Antimicrobial Activity of Minocycline-Loaded Genipin-Crosslinked Nano-Fibrous Chitosan Mats for Guided Tissue Regeneration

doi:10.4236/jbnb.2012.324054

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Journal of Biomaterials and Nanobiotechnology, 2012, 3, 528-532

http://dx.doi.org/10.4236/jbnb.2012.324054 Published Online October 2012 (http://www.SciRP.org/journal/jbnb)

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.

Email: [email protected]

Received August 8th, 2012; revised September 17th, 2012; accepted September 27th, 2012

ABSTRACT

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

Antimicrobial Activity of Minocycline-Loaded Genipin-Crosslinked

Nano-Fibrous Chitosan Mats for Guided Tissue Regeneration

529

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

Membrane

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).

Antimicrobial Activity of Minocycline-Loaded Genipin-Crosslinked

Nano-Fibrous Chitosan Mats for Guided Tissue Regeneration

530

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-

spectively.

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

531

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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