Journal of Biomaterials and Nanobiotechnology, 2012, 3, 528-532 Published Online October 2012 (
Antimicrobial Activity of Minocycline-Loaded
Genipin-Crosslinked Nano-Fibrous Chitosan Mats for
Guided Tissue Regeneration
Peter A. Norowski1, Jegdish Babu2, Pradeep C. Adatrow3, Franklin Garcia-Godoy2,
Warren O. Haggard1, Joel D. Bumgardner1
1Department of Biomedical Engineering, Herff College of Engineering, University of Memphis, Memphis, USA; 2Department of
Bioscience Research, College of Dentistry, University of Tennessee Health Science Center, Memphis, USA; 3Department of Perio-
dontology, College of Dentistry, University of Tennessee Health Science Center, Memphis, USA.
Received August 8th, 2012; revised September 17th, 2012; accepted September 27th, 2012
Antimicrobial delivery has been advocated for guided tissue regeneration (GTR) or guided bone regeneration (GBR)
therapies involving patients with aggressive or unresolved periodontitis/peri-implantitis. Electrospun chitosan mem-
branes demonstrate several advantages over traditional GTR barrier membranes because they stimulate healing, mimic
the topology of the extracellular matrix, and allow for diffusion of nutrients and wastes into/out of the graft site, and
were shown to stimulate bone formation in a rabbit calvarial critical-size defect model. Previously, we have shown im-
provements in mechanical properties and degradation kinetics by crosslinking electrospun membranes with 5 mM or 10
mM genipin. We have also demonstrated the ability of elecrospun chitosan membranes to inhibit lippopolysaccharide
(LPS)-induced monocyte activation. In this study, minocycline was incorporated into the chitosan membrane by passive
absorption at 5 or 10 mg/mL. The minocycline-loaded membranes and control membranes (carrier only) were tested
against Porphyromonas gingivalis (P. gingivalis) by repeated zone of inhibition (ZOI) measurements. Testing showed
that uncrosslinked and genipin-crosslinked membranes have similar capacity to absorb aqueous solutions (swelling ratio
1.7 - 2.2). Minocycline loading resulted in bacterial inhibition for up to 8 days from crosslinked membranes (with 11
mm initial ZOI) whereas uncrosslinked membranes loaded with minocycline only inhibited bacteria for 4 days (with 8
mm initial ZOI). These in vitro results suggest that genipin-crosslinked electrospun chitosan membranes loaded with
minocycline may be able to reduce early bacterial contamination of GTR graft sites.
Keywords: Chitosan; Nanofiber; Genipin; Minocycline; Guided Tissue Regeneration
1. Introduction
Bone graft materials are widely used to help repair bone
defects in periodontitis. Guided bone regeneration (GBR)
and guided tissue regeneration (GTR) barrier membranes
are often used to help maintain space for newly forming
bone and to prevent the infiltration of soft tissues into the
bone graft space. However, a major problem with current
GBR/GTR membranes is that they often become exposed
and are susceptible to infection which can reduce amount
of regenerated bone. Hence, there is a need for new
method to prevent infection of GBR/GTR membranes
and regenerating alveolar bone. Electrospun chitosan and
other chitosan membrane materials have been advocated
for GBR/GTR barrier membranes [1-3]. Chitosan is ad-
vantageous because it has been reported to stimulate the
healing of dental pulp wounds, [4] is osteogenic, and has
been shown to inhibit lippopolysaccharide (LPS)-induced
monocyte activation [5,6]. Electrospun material are ad-
vantageous because they mimic the topology of the ex-
tracellular matrix, [7] allow the diffusion of nutrients and
waste, have a small enough pore size to limit cellular
infiltration, and also have the capability to regenerate
dense cortical bone in animal models [8].
We have developed electrospun chitosan membranes
with genipin cross-linking (natural crosslinker) that have
resulted in 12% reduction in mass loss over 16 weeks,
and increased the tensile strength of the membranes three
fold [9]. We have also demonstrated that the genipin
crosslinked chitosan membranes were not cytotoxic to
osteoblast or fibroblast cells, and did not cause monocyte
activation. We have also shown the ability of genipin
Copyright © 2012 SciRes. JBNB
Antimicrobial Activity of Minocycline-Loaded Genipin-Crosslinked
Nano-Fibrous Chitosan Mats for Guided Tissue Regeneration
crosslinked electropun chitosan membranes to inhibit the
LPS-induced release of nitric oxide (NO) from RAW
264.7 monocyte cells over a 3 day period [10]. NO ex-
pression is elevated in the periodontal and gingival tis-
sues of patients with periodontitis and its inhibition is a
potential therapeutic target [11,12].
Because of the hydrophilic nature of chitosan and the
high surface area of electrospun fibers, electrsopun chi-
tosan membranes may also serve as an effective antibi-
otic delivery vehicle. The ability of the membranes to
prevent infection through the local delivery of antibiotics
during bone healing of the site while also preventing soft
tissue penetration may lead to a significant improvement
in clinical bone healing in patients. Minocycline is com-
monly used in periodontal therapy as an antimicrobial
agent but it also has the ability to limit tissue destruction,
by inhibition of tissue destroying enzymes such as colla-
genase, MMP-2 and MMP-9 [13].
In this study, uncrosslinked and genipin crosslinked
electrospun chitosan membranes were impregnated by
immersion in 10 mg/mL minocycline or 5 mg/mL mino-
cycline and tested against Porphyromonas gingivalis (P.
gingivalis) by zone of inhibition. P. gingivalis is an im-
portant and extensively studied periodontal pathogen
involved in the pathogenesis of periodontitis. In addition,
swelling was measured to assess the capacity of the elec-
trospun membranes to absorb aqueous solutions.
2. Materials and Methods
2.1. Electrospinning Procedure
Electrospun chitosan nanofibrous mats were fabricated as
previously described (Norowski et al., in review). Briefly,
a 5.50 wt% chitosan solution in 70(v/v)% trifluoroacetic
acid and 30(v/v)% methylene chloride was mixed with
genipin for 30 minutes prior to the start of electrospin-
ning. The genipin concentrations investigated were 0, 5,
or 10 mM. The solution was electrospun at 25 kV and the
fibers were collected on a non-stick aluminum foil target
(Reynolds wrap®) rotated at 8.4 RPM by an AC motor to
ensure even and random distribution of fibers. After
electrospinning, the nano-fibrous mat was put under
vacuum overnight to remove residual solvent, removed
from the foil, and then neutralized at room temperature in
5 M Na2CO3 (saturated solution) for 3 hours [14]. Mem-
branes were sterilized by ethylene oxide gas.
2.2. Characterization of Nano-Fibrous
2.2.1. Swelling
The swelling index of the nanofibrous membranes was
determined by a swelling test. Swelling in phosphate
buffered saline (PBS) was evaluated to estimate the
amount of antibiotic solution that could be absorbed by
the electrospun membranes. To determine the dry weight,
membranes were maintained at 40˚C overnight in a dry-
ing oven. After measuring the dry weight, membranes
were submerged in PBS for 1 hr (±15 minutes) to ensure
complete swelling (n = 4 - 5). Swelling index was calcu-
lated by (WtWET - WtDRY)/WtDRY.
2.2.2. Minocy cli n e Loadi ng
Minocycline was loaded into chitosan nano-fibrous mem-
branes by passive absorption. Pre-cut, pre-sterilized cir-
cular specimens (10 mm diameter), were submersed in
minocycline solution (10 or 5 mg/mL in de-ionized water)
for 15 minutes. Negative controls were submersed in de-
ionized water only. Minocycline solutions were weighed
before and after membrane swelling to determine the
amount of antibiotic solution absorbed.
2.2.3. P. gingivalis Zone of Inhibition (ZOI)
The model periodontal pathogen used in this study was
Porphyromonas gingivalis (ATCC No. 33277) which
was originally isolated from human gingival sulcus. Bac-
teria were maintained as frozen stock cultures and grown
anaerobically at 37˚C in trypticase soy broth (BD BBL,
Franklin Lakes, NJ, USA) supplemented with 1 g of
yeast extract per liter, 5 mg of hemin per liter, and 1 mg
of menadione per liter. After 72 hours of growth, bacteria
were collected and resuspended to contain 1 × 107 cells/ml.
A suspension (0.5 ml) of this stock suspension was
spread on a blood agar plate (BD BBL, Franklin Lakes,
NJ, USA) and the electrospun chitosan mats loaded with
minocycline were placed onto the agar and incubated in
an anaerobic jar with an anaerobic pack. Plates were
checked development of a ZOI by sequentially placing
membranes on freshly seeded bacterial lawns and re-
cording ZOI at days 1, 4, 6, 8 and 11 (n = 5). Zones were
measured in mm. For swelling and ZOI tests, statistical
differences were detected by ANOVA with Tukey’s test
used for post-hoc analysis (α = 0.05).
3. Results
3.1. Swelling
Swelling experiments demonstrated that the electrospun
membranes have a swelling ratio 2.24 ± 0.57, 1.71 ± 0.47,
and 1.85 ± 0.41 for uncrosslinked, 5 mM crosslinked and
10 mM genipin-crosslinked membranes, respectively.
The amount of swelling that occurred was not signifi-
cantly affected by crosslinking (p = 0.29). Although
swelling was allowed to occur for 1 hour to ensure com-
plete swelling, the membranes appeared to be fully hy-
drated within 5 minutes (data not shown).
Copyright © 2012 SciRes. JBNB
Antimicrobial Activity of Minocycline-Loaded Genipin-Crosslinked
Nano-Fibrous Chitosan Mats for Guided Tissue Regeneration
Copyright © 2012 SciRes. JBNB
3.2. Minocycline Loading
Submersion in 10 mg/mL minocycline solution resulted
in a range of 0.12 - 0.52, 0.53 - 0.54, and 0.38 - 0.57 mg
of minocycline uptake for uncrosslinked, 5 mM cross-
linked and 10 mM genipin-crosslinked membranes, re-
3.3. P. gingivalis Zone of Inhibition (ZOI)
ZOI testing demonstrated extended release of mino-
cycline from the barrier membrane in vitro for up to 8
days after soaking in 10 mg/mL (Figure 1) or 5 mg/mL
(Figure 2) minocycline for 15 minutes. It was noted that
uncrosslinked membranes only remained bacteriostatic
for 4 days as compared to the 5 mM and 10 mM
crosslinked membranes which remained bacteriostatic for
8 days. The ZOI from 5 mM and 10 mM crosslinking
were similar although 10 mM crosslinking did result in a
larger ZOI than 5 mM on day 4 when loaded with 10
mg/mL minocycline. None of the negative controls (car-
Figure 1. Inhibition of P. gingivalis over an 11 day period by electrospun chitosan membrane loaded with 10 mg/mL mino-
cycline. Error bars represent the standard deviation (n = 5). * and # denote statistical differences (α = 0.05).
Figure 2. Inhibition of P. gingivalis over an 11 day period by electrospun chitosan membrane loaded with 5 mg/mL mino-
ycline. Error bars represent the standard deviation (n = 5). *denotes statistical difference (α = 0.05). c
Antimicrobial Activity of Minocycline-Loaded Genipin-Crosslinked
Nano-Fibrous Chitosan Mats for Guided Tissue Regeneration
rier only) produced zones of inhibition.
4. Discussion
The swelling study demonstrated that membranes have
similar capacity to absorb fluid and any differences in
swelling capacity were not significant. Therefore, the
crosslinked membrane should have similar capacity to
absorb drugs, antibiotics, or growth factor solutions.
Unlike vapor crosslinking and solution crosslinking me-
thods, where materials are exposed to a crosslinking
agent that crosslinks polymer chains primarily on the
surface, in this study, the crosslinking agent is dispersed
within the polymer solution used for scaffold fabrication.
This method creates a more uniformly and thoroughly
crosslinked membrane, which likely contributed to im-
proved antibiotic retention as observed in this study and
more uniform degradation kinetics. A previous examina-
tion using x-ray diffraction showed that crystallinity was
decreased during crosslinking. This may have contrib-
uted to slightly lower swelling volumes, however this
difference was not significant.
In this study, genipin-crosslinked electrospun chitosan
was able to absorb minocycline and release it in an ex-
tended manner that remained bacteriostatic for longer
periods than uncrosslinked membranes (8 days as com-
pared to 4 days). These results are similar to reports by
others who have loaded biodegradable GTR membranes
with antibiotics/antiseptics such as tetracycline, doxycy-
cline or chlorhexidine [15-17]. Delayed degradation ki-
netics (Norowski et al., in review) contributed to the ex-
tended release seen from crosslinked membranes. Thus,
uncrosslinked chitosan membranes degraded faster and
resulted in lower minocycline loading and release levels.
One clinical investigation reported no improvements
in clinical parameters associated with the local applica-
tion of minocycline ointment before GTR therapy, but
this study did not investigate minocycline incorporation
into the GTR membrane itself, and only investigated the
use of type 1 collagen membranes. [18] Other investiga-
tions with a non-membrane local delivery system dem-
onstrated improvement in clinical parameters associated
with the use of minocycline microcapsules (Arestin®).
[19] This microcapsule study also showed that reduction
in periodontal pocket probing depth (improved clinical
outcomes) correlated strongly with the ability to inhibit
red complex bacteria in vitro, a sub-group of periodontal
pathogens that includes P. gingivalis, T. forsythia, and T.
denticola [19].
5. Conclusion
In this study, we have shown the ability of genipin-cross-
linked electrospun chitosan to deliver clinically relevant
levels of minocycline over an 8 day period. The eluted
minocycline was able to inhibit growth of P. gingivalis, a
model periopathogen, in vitro. Crosslinked membranes
released inhibitory levels of minocycline for 8 days while,
uncrosslinked membranes only inhibited growth for 4 days.
This prolonged minocycline elution profile suggests that
genipin-crosslinking improved the drug-carrier properties
of electrospun chitosan.
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