Biocompatibility and cytotoxic evaluation of drug-loaded biodegradable guided tissue regeneration membranes.

BACKGROUND: In periodontology, Guided Tissue Regeneration (GTR) is based on the concept of providing a space for entry of cells with regenerative potential into the wound environment to initiate the regeneration of structures lost due to periodontal disease. First generation GTR membranes were primarily non-absorbable membranes like expanded polytetrafluorethylene which required a second surgery for its removal. This led researchers to explore absorbable materials like collagen and synthetic biodegradable polymers to fabricate GTR membranes. In the present study, biodegradable Polylactic acid (PLA) is used to fabricate membranes with the potential to be used for GTR therapy.
MATERIALS AND METHODS: Biocompatibility of the PLA membranes were evaluated in a subcutaneous guinea pig model. Antimicrobial effect of the drug-loaded PLA membranes were assessed against a drug-resistant Staphylococcus aureus bacterial isolate. The cytocompatibility of the drug-loaded membranes were evaluated using HeLa cell lines.
RESULTS: The PLA membranes were shown to be biocompatible. The drug-loaded PLA membranes showed significant activity against the bacterial isolate. Among the drug-loaded membranes, tetracycline-loaded membrane showed minimal cellular toxicity.
CONCLUSION: The results of this study indicate that biodegradable drug-releasing polylactide membranes have the potential to be used for periodontal regeneration. It has the necessary characteristics of a GTR membrane like biocompatibility, space maintaining ability, and tissue integration. Among the various antimicrobial agents loaded in the PLA membranes, tetracycline-loaded membranes exhibited minimal cellular toxicity against HeLa cells; at the same time showing significant activity against a pathogenic bacterium.

INTRODUCTION

Conventional periodontal surgical treatment like surgical debridement and resective procedures aim at treating periodontal disease and preservation of the existing structures.[12] Healing at the surgical site typically occurs by repair with combination of connective tissue adhesion, formation of long junctional epithelium with very little regeneration.[3-5]

In contrast to the above approach, regenerative periodontal therapy attempts to restore lost periodontal structures through regeneration of cementum, periodontal ligament, and alveolar bone. Guided tissue regeneration (GTR) treatment concept is based on providing a barrier between the tissue compartment and bone defect site, allowing the entry of cells with regenerative potential like periodontal ligament cells, bone cells, and cementoblast into bone defect site first.[6]

First generation GTR membranes were made from non-absorbable materials like polytetrafluorethylene.[7] Use of these membranes for periodontal regeneration was associated with a second surgical intervention for barrier membrane removal, which led researchers to explore a variety of natural or synthetic bioabsorbable materials for fabrication of GTR membranes. Polylactic acid (PLA) is a synthetic biodegradable polymer with characters suitable to be fabricated as GTR membranes.[8] The biocompatibility and biodegradability of PLA has been studied in detail in numerous studies and clinical applications, whereby it has been shown that they degrade in vivo into carbon dioxide and water.[9] PLA membranes also offer sufficient mechanical strength to provide space maintenance and cell occlusiveness to be successful for periodontal regeneration.[10] In the present work, PLA-based GTR membranes are fabricated using a novel process (patent pending) which is discussed in the article. These membranes also offer the additional advantage of easy loading of drugs.

The aims of the present study were as follows:

Fabrication of polylactide membranes and evaluation of its biocompatibility in a subcutaneous animal model

Drug loading of the PLA membranes and their antimicrobial evaluation

Evaluation of cytocompatibility of the drug-loaded PLA membranes.

MATERIALS AND METHODS

Materials

Poly-D, L-Lactic acid (PDLLA, 45kDa) was purchased from Durect Corporation, Pelham, USA. Other chemicals were purchased from Himedia, Mumbai, India. Guinea pigs used for the experiments were maintained in cages kept in the animal house and were fed with standard animal feed and pure water. Anesthetic drugs used for the animal experiments were ketamine and lignocaine.

Fabrication of polylactide membranes

Fabrication of the polylactide membranes was carried out using an innovative and patent pending process (US patent application no: 12/739,588). Initially, polylactide particles were formulated using a modified double emulsion solvent evaporation technique.[11] To fabricate polylactide membranes, particles were spread on sterile plastic Petri dishes and wetted with ethanol. Ethanol effects partial solubilization of the polylactide particles resulting in fusion of the particles at their points of contact and formation of the polymer membrane. The membrane was repeatedly washed with sterile water to remove residual ethanol. To check the sterility of the polylactide membranes, they were kept on nutrient agar plates and observed for five days for any sign of growth indicating bacterial contamination. The morphology of the polylactide particles and membranes were visualized through optical microscope (Magnus).

Biocompatibility characterization of the membranes

Institutional Ethics committee clearance was obtained for conducting the animal experiments. All surgical procedures were carried out under anesthesia by ketamine injection. Eight guinea pigs were used for the implantation studies with the polylactide membrane to assess biocompatibility, which is the usual number used in such studies.[12] A small area on the dorsum of the guinea pigs were shaved, wiped with surgical spirit, and anesthetized with lignocaine injection. A full thickness skin incision was made and a skin flap was reflected to expose the subcutaneous tissue, polylactide membrane was implanted subcutaneously, and the flap was sutured to retain the membrane in the subcutaneous pouch. The skin pouch was opened on day 12 to assess the effect of polylactide membranes. Visual inspection of the wound bed was carried out to identify any signs of inflammation caused by the membrane. Overlying skin as well as the remnant polylactide membrane with attached subcutaneous tissue was excised for histological estimation. The wound was then allowed to heal naturally.

Drug loading and evaluation of antibacterial activity of the polylactide membranes

Methicillin-resistant Staphylococcal aureus (MRSA) bacterial isolate was obtained from the supragingival plaque of a patient with chronic periodontitis and was used only as a model organism to assess the efficacy of the drug-loaded polylactide membranes. Sampling was done using a sterile swab and sample collection was carried out in the morning before daily oral hygiene procedures. Identification was done based on colony characteristics, Gram staining, tube coagulase test, growth on Cystine-Lactose-Electrolyte-Deficient (CLED) agar, and β hemolysis on sheep blood agar. Sensitivity to antibiotics was determined using agar diffusion method (CLSI 2006) and the isolate was confirmed to be MRSA.

For drug loading of the membranes, the polylactide membranes were dipped in 1 ml of 0.2% Chlorhexidine, 0.1% Silver nitrate, and tetracycline antibiotic solution (1 mg/ml) for five minutes. Three wells of standard size (8 mm) were incised at specified distances in Mueller Hinton agar and 18-hour-old nutrient broth culture of the MRSA isolate was swabbed on the Mueller Hinton agar plate using a sterile cotton swab. 0.1 ml of the three drug solutions were added into separate wells. Circular polylactide membranes were prepared with a central space, so that bacterial penetration through the membranes could be easily observed during experimentation. The drug-loaded polylactide membranes as well as the untreated membrane were placed on the media surface at equidistance using a sterile forceps and pressed gently. After incubation at 37°C for 24 hours, the diameters of zones of inhibition were measured.

Cytotoxic evaluation of the drug-loaded membranes

The cytotoxic evaluation of the drug-loaded membranes was assessed using cell lines (HeLa cervical cancer cells). Polylactide membranes loaded with the drugs (chlorhexidine, silver nitrate, and tetracycline) at different concentrations (0.2%, 0.1%, and 0.05%) were transferred to six well culture plates containing 0.2 million HeLa cells and kept in a CO2 cell culture incubator. The optical microscope was used to observe the morphology of the cells at 12 hours. After overnight incubation with the drug-loaded membranes, the different wells were trypsinized and the dissociated cells were counted for number and viability in a Neubauer counting chamber after staining with trypan blue dye, which selectively stains dead cells.

RESULTS

Physical characterization and biocompatibility evaluation of polylactide membrane

The polylactide particles are typically of spherical morphology [Figure 1a]. The polylactide particles are of porous nature, earlier confirmed by electron microscopic studies.[13] The process of fabricating the membranes results in fusion of the PLA particles at the points of their contact [Figure 1b]. The polylactide membrane in a 50:50 mixture of alcohol and water remains flexible till the time of use [Figure 2a]. This property makes it easy to handle the membrane and to place it in the intended site, where it adapts to the contour of the site [Figure 2b]. At the site, in presence of an aqueous environment, the polylactide membrane becomes rigid which is an important characteristic to maintain a stable space for periodontal regeneration.

Figure 1

Optical image of polylactide particle and membrane. (a) Image of a single spherical polylactide particle; (b) Image of polylactide membrane formed by fusion of polylactide particles

Figure 2

Images of a polylactide membrane suitable as barrier membranes. (a) The polylactide membrane can be fabricated to the desired shape and is flexible; (b) Polylactide membrane adapts well at the site of placement and becomes rigid to exhibit sufficient strength as a barrier membrane

Opening of the subcutaneous pouch in the guinea pigs was done on day 12 and it was seen that the polylactide membrane had integrated well to the underlying subcutaneous tissue. Visual inspection showed no signs of inflammation. Histology of the excised skin overlying the membrane showed no signs of inflammatory infiltration induced by the membrane [Figure 3a and b]. Histological analysis of the interface between the polylactide membrane and the underlying subcutaneous tissue showed that there was no evidence of overt inflammatory reaction, but for the presence of inflammatory cells [Figure 3c and d]. Mild inflammatory reaction induced by polylactide devices is normal and this reaction usually tapers off uneventfully as the implant or membrane integrates into the tissue.[9]

Figure 3

Histological analysis of subcutaneously implanted PLA membrane. (a) No signs of inflammatory cells in presence of PLA membrane; (b) Histology of normal guinea pig skin; (c) Histology of subcutaneous tissue adhering to the PLA membrane, arrow shows the interface between the particles of the membrane and tissue; (d) Magnified image of the same with the arrow showing presence of inflammatory cells at the interface

Evaluation of antibacterial activity of drug-loaded polylactide membranes

The result of the disc diffusion method showing the strain to be MRSA is shown in Table 1. The drugs chosen to be loaded in the membranes were tetracycline, chlorhexidine, and silver nitrate. Chlorhexidine and tetracycline are already in use for controlled delivery applications in GTR therapy. Antibacterial activity of the individual drugs and the polylactide membranes loaded with antibacterial agents is shown in Table 2. The results showed that 0.2% Chlorhexidine, 0.1% silver nitrate, and tetracycline (1 mg/ml) as well as the polylactide membranes loaded with these drugs were effective against the MRSA, as zones of inhibition were seen around all the wells and the drug-loaded membranes. Chlorhexidine showed the maximum activity, followed by silver nitrate and tetracycline and this was faithfully replicated by the drug-loaded membranes [Figure 4]. Also, bacterial penetration across the plain membrane toward the central space was clearly visible, while the drug-loaded membranes showed a clear central space free of any bacterial penetration [Figure 4].

Table 1

Result of the sensitivity extended by S. aureus isolate to antibiotics

Table 2

Result of the antibacterial efficacy of the drug-loaded membrane against S. aureus isolate

Figure 4

Antibacterial efficacy of the drugs and drug-loaded polylactide membranes to Staphylococcal aureus (1) 0.2% Chlorhexidine gluconate, (2) 0.1% Silver Nitrate, (3) Tetracycline (1 mg/ml), (4) Polylactide membrane + Chlorhexidine gluconate, (5) Polylactide membrane + Silver Nitrate, (6) Polylactide membrane + Tetracycline, and (7) Plain polylactide membrane showing bacterial invasion to its central space

Evaluation of cytocompatibility of polylactide membrane

Evaluation of cytocompatibility of the membrane with B16 melanoma cell lines earlier showed that they were non-toxic.[14] The objective of the present experiment was to evaluate the cytocompatibility of the drug-loaded polylactide membranes which had showed activity against the bacterial isolate using HeLa cells. Toxic cellular effect was seen with the lowest concentration of both chlorhexidine and silver nitrate [Figure 5a and b]. Tetracycline showed the best cellular compatibility across all concentrations used; even the effective antimicrobial concentration of 1 mg/ml exhibited minimal cellular toxic changes after overnight incubation [Figure 5c and d]. The results of the morphological observations were correlated by dissociating the cells by trypsinization after overnight incubation and checking the cell number and viability after trypan blue staining. There were no viable cells after overnight incubation with various concentrations of chlorhexidine and silver nitrate (0.2%, 0.1%, and 0.05%). Tetracycline-incubated HeLa cells showed 90% viability as compared to control HeLa cells.

Figure 5

Cytocompatibility of drug-loaded PLA membranes with HeLa cells. (a) Vacuole formation in cells with 0.05% Chlorhexidine; (b) Lysis of cells with 0.05% Silver Nitrate; (c) Minimal changes in cell morphology after overnight treatment with 1 mg/ml Tetracycline; (d) Control untreated HeLa cells

DISCUSSION

Healing after periodontal surgical techniques is achieved usually by formation of long junctional epithelium with slight or no new connective tissue attachment and negligible new cementum formation.[1516] On the contrary, regenerative periodontal therapy attempts to restore lost periodontal structures through regeneration of these structures. It was suggested that cells that repopulate the root surface after periodontal surgery will determine the type of attachment that forms on the root surface during healing.[17] From this hypothesis came the development of procedures using barrier membranes to allow selective cellular repopulation of the root surface during regenerative therapy. Barrier membranes will retard apical migration of epithelium and exclude gingival connective tissue from the healing wound.[16] Studies show that cells originating from periodontal ligament have regenerative potential and the barrier membranes used in periodontal regeneration aids in the selective growth of these cells at the wound site.[18] Much of the earlier studies used non-absorbable Millipore filters and expanded polytetrafluorethylene (ePTFE) membranes.[1920] A major advantage of barrier membranes made of absorbable materials like collagen and synthetic biodegradable polymers like PLA is that a second surgical intervention can be avoided.[21-24]

The important characteristics desirable for a bioabsorbable GTR membrane are biocompatibility, space maintaining ability, and tissue integration.[25-27] In the present study, we used clinical grade PLA to fabricate the membranes using a novel process and evaluated its biocompatibility in a subcutaneous guinea pig model. The tensile strength was tested using universal testing machine and showed sufficient strength to be used for GTR therapy.

The development of new membranes and barriers for GTR therapy are taken through different phases of development before reaching the clinic for GTR therapy.[2829] Primarily, the initial phase of development concentrates on fabrication and assessing biocompatibility and cytotoxicity, followed by evaluation of efficacy in GTR therapy in animal models. Though polylactide-based membranes are available for GTR therapy, in the present study the membranes are made using a new process; therefore, it is needed to evaluate its biocompatibility and cytotoxicity.

Results of the study showed that the PLA membranes did not induce any adverse inflammatory reaction at the site of implantation. Space maintenance by the barrier membrane is necessary to withstand the forces exerted by overlying soft tissue flaps, to prevent collapse of the soft tissue, and maintain adequate wound space for regeneration to take place.[9] PLA-based membranes are more rigid than collagen, which will provide optimum space for regeneration. Studies have shown that PLA membranes[3031] offer similar clinical effectiveness as compared to collagen membranes and non-absorbable ePTFE membranes.[3233]

Membrane exposure is a common phenomenon in GTR treatment, which provides an environment for bacterial adherence and multiplication.[34] Yoshinari et al. demonstrated that numerous bacteria adhered and invaded membranes accompanied by bacterial infection.[35] Chlorhexidine and tetracycline-loaded GTR membranes have been shown to tackle this problem and showed better efficacy during GTR therapy.[3637] Shan –Ling Hung et al. showed that amoxicillin and tetracycline-loaded membrane can liberate antibiotics to concentration high enough to eliminate periodontal pathogens and these drugs will enhance the periodontal ligament cells attachment.[38]

In the present study, Chlorhexidine, tetracycline, and silver nitrate-loaded PLA membranes showed significant antimicrobial activity against MRSA bacteria isolated from a case of periodontitis. A major concern for drug-releasing GTR membranes is that the released drugs should not interfere with the actively dividing cells of the periodontium, at the same time showing adequate activity against any possible infectious microorganisms. Chlorhexidine and silver nitrate showed cell toxicity against HeLa cells in all concentrations used, with 0.05% being the lowest concentration. It has been shown that 0.0015% Chlorhexidine can reduce the relative viability of periodontal ligament cells by 50%.[39] At the same time, tetracycline-loaded PLA membranes showed minimal toxic effects to cells, even at concentrations showing effective antibacterial activity. In view of the bone regenerative capacity of tetracycline[40] and also the minimal cell toxicity exhibited by tetracycline, it seems to be an attractive choice to be loaded onto polylactide membranes fabricated using our process to be used for periodontal regeneration. In our case, the loading of the polylactide membrane with drugs is very easy due its unique architecture. The membrane consists of fused porous polylactide particles which when immersed in a drug solution, absorbs the drug molecules into the particles and when placed in the tissue site, releases them for therapeutic action. The polylactide membrane can also be explored to be loaded with growth factors that aid in faster regeneration of tissues.

CONCLUSION

Polylactide membranes were fabricated suitable to be used as GTR barrier membranes

The PLA membranes were found to be biocompatible and elicited minimal inflammatory reaction in a subcutaneous implant model

Drug-loaded (chlorhexidine, silver nitrate, and tetracycline) PLA membranes showed significant antibacterial activity against a drug-resistant Staphylococcal aureus strain

Tetracycline-loaded PLA membranes showed the minimal cytotoxicity against HeLa cells.