‘Pre-prosthetic use of poly(lactic- co-glycolic acid) membranes treated with oxygen plasma and TiO 2nanocomposite particles for guided bone regeneration processes’

Abstract

Objectives

Guided bone regeneration (GBR) processes are frequently necessary to achieve appropriate substrates before the restoration of edentulous areas. This study aimed to evaluate the bone regeneration reliability of a new poly-lactic- co -glycolic acid (PLGA) membrane after treatment with oxygen plasma (PO 2 ) and titanium dioxide (TiO 2 ) composite nanoparticles.

Methods

Circumferential bone defects (diameter: 10 mm; depth: 3 mm) were created on the parietal bones of eight experimentation rabbits and were randomly covered with control membranes (Group 1: PLGA) or experimental membranes (Group 2: PLGA/PO 2 /TiO 2 ). The animals were euthanized two months afterwards, and a morphologic study was then performed under microscope using ROI (region of interest) colour analysis. Percentage of new bone formation, length of mineralised bone formed in the grown defects, concentration of osteoclasts, and intensity of osteosynthetic activity were assessed. Comparisons among the groups and with the original bone tissue were made using the Kruskal–Wallis test. The level of significance was set in advance at a = 0.05.

Results

The experimental group recorded higher values for new bone formation, mineralised bone length, and osteoclast concentration; this group also registered the highest osteosynthetic activity. Bone layers in advanced formation stages and low proportions of immature tissue were observed in the study group.

Conclusions

The functionalised membranes showed the best efficacy for bone regeneration.

Clinical significance

The addition of TiO 2 nanoparticles onto PLGA/PO 2 membranes for GBR processes may be a promising technique to restore bone dimensions and anatomic contours as a prerequisite to well-supported and natural-appearing prosthetic rehabilitations.

Introduction

The concept of guided bone regeneration (GBR) is based on the use of physical barriers to prevent the gingival epithelium and connective tissue cells from invading the bone cavity during the healing process . Tissue engineering involves designing modified biomaterials to mimic the structural properties of the original tissues and to provide a stable support for the extracellular matrix . Advanced synthetic biodegradable polyesters such as poly-lactic- co -glycolic acid (PLGA) show good adhesion with bone, improve vascularisation, and can be resorbed by the human body without generating an immune rejection reaction .

However, PLGA polymers are hydrophobic , which decreases their mechanical resistance, facilitates the release of acid residues, diminishes the pH, and stimulates the bacterial proliferation and inflammatory responses . Ideally, the use of cold plasmas would improve these polymers’ surface roughness, stimulate the adhesion of osteogenic mediators and cells, and accelerate the membranes’ biodegradation . The incorporation of thin layers of nanocomposite particles as metallic oxides may also optimise the osteoinductive capacity of the barriers by stimulating osteoblastic adherence, causing functional differentiation, and forming new bone .

In this regard, the originality of our research line focuses on the description of a new method based on the addition of titanium dioxide (TiO 2 ) composite nanoparticles (through ‘dry way’ apposition) onto PLGA membranes modified with oxygen plasma (PO 2 ) to increase the bone-regeneration capacity of this biomaterial. Notwithstanding that our pilot evaluation logically included a group of PLGA/PO 2 /TiO 2 barriers, the present research represents a major advance over the previous one . Despite the importance of presenting customised membranes with positive results for GBR processes, our pilot study only showed data trends without statistical significance ( p ≥ 0.05). In the present investigation, the bone defects were bigger (and thus more difficult to fill), which made the histological differences between the groups clearer. A greater sample size of rabbits was used (almost three times larger than the number used in the pilot study). Unlike in the pilot experiment, a double marking technique was applied (using calcein) to quantify the length of mineralised bone formed in the grown defects, and two fundamental parameters of bone regeneration ( i.e. , percentage of new bone formation and concentration of osteoclasts/mm 2 ) were compared with those of the original bone tissue for the first time. Furthermore, statistically significant results were obtained, providing scientific evidence in favour of the newly designed membranes.

In summary, the aim and novelty of this prospective study were to evaluate the bone-regeneration efficacy of PLGA membranes treated with PO 2 and sputtered with nanometric particles of TiO 2 , which may act as osteogenic mediators .

The null hypothesis tested was that the functionalisation of PLGA membranes with the described method does not modify their potential for bone regeneration.

Materials and methods

Preparation of the membranes

Sixteen 40-μm-thick, resorbable inert PLGA scaffolds based on poly-lactic- co -glycolic acid were fabricated using polycondensation (Institute of Materials Science, Seville, Spain). The membranes were used to cover bone defects prepared on the skulls of eight experimental rabbits. Two groups of regenerative membranes ( n = 8 each) were prepared and tested for GBR processes: 1: PLGA (control) and 2: PLGA/PO 2 /TiO 2 .

The control group of PLGA membranes was characterised by X-ray photoemission spectroscopy (XPS) so that the following chemical composition was registered on the membranes’ surfaces: 54% carbon and 46% oxygen. The molecular weight of the PLGA copolymer was 12 kDa. The PLGA was synthesised by means of the ring-opening copolymerisation of two different monomers, the cyclic dimers (1,4-dioxane-2,5-diones) of glycolic acid and lactic acid. During polymerisation, successive monomeric units (of glycolic or lactic acid) were attached in PLGA using ester linkages, thus yielding linear, aliphatic polyester. The C1s spectrum of the bare PLGA consisted of three well-defined bands at 284.6, 287.5 and 288.5 eV; these bands are attributed to C C/C H, COO, and the COR functional groups of the composite polymer .

The Group 2 membranes’ surfaces were first exposed to pure PO 2 at 400 W for 1 min in a plasma reactor with a remote configuration (Flarion PECVD, Plasmionique, France); this was an intermediate step to enhance the retention of the TiO 2 composite nanoparticles . The system, supplied with an external microwave plasma source (SLAN, Plasma Consult, GmbH, Germany) in a downstream arrangement, was coupled to the reaction chamber and separated from it by a 10-cm grounded grid to avoid the microwave heating of the barriers. This technique generated an etching effect that improved the surface roughness and favoured the adhesion of oxide layers to the membranes . The treatment of PLGA substrates with PO 2 took place at close to ambient temperatures (RT: 23.0 ± 1.0 °C) and did not affect their structural integrity .

Subsequently, the membranes of Group 2 were coated with bioactive layers of TiO 2 (by means of ‘dry way’ procedures) to prevent contact between the polymer and liquid. Thus, the inorganic coating was directly precipitated onto the membranes’ surfaces using evaporation technologies (Physical Vapor Deposition, PVD) . The TiO 2 particles were therefore deposited on special substrate plates (Goodfellow, Oakdale, PA, USA) by Plasma-Enhanced Chemical Vapor Deposition (PECVD) in the plasma reactor (Flarion PECVD). The synthesis of the films was carried out at RT using nanocomposite particles of titanium tetrakis isopropoxide (TTIP) as precursors . To avoid any damage to the PLGA substrate, before the deposition of the TiO 2 nanoparticles, the barrier was protected with a shutter until the plasma discharge was stabilised in the presence of the precursors . The total pressure during deposition was 4 × 10 −3 Torr . The TiO 2 particles were characterised under XPS by analysing the shapes of the background spectra behind the elastic photoemission peaks. The XPS Ti2p spectrum in the experimental samples was characterised by a Ti2p3/2 BE of 458.4 eV, typical of the Ti4+ oxidation state of this element, proving that titanium was deposited in the form of TiO 2 . Meanwhile, the O1s spectrum of the investigated samples was characterised by a first peak at 529.8 eV; the oxygen ions of TiO 2 were convoluted with a broad shoulder at approximately 532.5 eV because of the different oxygen groups in the polymeric mixture . The working conditions for the functionalisation with PO 2 , as well as the sputtering of inorganic nanometric particles, were detailed from an engineering point of view . Some of the co-authors patented this new formula for GBR processes .

Animal experimentation specimens

An a priori power calculation made from the data obtained in the pilot study provided the sample size required to achieve statistical significance in the main investigation ( α = 0.05, β = 0.2). Thus, eight white, New Zealand-breed experimentation rabbits with identical characteristics (age: 6 months; weight: 3.5–4 kg) were selected for the study and fed daily with rabbit-maintenance Harlan-Teckland Lab Animal Diets (2030). Two lead dental surgeons, two collaborating dental surgeons, a veterinarian, a supporting anaesthesiologist, a biologist, nurses, and specialised clinical assistants participated in the surgery.

The surgical interventions were carried out at the Minimally Invasive Surgery Centre Jesús Usón (CCMI, Cáceres, Spain). The experiment was developed in accordance with the guidelines of the US National Institute of Health (NIH) and with European Directive 86/609/EEC regarding the care and use of animals for experimentation. The study also complies with European Directive 2010/63/EU about the protection of animals used for scientific purposes and with all local laws and regulations. This research obtained the approval of the Ethics Committee of the University of Seville (US, Spain).

As stated by the legislative framework, the minimum number of animals was utilised for ethical reasons . Comparable models have been published concerning the histological and animal experimentation methods.

Surgical procedure

The animals were immobilised, and their vital signs were checked. The anaesthesia used for induction was intravenous midazolam (0.25 mg/kg) and propofol (5 mg/kg); for maintenance, the animals inhaled 2.8% inspired sevoflurane gas. Analgesia was provided with ketorolac (1.5 mg/kg) and tramadol (3 mg/kg). After the rabbits were sedated and prepared, incisions between the bases of their ears and between their eyes were performed with a No. 15 scalpel blade. After the two incisions were connected with an incision that coincided with the skull midline, a triangular field was discovered ( Fig. 1 (a)). The epithelial, connective, and muscular tissues were displaced using a Pritchard periosteotome. The skull surface was washed with a sterile saline solution, keeping the aspiration. Two bone defects (diameter: 10 mm; depth: 3 mm) were created on the parietal bone, on each side of the skull midline, 3 mm apart, using a trephine (Helmut-Zepf Medical Gmbh, Seitingen, Germany) mounted on an implant micromotor operating at 2000 rpm under saline irrigation ( Fig. 1 (a)). The trephine had an internal diameter of 10 mm, a length of 30 mm, and teeth of 2.35 mm. As the defects were created in a symmetric fashion in relation to the longitudinal axis of each rabbit skull, possible between-groups variations in section thickness were minimised.

Fig. 1
Images of the surgical intervention. (a) Preparation of bone defects after the incision and displacement of the tissues. (b) Placement of a functionalised membrane to cover the bone defect.

For each defect, the outer table and the medullary bone were completely removed with piezosurgery, and the inner table was preserved to avoid damage to the brain tissue. The depth was controlled with a periodontal probe.

A randomly assigned membrane was used to cover each bone defect. The randomisation sequence was generated using specific software (Research Randomizer, V. 4.0, Urbaniak GC & Plous S, 2013) . The PLGA fibres of the barriers were also randomly oriented. The membranes were fixed with the fibrin tissue adhesive Tissucol (Baxter, Hyland S.A. Immuno, Rochester, MI, USA), which was placed on the bone rims adjacent to the defects ( Fig. 1 (b)). Proper adhesion and limited mobility of the membranes was confirmed when the flaps were moved back to their initial positions. Sutures were made on the following planes using resorbable material: periosteal (4/0), sub-epidermal (4/0), and skin (2/0). Simple stitches were used as close as possible to the edge. The wound was carefully cleaned with a sterile saline solution.

Anti-inflammatory analgesia (buprenorphine 0.05 mg/kg, and carprofen 1 mL/12.5 kg) was administered. The animals were sacrificed two months after surgery using an intravenous overdose of potassium chloride solution. Samples were obtained from the skull of each specimen, cutting them in an anatomical sagittal plane. After the brain mass was separated and the skull was washed with a sterile saline solution, the tissue samples were cut and marked individually.

Processing of samples

The skull samples were processed for histological analysis at the Dental School of the Paris-Descartes University (Montrouge, France). The protocols of Gallina et al. and Torres-Lagares et al. were strictly followed to complete the tissue fixation. After immersion in alcohol, the skull samples were fixed in a cold (4 °C) 70% ethanol solution . Once dehydrated, the samples were embedded without demineralisation in methyl methacrylate (MMA) blocks (Merck, Darmstadt, Germany) which were set perpendicularly to the sagittal axis and polished in a conventional machine . Histological 5-μm-thick sections were cut using the Jung Ultrafrase Polycut E microtome (Leica, Heidelberg, Germany). To minimise the variance in thickness among the specimens, the slices were obtained from the middle region of the bone defects in series of ten slices each, placed on glass sample holders pretreated with albumin, and set in a 80% alcohol solution. The original bone disks were prepared for histology.

Staining and morphometric study

For histological staining and rapid contrast tissue analysis (Merck Toluidine Blue-Merck, Darmstadt, Germany), a metachromatic dye was utilised to assess the percentage of new bone formation. A 1% toluidine blue (TB) solution with a pH of 3.6 was chosen and adjusted with HCl 1 N. The samples were exposed to the dye for 10 min at RT, rinsed with distilled water, and air-dried. The von Kossa (VK) silver nitrate technique (Sigma–Aldrich Chemical Co., Poole, UK) was applied to visualise the mineralised bone.

To quantify the length of mineralised bone formed in the grown defects per day, intravenous labelling with calcein/demeclocycline in a 5% physiologic serum was carried out eight days before sacrifice. The reactives were calcein disolved into 2% NaHCO 3 in serum (ref. SIGMA C0875 1 g) and demeclocycline hidroclorhide (ref. SIGMA A6140 5 g). The administration included 30 mg/kg of calcein (using 9 mg calcein/mL) for each rabbit ( i.e ., 45 mg for an animal of 1.5 kg) eight days before sacrifice. For each rabbit, 45 mg calcein was combined with 5 mL physiologic serum and 0.1 g NaHCO 3 ; the VT was 5 mL per animal. The administration also included 30 mg/kg of demeclocycline (using 9 mg demeclocycline/mL) for each rabbit ( i.e ., 45 mg for an animal of 1.5 kg) one day before sacrifice. For each rabbit, 45 mg demeclocycline was combined with 5 mL physiologic serum; the VT was 5 mL per animal. Each rabbit was injected with 5 mL of the solution intraperitoneally. The calceine supplied a green colour to the new bone, and the demeclocycline provided a yellow colour.

To examine the resorptive activity of the regenerated bone tissue, the enzymological technique for evidence of tartrate of sodium resistant acid phosphatase (TRAP) was utilised. This hydrolase served as a cytochemical marker for osteoclasts and pre-osteoclasts released into the surrounding environment during bone resorption to facilitate bone dissolution . The samples’ slices were incubated in Naphthol ASTR phosphate (Sigma–Aldrich), which was previously dissolved in NN dimethylformamide. Then, a 0.1 M buffer of acetate sodium tartrate and Fast Red TR salt hemi (zinc chloride) (Sigma–Aldrich) were added, the pH was adjusted to 5.2, and the final solution was filtered. The slices were incubated at 37 °C for 1 h in the dark.

The technique of evidence of alkaline phosphatase (ALP) was developed in osteoblasts and pre-osteoblasts to investigate the osteosynthetic activity of the regenerated tissues. The slices were pre-incubated for 10 min in 0.1% Tris Triton (Sigma–Aldrich), washed in 0.1 M Tris hydrochloride (pH 9), and incubated in the dark at 37 °C for 30 min in Naphthol ASTR phosphate together with the coloured reaction developer Fast Blue RR Salt (Sigma–Aldrich) as a coupler (also with adjusted pH 9). A weak colouring of 1% TB was diluted to 10% in distilled water to give the samples contrast. Cells marked in violet, which defined the dimensions of the samples’ osteogenic strip, were considered as positive results.

Following the protocol described in the previous subsection, the TRAP and ALP staining were performed on resin (methylmethacrylate) embedded samples to achieve better preservation of the trabecular bone structure . Compared to other plastic media such as water-soluble methacrylates ( e.g ., glycolmethacrylate, GMA), MMA embedding of undecalcified bone offers two important advantages: (a) MMA penetrates tissue better than GMA, and the histological quality of bone sections is generally higher in MMA-embedded bone samples, and (b) MMA can easily and completely be removed from tissue sections, which results in superior staining characteristics and excellent morphological detail. However, conventional MMA embedding causes almost complete loss of enzyme activity and protein antigenicity in the tissues. Hence, based on the MMA embedding technique described by Wolf et al. , we have followed the improved method of MMA embedding introduced by Erben , thus circumventing the decalcification procedure . Compared to the original technique, this method is different in several aspects: (a) Instead of dehydration of the bone samples with acetone , we used a standard dehydration protocol that is also employed by conventional MMA embedding . Overall, acetone fixation causes massive shrinkage artefacts and acetone dehydration results in inferior quality of the bone section. (b) The infiltration protocol allowed achieving optimal infiltration of the tissue with the plastic. Good infiltration is the basis for uniform polymerization and high section quality. (c) The addition of methylbenzoate to the methacrylate solution during infiltration and polymerization improved preservation of antigenicity of the tissue. (d) Methylbenzoate acts as a plasticizer in MMA embedding mixtures. (e) The amount of the accelerator N , N -dimethyl- p -toluidine used for chemical polymerization at low temperatures has been reduced as recommended by Erben , resulting in a lower tendency for bubble formation and more homogeneous polymerization of the blocks. (f) This method does not require purification and destabilization of the methacrylates used for infiltration and polymerization.

Finally, the regenerated bone was submitted to a morphometric study under a Zeis Axioscop 2 microscope (Axioscop 40, Goettingen, Germany) using 4×, 10×, and 20× lenses. Pictures were taken with a digital signal processor (DSP) 3CCD camera (Sony, Tokyo, Japan) and Intelicam 8.0 software (Smart Matrox Imaging, Montreal, Canada). To quantify the newly regenerated tissue and to compare it with the original bone tissue, an assessment from distal (external) to proximal (internal) was made using ROI (region of interest) colour analysis with specific software (Fiji Is Just ImageJ, Tokyo, Japan). The percentage of new bone formation, length of mineralised bone formed in the grown defects, concentration of osteoclasts, and intensity of bone osteosynthetic activity were evaluated by a single operator.

Statistical analysis

Means and standard deviations (SD) were calculated. The intra-examiner reliability was assessed using the Kappa test . Since the Kolmogorov–Smirnov test demonstrated that the data were not normally distributed, the Kruskal–Wallis test was run for post hoc comparisons . The level of significance was set in advance at a = 0.05 . The Statview F 4.5 Macintosh software (Abacus Concepts, Berkeley, CA, USA) was utilised for the analysis . All the statistical probes applied in this study adhere to the requirements of Hannigan and Lynch for oral and dental research.

Materials and methods

Preparation of the membranes

Sixteen 40-μm-thick, resorbable inert PLGA scaffolds based on poly-lactic- co -glycolic acid were fabricated using polycondensation (Institute of Materials Science, Seville, Spain). The membranes were used to cover bone defects prepared on the skulls of eight experimental rabbits. Two groups of regenerative membranes ( n = 8 each) were prepared and tested for GBR processes: 1: PLGA (control) and 2: PLGA/PO 2 /TiO 2 .

The control group of PLGA membranes was characterised by X-ray photoemission spectroscopy (XPS) so that the following chemical composition was registered on the membranes’ surfaces: 54% carbon and 46% oxygen. The molecular weight of the PLGA copolymer was 12 kDa. The PLGA was synthesised by means of the ring-opening copolymerisation of two different monomers, the cyclic dimers (1,4-dioxane-2,5-diones) of glycolic acid and lactic acid. During polymerisation, successive monomeric units (of glycolic or lactic acid) were attached in PLGA using ester linkages, thus yielding linear, aliphatic polyester. The C1s spectrum of the bare PLGA consisted of three well-defined bands at 284.6, 287.5 and 288.5 eV; these bands are attributed to C C/C H, COO, and the COR functional groups of the composite polymer .

The Group 2 membranes’ surfaces were first exposed to pure PO 2 at 400 W for 1 min in a plasma reactor with a remote configuration (Flarion PECVD, Plasmionique, France); this was an intermediate step to enhance the retention of the TiO 2 composite nanoparticles . The system, supplied with an external microwave plasma source (SLAN, Plasma Consult, GmbH, Germany) in a downstream arrangement, was coupled to the reaction chamber and separated from it by a 10-cm grounded grid to avoid the microwave heating of the barriers. This technique generated an etching effect that improved the surface roughness and favoured the adhesion of oxide layers to the membranes . The treatment of PLGA substrates with PO 2 took place at close to ambient temperatures (RT: 23.0 ± 1.0 °C) and did not affect their structural integrity .

Subsequently, the membranes of Group 2 were coated with bioactive layers of TiO 2 (by means of ‘dry way’ procedures) to prevent contact between the polymer and liquid. Thus, the inorganic coating was directly precipitated onto the membranes’ surfaces using evaporation technologies (Physical Vapor Deposition, PVD) . The TiO 2 particles were therefore deposited on special substrate plates (Goodfellow, Oakdale, PA, USA) by Plasma-Enhanced Chemical Vapor Deposition (PECVD) in the plasma reactor (Flarion PECVD). The synthesis of the films was carried out at RT using nanocomposite particles of titanium tetrakis isopropoxide (TTIP) as precursors . To avoid any damage to the PLGA substrate, before the deposition of the TiO 2 nanoparticles, the barrier was protected with a shutter until the plasma discharge was stabilised in the presence of the precursors . The total pressure during deposition was 4 × 10 −3 Torr . The TiO 2 particles were characterised under XPS by analysing the shapes of the background spectra behind the elastic photoemission peaks. The XPS Ti2p spectrum in the experimental samples was characterised by a Ti2p3/2 BE of 458.4 eV, typical of the Ti4+ oxidation state of this element, proving that titanium was deposited in the form of TiO 2 . Meanwhile, the O1s spectrum of the investigated samples was characterised by a first peak at 529.8 eV; the oxygen ions of TiO 2 were convoluted with a broad shoulder at approximately 532.5 eV because of the different oxygen groups in the polymeric mixture . The working conditions for the functionalisation with PO 2 , as well as the sputtering of inorganic nanometric particles, were detailed from an engineering point of view . Some of the co-authors patented this new formula for GBR processes .

Animal experimentation specimens

An a priori power calculation made from the data obtained in the pilot study provided the sample size required to achieve statistical significance in the main investigation ( α = 0.05, β = 0.2). Thus, eight white, New Zealand-breed experimentation rabbits with identical characteristics (age: 6 months; weight: 3.5–4 kg) were selected for the study and fed daily with rabbit-maintenance Harlan-Teckland Lab Animal Diets (2030). Two lead dental surgeons, two collaborating dental surgeons, a veterinarian, a supporting anaesthesiologist, a biologist, nurses, and specialised clinical assistants participated in the surgery.

The surgical interventions were carried out at the Minimally Invasive Surgery Centre Jesús Usón (CCMI, Cáceres, Spain). The experiment was developed in accordance with the guidelines of the US National Institute of Health (NIH) and with European Directive 86/609/EEC regarding the care and use of animals for experimentation. The study also complies with European Directive 2010/63/EU about the protection of animals used for scientific purposes and with all local laws and regulations. This research obtained the approval of the Ethics Committee of the University of Seville (US, Spain).

As stated by the legislative framework, the minimum number of animals was utilised for ethical reasons . Comparable models have been published concerning the histological and animal experimentation methods.

Surgical procedure

The animals were immobilised, and their vital signs were checked. The anaesthesia used for induction was intravenous midazolam (0.25 mg/kg) and propofol (5 mg/kg); for maintenance, the animals inhaled 2.8% inspired sevoflurane gas. Analgesia was provided with ketorolac (1.5 mg/kg) and tramadol (3 mg/kg). After the rabbits were sedated and prepared, incisions between the bases of their ears and between their eyes were performed with a No. 15 scalpel blade. After the two incisions were connected with an incision that coincided with the skull midline, a triangular field was discovered ( Fig. 1 (a)). The epithelial, connective, and muscular tissues were displaced using a Pritchard periosteotome. The skull surface was washed with a sterile saline solution, keeping the aspiration. Two bone defects (diameter: 10 mm; depth: 3 mm) were created on the parietal bone, on each side of the skull midline, 3 mm apart, using a trephine (Helmut-Zepf Medical Gmbh, Seitingen, Germany) mounted on an implant micromotor operating at 2000 rpm under saline irrigation ( Fig. 1 (a)). The trephine had an internal diameter of 10 mm, a length of 30 mm, and teeth of 2.35 mm. As the defects were created in a symmetric fashion in relation to the longitudinal axis of each rabbit skull, possible between-groups variations in section thickness were minimised.

Jun 19, 2018 | Posted by in General Dentistry | Comments Off on ‘Pre-prosthetic use of poly(lactic- co-glycolic acid) membranes treated with oxygen plasma and TiO 2nanocomposite particles for guided bone regeneration processes’
Premium Wordpress Themes by UFO Themes