4
102.0
91.2
98.0

91.2
93.0

0.07 and 0.08. All

are

After Elution'
Mean

111.0
95.0
98.0
101.0

significant at the

1%

Volume 66
Number 11

MARKMAN, FRACALANZZA, NOVAES JR, NOVAES

Table 3. Determination of the Concentrations of Te-HCl in the

Chambers Implanted in the Peritoneal Cavity Following the Indicated Period
the Chambers
Remained

Days
1
2
3
4
5
6
7
8
9
10
11
12
13
14

Concentration of Te-HCl

981

1000

(in g/ml)

218
91
114
91
91
89
44
38
54
39
22
21

<3-l*
<3.1

Smallest concentration of Te-HCl tested in the standard

curve.

After recovery, the fluid inside the chambers was asand tested the same day using the previously described bioassay.
The concentrations of the antibiotic obtained during the
14 days of the experiment are reported in Table 3. The
concentrations of Te-HCl were calculated as described for
the in vitro study.
After 1 day the concentration of the antibiotic within
the chamber was 218 g/ml. The concentrations fell progressively to 20.8 g/ml after 12 days and then the antibiotic could no longer be detected with the methodology
used in this study.
An analysis of Figure 2 reveals that the high concentrations found after 24 hours fell to values close to 100
g/ml after 48 hours remaining at this level up to the 6th
day. From the 7th to the 10th day the concentration stabilized between 40 to 50 g/ml. On the 11th and 12th
days the concentrations of the antibiotic within the chambers remained around 20 g/ml.

pirated

DISCUSSION

Tetracycline hydrochloride was one of the first antibiotics

to be used in periodontal therapy and it acts by impeding
the union of the complex tRNA-amino acid to the ribosome, thus affecting protein synthesis.20 In vitro studies
have shown that tetracycline hydrochloride is highly effective against the majority of periodontopathic microorganisms related to chronic Periodontitis, juvenile Periodontitis,78 and refractory Periodontitis.21
Antibiotics can be delivered topically in a variety of
ways. They can be administered through daily irrigation
of the pockets, through direct conditioning of the tooth
structures, in rinses, and through slow release devices
placed in the pocket.21-23 Slow release devices seem to be
more beneficial when compared to other forms of application. They can be used as an alternative to systemic
delivery and allow the utilization of doses lower than

_u
0

9 10 11 12 13 14

Days in the Peritoneal Cavity

2. Curve representing the behavior of the concentrations
HCl within the chambers in periods up to 14 days.

Figure

of Te-

those usually employed systemically. The concentration

of the antibiotic in the pocket has been shown to be 100
times higher than the concentration obtained when administered systemically.24
The advantages of local delivery of antibiotics, the efficacy of tetracycline against periodontopathic bacteria,
and the results obtained by GTR led us to this study,
where the main objective was to combine all these properties into one product.
The first step was to evaluate the capacity of the cellulose membrane to be impregnated without damage to
its physical structure. The results showed that the membrane can be easily impregnated without apparent alteration to its structure.
The antibiotic impregnating the membrane has to be
liberated when coming in contact with an aqueous medium in order to be effective. To test this property an in
vitro and an in vivo experiment were carried out.
The in vitro phase of the study had the objective of
evaluating if the impregnation occurred homogeneously
throughout the membrane, and if the tetracycline was liberated when placed in an aqueous medium. Out of an
expected concentration of 400 g of tetracycline in each
0.5 cm2 membrane, concentrations close to that were obtained (Table 2). This meant that the antibiotic was easily

982

TETRACYCLINE-TREATED CELLULOSE MEMBRANE USE IN GTR

released into an aqueous medium, similar to the medium

found in the periodontal pocket.
In the in vivo phase, an experimental model with the
liberation of the antibiotic, in the presence of a "sterile"
inflammatory process, was successfully created. As expected, the thioglycolate medium induced a local inflammatory process, causing the formation of a capsule of
highly vascularized newly formed tissue around and attached to the extremities of the chamber. The nitrocellulose filters at the extremities of the chamber allowed the
diffusion of the antibiotic from within the chamber to the
tissues around the chamber, similar to that which occurs
when a slow-release device is introduced into a periodontal pocket.
It is important to determine how long effective concentrations of the antibiotic can be detected. Fourteen animals received one chamber each and were sacrificed daily so that the concentration of the drug could be determined during this period.
A decrease in the concentration of Te-HCl from 218
g/ml after 24 hours to 20.8 g/ml after 12 days was
observed. These results are highly significant due to the
high concentration of the antibiotic that was obtained and
by the way it was slowly liberated from the membrane.
If used as a slow-release device in a periodontal pocket
it would have been effective against the bacteria involved
in periodontal disease. The microorganisms commonly
found in association with Periodontitis are generally susceptible to a concentration of 8 g/ml of Te-HCl.9 Even
microorganisms such as S. sputigena, non-pigmented
Bacteroides species, and some Gram-negative rods, which
have a minimal inhibitory concentration less than or equal
to 16 g/ml, could have been inhibited up to the 12th
day by the impregnated cellulose membrane.
Although a comparison between the cellulose membrane impregnated with Te-HCl with slow-release devices
described in the literature is inadequate due to differences
in the concentrations used, in the type of material employed, and due to differences in the treatment protocol,
the concentrations obtained and maintained for up to 12
days in this study are equal to or better than the results
obtained in some studies with slow-release devices.22 24"27
The results obtained with the methodology employed
in this study indicate that concentrations between 218 and
20 g/ml up to 12 days allow adequate control of the
bacteria involved in the disease and the microorganisms
that might adhere to the membrane in case of membrane
exposure during the healing period.

Conclusions
The methodology employed for the impregnation of the
cellulose membrane was adequate, reproducible, and easy
to perform, important factors to make serial production
feasible.
The cellulose membrane impregnated with tetracycline

J Periodontol
November 1995

be utilized in periodontal procedures, because in addition to being a biocompatible membrane, it acts as a

barrier during GTR and has the capability of liberating
Te-HCl up to 12 days in concentrations more than sufficient to eliminate periodontopathic microorganisms.
can

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Send reprint requests to: Dr. Arthur B. Novaes, Jr., Av. das Americas
1155, Room 1002, Rio de Janeiro, RJ, Brazil 22631-000.
Accepted for publication May 11, 1995.