Guided Bone Regeneration Processes Research Articles

β-Tricalcium phosphate incorporated natural rubber latex membranes for calvarial bone defects: Physicochemical, in vitro and in vivo assessment

Natural rubber latex membrane (NRL) is a biocompatible macromolecule that stimulates angiogenesis and promotes bone repair. Similarly, β-tricalcium phosphate (β-TCP) is an osteoconductive and osteoinductive bioceramic widely used as a bone substitute. Here, we investigated the combined use of these biomaterials in the guided bone regeneration process for calvarial defects in rats. Physicochemical characterization was performed to evaluate the interaction between β-TCP and NRL. Membrane toxicity was assessed using MC3T3 osteoblasts culture and in vivo assays with Caenorhabditis elegans. Lastly, NRL membranes, NRL incorporated with β-TCP membranes (NRL-β-TCP), and a periosteum-only (control group) were tested on rodents. MC3T3 cells adhered to membranes, preserving their morphology and intercellular connections. NRL-β-TCP membranes demonstrated no toxicity in larvae, which maintained their sinusoidal wave shape. Tests results on rodents revealed statistical difference between the groups at 60 days post-operation. NRL-β-TCP (56.1 ± 14.0 %) had an average 1.48-fold higher than the control group (38.0 ± 9.1 %), with tissue production and bone remodeling. Our qualitative histological analyses revealed that membranes significantly accelerated bone formation without any signs of inflammatory reactions. We conclude that NRL-β-TCP has potential to be used for flat bone regeneration, with osteoconductive properties, being a cheap, biocompatible, and effective occlusive barrier.

Oxygen plasma-modified polycaprolactone nanofiber membrane activates the biological function in cell adhesion, proliferation, and migration through the phosphorylation of FAK and ERK1/2, enhancing bone regeneration

Bone defects present significant clinical challenges in orthopedics, orthodontics, and maxillofacial surgeries. Accelerated bone regeneration can be facilitated by incorporating mesenchymal stem cells, but limitations in cell survival and growth remain concerns for complete bone repair. Biomaterial-based membranes have been developed to form biocompatible, degradable, and mechanically stable barriers in guided bone regeneration processes. Polycaprolactone (PCL) is a biodegradable polymer widely used as a bone regenerative material because of its favorable mechanical properties. However, its inherent hydrophobicity limits cell adhesion and proliferation, which are necessary for effective bone regeneration. To address this, we fabricated cell-regulatory PCL nanofiber membranes using plasma treatment to enhance induced pluripotent stem cell-derived mesenchymal stem cells and osteoblast growth. The plasma treatment parameters (gas type, flow rate, power, and exposure time) were optimized to enhance PCL’s surface hydrophilicity and protein adsorption properties while maintaining its mechanical integrity. The optimized oxygen plasma-modified PCL membrane demonstrated notable improvements in cell viability, adhesion, and proliferation. In addition, there was improved migration, regulated by cell surface markers and signaling proteins, of the induced pluripotent stem cell-derived mesenchymal stem cells and osteoblasts. In vivo studies in a rat calvarial defect model showed that the plasma-modified PCL membrane dramatically improved new bone formation, facilitating bone regeneration. These findings highlight the potential of plasma treatment of PCL nanofibers to produce effective cell-regulatory membranes for bone defect reconstruction.

Effect of Applying 1% Metformin on Guided Bone Regeneration Processes with Bovine-Derived Xenografts.

Background: Although xenografts have shown successful results in GBR procedures due to their osteoconductive properties, many authors have opted to add co-adjuvant drugs to favor osteogenesis and differentiate cells into an osteoblastic lineage. Metformin has been shown to have bone-protective properties, regulating osteoclast differentiation, as well as the ability to promote osteoblast mineralization and differentiation. The present study aimed to evaluate the effect of the local application of a 1% metformin solution on bone neoformation in the treatment of an experimental bone defect in a guided bone regeneration animal model with a particulated bovine hydroxyapatite xenograft with hyaluronate. Methods: With this purpose in mind, two critical defects with 8 mm diameter and 0.5 mm depth were created in eight male New Zealand rabbit calvarias. Titanium cylinders were fixed in each defect and filled with particulate hydroxyapatite of bovine origin and sodium hyaluronate, with sterile injectable saline added to the control group and sterile 1% metformin solution added to the test group. At 6 weeks, the animals were euthanized, and samples were obtained and prepared for histomorphometric analysis. Results: A higher percentage of new bone formation was observed in the metformin samples than in the control samples, both in the region closest to the animal's calvaria and in the most distal region analyzed. A higher average bone-biomaterial contact percentage was observed in the samples, with metformin in both the proximal and distal regions. There was no statistically significant difference in the mean value in either region in both parameters. Conclusion: The local application of a 1% metformin solution in an animal model of guided bone regeneration with particulate bovine hydroxyapatite and hyaluronate resulted in greater bone neoformation and xenograft osseointegration than in the control group.

THE ADVERSE EFFECT OF HEALING COMPLICATIONS TO THE OUTCOME OF GBR – A 'SPLIT MOUTH' CASE REPORT

Inadequate alveolar process width and height can be overcome by the utilization of distraction osteogenesis, split crest technique or a sinus lift, however many clinical scenarios require the use of guided bone regeneration (GBR). When vertical deciencies are present, the collective opinion is that non-resorbable membranes should be employed in the augmentation procedure. While remaining form stable throughout the healing period is advantageous for vertical regeneration, the non-resorbable membranes' biological properties do not aid the primary intention closure over the grafted region in any way. As a result, membrane exposures occur which have emphatically negative effect on the regeneration process. There certainly is an abundance of evidence on the detrimental effect of healing complications on the GBR process, however the information regarding the outcome of the bone regeneration process involving exposed vs unexposed sites in the same individual is scarce.

Tailored alginate/PCL-gelatin-β-TCP membrane for guided bone regeneration

Membranes prepared for guided bone regeneration (GBR) signify valued resources, inhibiting fibrosis and assisting bone regenration. However, existing membranes lack bone regenerative capacity or adequate degradation profile. An alginate-casted polycaprolactone-gelatin-β-tricalcium phosphate dual membrane was fabricated by electrospinning and casting processes to enhance new bone formation under a GBR process. Porous membranes were synthesized with suitable hydrophilicity, swelling, and degradation behavior to confirm the compatibility of the product in the body. Furthermore, osteoblast-type cell toxicity and cell adhesion results showed that the electrospun membrane offered compatible environment to cells while the alginate sheet was found capable enough to supress the cellular attachment, but was a non-toxic material. Post-implantation, the in-vivo outcomes of the dual-layered membrane, showed appreciable bone formation. Significantly, osteoid islands had fused in the membrane group by eight weeks. The infiltration of fibrous tissues was blocked by the alginate membrane, and the ingrowth of new bone was enhanced. Immunocytochemical analysis indicated that the dual membrane could direct more proteins which control mineralization and convene osteoconductive properties of tissue-engineered bone grafts.

Photobiomodulation Therapy on the Guided Bone Regeneration Process in Defects Filled by Biphasic Calcium Phosphate Associated with Fibrin Biopolymer.

The aim is to evaluate the effects of photobiomodulation therapy (PBMT) on the guided bone regeneration process (GBR) in defects in the calvaria of rats filled with biphasic calcium phosphate associated with fibrin biopolymer. Thirty male Wistar rats were randomly separated: BMG (n = 10), defects filled with biomaterial and covered by membrane; BFMG (n = 10), biomaterial and fibrin biopolymer covered by membrane; and BFMLG (n = 10), biomaterial and fibrin biopolymer covered by membrane and biostimulated with PBMT. The animals were euthanized at 14 and 42 days postoperatively. Microtomographically, in 42 days, there was more evident bone growth in the BFMLG, limited to the margins of the defect with permanence of the particles. Histomorphologically, an inflammatory infiltrate was observed, which regressed with the formation of mineralized bone tissue. In the quantification of bone tissue, all groups had a progressive increase in new bone tissue with a significant difference in which the BFMLG showed greater bone formation in both periods (10.12 ± 0.67 and 13.85 ± 0.54), followed by BFMG (7.35 ± 0.66 and 9.41 ± 0.84) and BMG (4.51 ± 0.44 and 7.11 ± 0.44). Picrosirius-red staining showed greater birefringence of collagen fibers in yellow-green color in the BFMLG, showing more advanced bone maturation. PBMT showed positive effects capable of improving and accelerating the guided bone regeneration process when associated with biphasic calcium phosphate and fibrin biopolymer.

In Vivo Comparative Evaluation of Biocompatibility and Biodegradation of Bovine and Porcine Collagen Membranes.

Mechanical barriers prevent the invasion of the surrounding soft tissues within the bone defects. This concept is known as Guided Bone Regeneration (GBR). The knowledge about the local tissue reaction and the time of degradation of absorbable membranes favors the correct clinical indication. This study aimed to evaluate the biocompatibility and biodegradation of a bovine collagen membrane (Lyostypt®, São Gonçalo, Brazil) and compare it to a porcine collagen membrane (Bio-Gide®) implanted in the subcutaneous tissue of mice, following ISO 10993-6:2016. Thirty Balb-C mice were randomly divided into three experimental groups, LT (Lyostypt®), BG (Bio-Gide®), and Sham (without implantation), and subdivided according to the experimental periods (7, 21, and 63 days). The BG was considered non-irritant at seven days and slight and moderate irritant at 21 and 63 days, respectively. The LT presented a small irritant reaction at seven days, a mild reaction after 21, and a reduction in the inflammatory response at 63 days. The biodegradation of the LT occurred more rapidly compared to the BG after 63 days. This study concluded that both membranes were considered biocompatible since their tissue reactions were compatible with the physiological inflammatory process; however, the Bio-Gide® was less degraded during the experimental periods, favoring the guided bone regeneration process.

Delayed bone healing by collagen membrane in early phase of 4 weeks

Barrier membranes are important in maintaining space in guided bone regeneration process by preventing downgrowth of epithelial or connective tissue. In this study, the effects of resorbable membranes during the early stages of bone regeneration in rats with impaired bone healing capacity were investigated. Twenty-eight rats were selected for this study. Half of the animals were selected for radiation therapy before surgical procedure (G3, G4). Animals were assigned into 4 groups (G1-G4). A circular defect was created in the central parietal bone. It was covered with resorbable membrane in G2 and G4. After 4 weeks, the animals were sacrificed. At week 4, the new bone formation was observed around the margin of old bone in G1, G2 and G4 groups. Osteoclast was most abundant in the G1 group (18.3 ± 7.7) and least abundant in the G4 group (7.9 ± 4.7). The mean of osteocalcin levels in blood was the highest in the G2 group and lowest in the G3 group. Only G4 group showed significant difference in Runx2 levels between before-treatment and after- treatment. Bone healing is adversely affected after radiation therapy. In addition, resorbable membranes can delay healing in the early stages of bone regeneration.

PR581: In vitro comparative study of poly(lactic–co–glycolic) (PLGA) membranes treated with oxygen plasma and different nanostructured particles for guided bone regeneration processes

PR581: In vitro comparative study of poly(lactic–co–glycolic) (PLGA) membranes treated with oxygen plasma and different nanostructured particles for guided bone regeneration processes

Using biomimetically mineralized collagen membranes with different surface stiffness to guide regeneration of bone defects.

Because guided bone regeneration (GBR) process is pronouncedly affected by the micro-environment in the defect, the surface stiffness of collagen membranes as a constituent part of the micro-environment was investigated in this study. The objective of this study was to manufacture biomimetically mineralized collagen membranes with controllable surface stiffness based on biomimetic strategy and to investigate the influences of surface stiffness on GBR process. The characterization and biocompatibility of membranes were examined in vitro. The mechanical properties of membranes were evaluated on macro and micro levels using tensile test and atomic force microscope, respectively. The critical-size cranial defect model and ectopic osteogenesis were chosen to employ their performances in vivo. The results indicated that the biomimetically mineralized collagen membranes with controllable surface stiffness were manufactured based on the biomimetic theory. The in vitro experiments showed that the mineralized collagen membrane with satisfactory surface stiffness can better promote the adhesion, proliferation, and osteogenic differentiation of mesenchymal stem cells. The membranes can perform excellently in both osteoinduction and osteoconduction, which results in effective manifestations in aspects of ectopic osteogenesis and GBR in vivo. Therefore, this biomimetically mineralized collagen membrane is a promising candidate for GBR treatment in future.

In Vitro Comparative Study of Oxygen Plasma Treated Poly(Lactic⁻Co⁻Glycolic) (PLGA) Membranes and Supported Nanostructured Oxides for Guided Bone Regeneration Processes.

(1) Background: The use of physical barriers to prevent the invasion of gingival and connective tissue cells into bone cavities during the healing process is called guided bone regeneration. The objective of this in-vitro study was to compare the growth of human osteoblasts on Poly(Lactic–co–Glycolic) (PLGA) membranes modified with oxygen plasma and Hydroxyapatite (HA), silicon dioxide (SiO2), and titanium dioxide (TiO2) composite nanoparticles, respectively. (2) Methods: All the membranes received a common treatment with oxygen plasma and were subsequently treated with HA nanostructured coatings (n = 10), SiO2 (n = 10) and TiO2 (n = 10), respectively and a PLGA control membrane (n = 10). The assays were performed using the human osteoblast line MG-63 acquired from the Center for Scientific Instrumentation (CIC) from the University of Granada. The cell adhesion and the viability of the osteoblasts were analyzed by means of light-field microphotographs of each condition with the inverted microscope Axio Observer A1 (Carl Zeiss). For the determination of the mitochondrial energy balance, the MitoProbe™ JC-1 Assay Kit was employed. For the determination of cell growth and the morphology of adherent osteoblasts, two techniques were employed: staining with phalloidin-TRITC and staining with DAPI. (3) Results: The modified membranes that show osteoblasts with a morphology more similar to the control osteoblasts follow the order: PLGA/PO2/HA > PLGA/PO2/SiO2 > PLGA/PO2/TiO2 > PLGA (p < 0.05). When analysing the cell viability, a higher percentage of viable cells bound to the membranes was observed as follows: PLGA/PO2/SiO2 > PLGA/PO2/HA > PLGA/PO2/TiO2 > PLGA (p < 0.05), with a better energy balance of the cells adhered to the membranes PLGA/PO2/HA and PLGA/PO2/SiO2. (4) Conclusion: The membrane in which osteoblasts show characteristics more similar to the control osteoblasts is the PLGA/PO2/HA, followed by the PLGA/PO2/SiO2.

In Vitro and in Vivo Study of Poly(Lactic⁻co⁻Glycolic) (PLGA) Membranes Treated with Oxygen Plasma and Coated with Nanostructured Hydroxyapatite Ultrathin Films for Guided Bone Regeneration Processes.

The novelty of this study is the addition of an ultrathin layer of nanostructured hydroxyapatite (HA) on oxygen plasma modified poly(lactic–co–glycolic) (PLGA) membranes (PO2) in order to evaluate the efficiency of this novel material in bone regeneration. Methods: Two groups of regenerative membranes were prepared: PLGA (control) and PLGA/PO2/HA (experimental). These membranes were subjected to cell cultures and then used to cover bone defects prepared on the skulls of eight experimental rabbits. Results: Cell morphology and adhesion of the osteoblasts to the membranes showed that the osteoblasts bound to PLGA were smaller and with a lower number of adhered cells than the osteoblasts bound to the PLGA/PO2/HA membrane (p < 0.05). The PLGA/PO2/HA membrane had a higher percentage of viable cells bound than the control membrane (p < 0.05). Both micro-CT and histological evaluation confirmed that PLGA/PO2/HA membranes enhance bone regeneration. A statistically significant difference in the percentage of osteoid area in relation to the total area between both groups was found. Conclusions: The incorporation of nanometric layers of nanostructured HA into PLGA membranes modified with PO2 might be considered for the regeneration of bone defects. PLGA/PO2/HA membranes promote higher osteosynthetic activity, new bone formation, and mineralisation than the PLGA control group.

Reliability of new poly (lactic-co-glycolic acid) membranes treated with oxygen plasma plus silicon dioxide layers for pre-prosthetic guided bone regeneration processes.

BackgroundThe use of cold plasmas may improve the surface roughness of poly(lactic-co-glycolic) acid (PLGA) membranes, which may stimulate the adhesion of osteogenic mediators and cells, thus accelerating the biodegradation of the barriers. Moreover, the incorporation of metallic-oxide particles to the surface of these membranes may enhance their osteoinductive capacity. Therefore, the aim of this paper was to evaluate the reliability of a new PLGA membrane after being treated with oxygen plasma (PO2) plus silicon dioxide (SiO2) layers for guided bone regeneration (GBR) processes.Material and MethodsCircumferential bone defects (diameter: 11 mm; depth: 3 mm) were created on the top of eight experimentation rabbits’ skulls and were randomly covered with: (1) PLGA membranes (control), or (2) PLGA/PO2/SiO2 barriers. The animals were euthanized two months afterwards. A micromorphologic study was then performed using ROI (region of interest) colour analysis. Percentage of new bone formation, length of mineralised bone, concentration of osteoclasts, and intensity of ostheosynthetic activity were assessed and compared with those of the original bone tissue. The Kruskal-Wallis test was applied for between-group com Asignificance level of a=0.05 was considered.ResultsThe PLGA/PO2/SiO2 membranes achieved the significantly highest new bone formation, length of mineralised bone, concentration of osteoclasts, and ostheosynthetic activity. The percentage of regenerated bone supplied by the new membranes was similar to that of the original bone tissue. Unlike what happened in the control group, PLGA/PO2/SiO2 membranes predominantly showed bone layers in advanced stages of formation.ConclusionsThe addition of SiO2 layers to PLGA membranes pre-treated with PO2 improves their bone-regeneration potential. Although further research is necessary to corroborate these conclusions in humans, this could be a promising strategy to rebuild the bone architecture prior to rehabilitate edentulous areas. Key words:Guided bone regeneration (GBR), poly(lactic-co-glycolic acid) (PLGA), membrane; oxygen plasma (PO2), nanocomposite, silicon dioxide layers.

The Gephi Network Visualisation of the Guided Bone Regeneration Process Induced with Tissue Engineered Grafts

The goal of this study was to create a Gephi network visualisation of the guided bone regeneration induced by tissue-engineered grafts using a histological score. Bioengineered bone grafts were obtained using CD 1 mice mesenchymal stem cells seeded on red deer antler scaffolds. Both basal and complex osteogenic media were used as osteogenic inducers of mesenchymal stem cells in the bioengineering process.Bone defects (5 mm in diameter) in the cranial bone of CD1 mice were surgically induced. Bone reconstruction was not performed in surgically induced bone defects in the control group, consisting of 30 subjects. In the study group, consisting of 60 subjects, bone reconstruction was performed using tissue engineered bone grafts. Subjects were sacrificed 2 and 4 months after surgery. Bone regeneration was assessed using a histological record.Comparative analysis of the bone regeneration process between the groups was performed using SPSS (Statistical Package for the Social Sciences) , network analysis and the visual results were performed using Gephi software.The created Gephi network indicates a more advanced bone regeneration stage for subjects in the study group sacrificed after 4 months.The Gephi networks reveal the time evolution of the bone regeneration process, the nodes size and edges complexity increasing from subjects sacrificed at 2 months to those sacrificed at 4 months.At each stage, two and four months after surgery, bone regeneration was more advanced in the study group than in the control group and it was different for the two osteogenic media. We conclude that bone regeneration in critical size defects, as in our research, cannot be obtained without bone reconstruction.

‘Pre-prosthetic use of poly(lactic-co-glycolic acid) membranes treated with oxygen plasma and TiO2 nanocomposite particles for guided bone regeneration processes’

ObjectivesGuided 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 (PO2) and titanium dioxide (TiO2) composite nanoparticles. MethodsCircumferential bone defects (diameter: 10mm; depth: 3mm) 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/PO2/TiO2). 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. ResultsThe 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. ConclusionsThe functionalised membranes showed the best efficacy for bone regeneration. Clinical significanceThe addition of TiO2 nanoparticles onto PLGA/PO2 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.

In vivo comparative model of oxygen plasma and nanocomposite particles on PLGA membranes for guided bone regeneration processes to be applied in pre-prosthetic surgery: A pilot study

ObjectivesTo evaluate the bone regeneration potential of a new membrane fabricated with polyglycolide acid (PLGA) after being treated with oxygen plasma (PO2), and/or being functionalized with silicon dioxide (SiO2) or titanium dioxide (TiO2) nanoparticles. MethodsBone defects (5mm×3mm) were produced on the top of 3 experimentation rabbits’ skulls and were covered with variously modified PLGA scaffolds. After the animals were sacrificed, neoformed bone (%), mineralized bone (mm), bone resorption (%), osteoclasts/mm2, and intensity of osteosynthetic activity, were assessed under microscope. ResultsThe following groups were formed depending on the type of membrane: PLGA (control); PLGA/PO2; PLGA/SiO2; PLGA/TiO2; PLGA/PO2/SiO2; and PLGA/PO2/TiO2. The histological sections showed bone layers in advanced stages of formation. The highest percentages of neoformed bone corresponded to PLGA/PO2/SiO2 membranes (59.07%; p=0.31) followed by PLGA/PO2 barriers (50.27%). The controls showed the lowest mineralization (13.89mm; p=0.24). PLGA/TiO2 scaffolds exhibited the least bone resorption (4.45%; p=0.77) and osteoclasts/mm2 (1.58; p=0.86). PLGA/SiO2 and PLGA/TiO2 membranes stimulated the maximum osteosynthetic activity. ConclusionsThe treatment of PLGA barriers with PO2 increased bone regeneration in rabbits. When comparing the effect of PO2/SiO2 and PO2/TiO2, higher percentages of neoformed bone were encountered after silicon-dioxide coating. Clinical significanceThe incorporation of SiO2 nanoparticles onto PO2-treated PLGA membranes was the most promising technique out of those investigated to promote bone formation in rabbits. The addition of SiO2 or TiO2 layers to PLGA substrates may stimulate the osteosynthetic activity, which might be useful to restore bone dimensions in preparation for naturally appearing dental prostheses.