Results and Discussion

It could be observed by the electronic microscope that, in Figure 3-2(a), the fiber diameter of PLLA was approximately 3~5μm and on the surface of the fibers were pores at the size of around 10nm. In Figure 3-2(b), the fiber diameter of PLLA/CHX was approximately 600~900nm and the surface was smooth. It was further observed that PLLA/CHX fibers tended to overlap and formed a structure of 2~3 fiber tubes.

Figure 3- 2 Field Emission Scanning electron micrographs of the electrospinning fibers: (a) PLLA fibers, (b) PLLA/CHX fibers image (50:50).

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In this experiment, the original intent was to understand, by means of FTIR & Raman Spectroscopy, if CHX was successfully mixed with PLLA solution to fabricate via electrospinning biodegradable PLLA fiber membranes that contained CHX. FTIR spectra of analyzed polymers were demonstrated in Figure 3-3. The band originating from C=O stretching vibrations was situated at 1745 cm^-1 for Poly lactide[76]. In the range 1050-1250 cm^-1 C-O and C-O-C stretching vibrations could be attributed. There were three bands in the range 1300-1500 cm^-1 in PLLA spectrum that might attribute to symmetric and asymmetric deformational vibrations of C-H in CH3 groups[13].

Figure 3- 3 Fourier Transform Infrared Spectra of PLLA fibers and PLLA/CHX fibers (50:50).

However, in the FTIR experiment, the characteristic IR peaks of CHX were observed between 1500 cm^-1 and 1650 cm^-1 (C=N stretching and aromatic C=C bending vibrations, respectively)[36] and PLLA demonstrated a

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very strong peak νC=O at 1760 cm^-1, so we could not confirm via FTIR whether the biodegradable PLLA fiber membranes contained CHX. FTIR could at most confirm that PLLA was used in the experiment. (In the FTIR spectrum of the PLLA film, the most pronounced difference with the spectra of individual homopolymers was the disappearance of the absorption peak at 1270 cm^-1.[13, 76])

Therefore, to confirm the existence of CHX in the fiber membranes, Micro-Raman Spectroscope was utilized to conduct fiber membrane analyses at CH3 and CH bending region. The CH3 asymmetric deformation modes appeared at about 1450±2cm^-1 as intense Raman and IR bands in all the compounds[76-77].

Figure 3- 4 Micro-Raman spectra of PLLA fibers and PLLA/CHX fibers.

In the Raman spectra of the PLLA/CHX fibers, the characteristic CHX peak at 1604 cm^-1, as indicated by an arrow in Figure 3-4, was observed. The

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peak shifted 34 cm^-1 to higher wavenumbers compared to that of pure CHX powders (1570 cm^-1)[35-36], which could be attributed to the interaction between CHX and the polymer matrix in the fibers. As mentioned in previous literature, the I875/I1452 Raman intensity ratio signaled results of bio-degradation and structural differences[76-77]. In this experiment, although both were bio-degradable membranes, the I875/I1452 Raman intensity ratio of PLLA fiber membranes was 1.494 while that of PLLA/CHX fiber membranes was 1.304.

Figure 3-5(a)(b)(c) illustrated the interactions between the PLLA/CHX (50:50) drug releasing membranes and XL1-Blue. Figure 3-5(d) was the bacteria growth curves derived by observing the ODs (Optical Density 600nm) of the 1mL broth extracted from flasks of various conditions at one-hour intervals. As ampicillin, a form of antibiotics, could effectively inhibit the growth of XL1-Blue, no growth was observed (Optical Density 600nm = 0.002) in XL1-Blue added with ampicillin solution 100μg/mL. No obvious growth was observed in the zero to third hours in the Lag Phase. Only PLLA and PLLA/CHX (90:10) experienced some minor growth in OD (Optical Density 600nm = 0.018, 0.016) in the third hour. The OD of PLLX/CHX (50:50) in the zero to third hours was 0.002, which signaled that the growth of bacteria was effectively inhibited. PLLA/CHX (50:50) PLLA was non-toxic and biocompatible. Even though PLLA/CHX (90:10) contained CHX, the concentration is very low (0.05%). Therefore, bacteria also started to grow in the third hour. In the fourth to ninth hours, the growth curves of PLLA and PLLA/CHX (90:10) entered the Log Phase, a period featured by exponential multiple growth in terms of the number of XL1-Blue. During this phase,

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PLLA/CHX (50:50) still could release CHX steadily, maintaining the concentration level of CHX in the flasks and thus inhibiting the growth of XL1-Blue (average Optical Density 600nm = 0.006).，Figure 2-4(b) was a further illustration of the growth curves resulted from the interactions between PLLA/CHX (50:50) membranes and XL1-Blue in the fourth to ninth hours.

During the Log Phase, bacteria could grow in exponential multiples, but as PLLA/CHX (50:50) steadily released CHX, the concentration within the flasks was thus maintained at a level that could effectively inhibit XL1-Blue from growing. In the tenth to twelfth hours, it was observed from the growth curves of PLLA and PLLA/CHX (90:10) that the Stationary Phase was initiated.

During this period of time, PLLA/CHX (50:10) could no longer effectively inhibit the growth of XL1-Blue. The OD (600nm) was 0.065 in the tenth hour, 0.251 in the eleventh hour, and 0.461 in the twelfth hour. Figure 2-4(c), the growth curves of PLLA/CHX membranes in the tenth to twelfth hours, further manifested such results. Theoretically, if the concentration of CHX released by PLLA/CHX (50:50) into the flasks was high enough to inhibit bacteria, XL1-Blue would not grow. However, the ODs proved that CHX concentration in the flasks was not high enough to inhibit the growth of XL1-Blue (from the tenth hour and beyond).

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Figure 3- 5 (a) (b) (c) are illustrations of interaction between drug released by drug delivery membranes and growth rates of XL1-Blue in the (a) zero to third hours (b) fourth to ninth hours (c) tenth to twelfth hours (yellow membrane:PLLA/CHX(50:50 volume ratio), Green bacterial: Xl1-Blue) (d) Growth curves of XL1-Blue in LB medium inoculated with 10⁷ CFU of bacteria.

The presence of different concentrations of (▲) PLLA/CHX fibers (90:10) and (●) PLLA/CHX fibers (50:50). Another two curves: one is only (▼) poly(L-lactic acid)s fibers; the other is (■) ampicillin-added 100 µg/mL.

Figure 3-6(a)(b)(c) were the illustrations of the interaction between PLLA/CHX (50:50) drug release membranes and TOPO XL1-Blue. Figure 3-6(d) was the bacteria growth curves of TOPO XL1-Blue derived by

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observing the ODs of the 1mL broth extracted from flasks of various conditions at one-hour intervals. Although antibiotics, ampicillin, could effectively inhibit the growth of E. coli, a section of the genetic sequence of TOPO plasmid which was anti-ampicillion was inserted, and therefore TOPO XL1-Blue can still grow in broth with ampicillin.。Microbial strains with plasmid could stay in the Lag Phase for one to two hours more than those without plasmid before they entered into the Log Phase. This characteristic was what this experiment needed. If the drug delivery membranes fabricated via electrospinning simply released CHX into the flasks through diffusion or permeation at steady rates, the 4 bacteria growth curves that reflected their interactions with TOPO XL1-Blue as illustrated in Figure 3-6(d) should be in line with Figure 3-5(d). In the zero to fourth hours in the Lag Phase, no significant bacterial growth was observed. Only in the fourth hour were minor OD increases observed in PLLA, PLLA/CHX (90:10), and ampicillion solution 100μg/mL (Optical Density 600nm = 0.023, 0.016, 0.011); ODs of PLLA/CHX (50:50) from the zero to fourth hours were maintained at the same level (Optical density 600nm = 0.003). We can further refer such results to Figure 3-6(a), the growth curves derived from the interaction between PLLA/CHX membranes and TOPO XL1-Blue. Figure 3-6(d) showed that there were not many bacteria in the flasks in the zero to fourth hours, and the release of CHX by PLLA/CHX (50:50) effectively inhibited the growth of bacteria during this period. In the following fifth to tenth hours, it was observed from the growth curves derived from the interaction between TOPO XL1-Blue and PLLA, PLLA/CHX (90:10), and ampicillin solution 100μg/mL that the Log Phase was initiated and XL1-Blue E. coli with TOPO plasmid

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were growing exponentially. However, as PLLA/CHX (50:50) continued to release CHX, the concentration was maintained at a level that inhibited the growth of TOPO XL1-Blue (average Optical Density 600nm=0.002). We can further refer such results to Figure 3-6(b), the growth curves illustrating the interaction between the PLLA/CHX membranes and TOPO XL1-Blue in the fifth to tenth hours. If the PLLA/CHX (50:50) membranes released drug at a steady rate, CHX should be fully released from the membranes into the flasks with XL1-Blue or TOPO XL1-Blue in the ninth to tenth hours. The results should be similar to Figure 3-5(d), the illustration of the interaction between PLLA/CHX (50:50) and XL1-Blue, where XL1-Blue or TOPO XL1-Blue should start to grow in the tenth hour. However, this was not the case with TOPO XL1-Blue. It was observed in the eleventh to twelfth hours that the growth curves illustrating the interactions between TOPO XL1-Blue and PLLA, PLLA/CHX (90:10), and ampicillin solution 100μg/mL, still stayed in the Log Phase. TOPO XL1-Blue continued to grow in exponential multiples and ODs of PLLA/CHX (50:50) in this phase did not show significant increases (Optical density 600nm = 0.005), i.e. the growth of TOPO XL1-Blue was still inhibited. We can further refer the results to Figure 3-6(c), the illustration of the interactions between PLLA/CHX (50:50) and TOPO XL1-Blue. In addition to environmental factors, the concentration level of inhibiting drug was the main element contributing to the inhibiting capability.

Given the assumption that the drug was released at a steady rate, the reason PLLA/CHX (50:50) could still effectively inhibit the growth of TOPO XL1-Blue in the eleventh to twelfth hours was TOPO XL1-Blue grew slower than XL1-Blue. Therefore, when CHX was first released into the flasks, there

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was no TOPO XL1-Blue to interact with. CHX continued to stay in the flasks until XL1-Blue grew to a number that was too many for CHX to suppress 〔or there was not sufficient CHX to suppress TOPO XL1-Blue〕(or until CHX concentration was lower than the effective inhibiting level). Then, it became possible for TOPO XL1-Blue to grow.

This study made use of bacterial growth curves to evaluate on a real-time basis the impacts of drug delivery speeds on the growth rates of bacteria in different phases. The growth curves of XL1-Blue and TOPO XL1-Blue were used to observe the impacts of drug delivery speeds of PLLA/CHX (50:50).

The XL1-Blue growth curves showed that the concentration level of CHX in the ninth hour could no longer inhibit XL1-Blue, and XL1-Blue started to grow exponentially in the tenth to twelfth hours, demonstrating the commom characteristics of a drug delivery system. According to Fick‘s law of diffusion, PLLA/CHX (50:50) released CHX into the flasks at a steady pace. Even though TOPO XL1-Blue grew slower, PLLA/CHX (50:50) kept on releasing CHX at a steady speed. When TOPO XL1-Blue grew to a number that was too many for CHX to suppress 〔or there was not sufficient CHX to suppress TOPO XL1-Blue〕(or until CHX concentration was lower than the effective inhibiting level), it became possible for TOPO XL1-Blue to grow. there is not sufficient CHX to suppress TOPO XL1-Blue, it becomes possible for TOPO XL1-Blue to grow. Therefore, it could be concluded from this study that the biodegradable CHX delivery membranes fabricated via electrospinning were a rate-preprogrammed drug delivery system.

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Figure 3- 6 (a) (b) (c) are illustrations of interaction between drug released by PLLA/CHX(50:50) membranes and growth rates of TOPO XL1-Blue in the (a) zero to fourth hours(b) fifth to tenth hours(c) eleventh to twelfth hours (yellow membrane:PLLA/CHX(50:50 volume ratio), Green bacterial: TOPO Xl1-Blue).(d) Growth curves of TOPO XL1-Blue in LB medium inoculated with 10⁷ CFU of bacteria. The presence of different concentrations of (▲) PLLA/CHX fibers (90:10) and (●) PLLA/CHX fibers (50:50). Another two curves: one is only (▼) poly(L-lactic acid)s fibers; the other is (■) ampicillin-added 100 µg/mL.

Polymeric drug delivery systems have numerous advantages compared to conventional dosage forms, such as improved therapeutic effect, reduced toxicity, convenience, and so on. However, the application of drug delivery

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systems in dentistry is a comparatively new area of research. In addition to applications in wound dressing, PLLA/CHX is also added to mouth-wash and used in periodontal treatments as it not only is bio-degradable membrane but also contains CHX It has been confirmed in pharmacology, microbiology, and toxicology that CHX is very powerful in killing or suppressing germs, such as gram-positive bacteria, gram-negative bacteria, anaerobic or aerobic bacterial, various bacilli, pseudomonas aeruginosa, albicans, etc. Furthermore, since it is not absorbable by human‘s gastrointestinal system, it practically has no systemic toxicity. At present, the practice of guided tissue regeneration (GTR) in treating periodontal diseases aims to stimulate the regeneration of alveolar bone and periodontal tissue. However, the infection of guided tissue regeneration membranes by bacteria often leads to poor surgery results. This explains why anti-bacteria agents, such as antibiotics or CHX, are usually used after surgery. If PLLA/CHX fiber membranes generated via electrospinning can be applied in GTR, the treatment of periodontal diseases can be brought up to another level.

In previous studies, it is known that Chlorhexidine (CHX) can effectively inhibit E. coli and S. aureus[75]. This experiment works with E. coli because it has a simpler physiology than other bacteria, and uses TOPO plasmid as control group to confirm that there is no human error during the process.

Hence, future work can look into whether biodegradable PLLA/CHX membranes fabricated via electrospin have the same bacteria-inhibiting capability on the strains that CHX is known to inhibit.

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Chapter 4 Membranes for Guided Bone Regeneration