| Guided bone regeneration (GBR) is a technique placing a membrane material on the bone defects to block the rapid growing epithelial cells and fibroblasts migrating into the bone defects. This promotes the osteogenic cells to enter first and repair the defect area through bone regeneration. Barrier membrane materials play an important role in guided bone regeneration.But Barrier membrane materials as we know lack of osteoinductivity.To develop a new osteoinductivity of Barrier membrane material has important clinical significance on the bone defect and other diseases.Bioactive glasses (BG) are glasses with specific biological and physiological functions. Implantation of BG into human bone defects results in BG binding to the bone tissue. It plays an important role in bone repair and functional restoration. BG degradation products and its released ions promote the proliferation, metabolism, and cell connection of osteoblasts. This mediates gene expression of some important proteins (e.g., osteocalcin, bone adhesion protein, and osteopontin) and their functions. This also improves the activity of alkaline phosphatase (ALP) and promotes the formation of type I collagen to accelerate osteogenesis, which has a higher osteogenic rate than the hydroxyapatite.It has more superior performance which is used with inorganic or organic materials and polymer materiasls.It has been widely used in tissue engineering and repair of bone.PLLA has many advantages such as its good biocompatibility, non-toxicity and biodegradable features. However, the PLLA degradation product is acidic and causes painless inflammation inside the body. It also lacks reactive functional groups in the chemical structure of PLLA—this results in poor hydrophilicity and weak cell adhesion. So we try to composites of BG and PLLA combine the advantages of each.To develop a new of Barrier membrane material combines more superior performance, good physical properties, biodegradation and osteoinduction.The low-temperature plasma technology is widely used to change the surface of materials.It makes the physiochemical reactions on the surface of matericals with high emgery particales and active species.The advantages of this technology is that process is simple, easy to control, no pollution and no affection on the properties of materials.Composites of BG and PLLA combine the advantages of each. For example, PLLA has good mechanical and physical properties, good compatibility and biodegradability, good tensile strength, and high degree of elongation to compensate for the defect of BG The surface of PLLA has no cell recognition sites, but BG in the composite membrane provides cell recognition sites to improve cell adhesion and proliferation. In addition, BG buffers the acidic degradation products of PLLA. The low-temperature plasma technology for the modification of PLLA surface improves the hydrophilicity, adhesion, and biocompatibility of PLLA surface without affecting the PLLA materials. This further promotes cell adhesion against the material surface.There is no study reported the same.Chapter One Preparation and characterization of poly-1-lactic acid (PLLA)/ bioactive glass (BG) guided bone regeneration membranesObjectiveTo prepare a new type of poly-1-lactic acid/bioactive glass (PLLA/BG) hydrophilic nanofiber membrane and investigate its feasibility as a barrier membrane for guided bone regeneration.Methods1. Medical PLLA (PLLA:BG ratio= 9:1) was weighted and added to the tetrahydrofuran solution containing BG. We prepare poly-1-lactic acid/bioactive glass (PLLA/BG) hydrophilic nanofiber membrane by thermally induced phase separation (TIPS),and obtain PLLA/BG composite membrane containing an oxygen-rich surface with polar functional groups by the low-temperature plasma technology.2. The gross morphological features of the composite membrane were observed with visual inspection and digital photography.3. JSM-6330F cold cathode field emission scanning electron microscope (SEM) was used to analyze the surface morphology of gold-coated PLLA/BG composite membranes. The fiber diameter and pore size of the PLLA/BG composite membranes were measured using Adobe Photoshop.4. A liquid (absolute ethanol) replacement method was used to measure the porosity. The proportion pycnometer was filled with ethanol to a value of W1 (g). Sample (with the quality set as Ws (g) was immersed in the pycnometer filled with ethanol to deaerate and allow the ethanol to fill in the membrane’s pores, followed by refilling of the ethanol. The value of the pycnometer containing sample and ethanol was defined as W2 (g). After removing the sample from the ethanol, the value of the pycnometer containing the remaining ethanol was W3 (g).The porosity of the sample scaffold was calculated based on the following equation. Here, P represents the porosity (%) of the material; Vo is the volume of material in a natural condition, or named as apparent volume (cm3); and V represents the volume of an absolutely compacting material (cm3).5. a contact angle meter was used to measure the surface contact angle of each sample. Ten microliters of double distilled water were placed in 3 different positions of the sample to measure the average contact angle.6. Material was tailored into rectangular shape (40 mm x 5 mm x 2 mm), held in a homemade framework between two fine sandpapers, and further loaded onto the mechanical property tester. The lateral sides of the scaffold were stretched for 3 mm/min to measure the sample at room temperature. Each sample was tested in 5 parallel experiments.Results1. The diameter of the material was 1.5 cm with 2 mm thickness. This material was white or yellowish with a rough surface and compressibility.2. The absolute PLLA obtained from experimentally-induced phase separation was in a three dimensional network structure. The fiber material diameter was 160-320 nm with uniform pore distribution and pore sizes of 1-4 um. When the mass ratio of PLLA and BG is 9:1, the BG particles adhere better on the PLLA matrix and uniformly disperse in the porous material. The fiber diameter and porosity of the composite material had no significant change. They formed nano-diameter fibers with micron sized pores.3. The medical grade PLLA membrane had a high porosity (93.926%). Addition of BG in either oxygen plasma treated or untreated PLLA/BG composite membranes resulted in a lower porosity than the PLLA only membrane, but the general structure of the membrane was not altered. A statistically significant difference was found in the porosity of the 3 membrane material groups (F= 49.412; P< 0.001). No significant difference was found in the pairwise comparison between untreated PLLA/BG composite membrane and oxygen plasma treated PLLA/BG composite membrane (P= 0.779); whereas a significant difference was found when comparing the porosities of PLLA and untreated PLLA/BG membranes (P< 0.001). This indicates a high porosity in the 3 membrane material groups4. The water contact angle of the PLLA/BG composite membrane after oxygen plasma treatment was 81.32 ± 5.77°. This was the lowest of the 3 membrane materials. A significant difference was found in the pairwise comparisons among the 3 groups (P< 0.001). Statistically significant differences were found when comparing the oxygen plasma treated composite membrane and the other 2 membrane material groups. This indicates that the hydrophilicity of the oxygen plasma treated PLLA/BG membrane was significantly improved5. The average tensile strength (tensile fracture stress) of the PLLA was 34.6 MPa. The average tensile strength of the PLLA/BG scaffold was 27.1 MPa indicating that the addition of BG affected the PLLA regularity and impeded the crystallization by reducing the degree of crystallinity to decrease the average tensile strength. Oxygen plasma treatment resulted in no decrease in tensile strength (>20 MPa), but the etched membrane surface did have a reduced average tensile strength, which was significantly lower than PLLA or untreated PLLA/BG material.ConclusionsThe PLLA/BG composite membrane prepared in this study had a nanofibrous three-dimensional network structure. The BG was diffusely distributed inside the scaffold. The PLLA/BG composite membrane had good porosity and tensile elasticity.lt can be used in clinical practice.Chapter Two in vitro cytological study of poly-1-lactic acid (PLLA)/bioactive glass (BG) guided bone regenerationObjectiveTo analyse biocompatibility and osteogenic properties of poly-1-lactic acid (PLLA)/ bioactive glass (BG) guided bone regeneration membranes.Methods1. A 100 μL cell suspension containing 1 × 106 cells were inoculated into a 24-well plate containing 3 membrane material groups of the pre-wet membranes. The cell adhesion rate of the membranes from the control and experimental groups after 1,3, and 6 h of inoculation were measured by collecting media from each well and measuring the number of cells in the suspension (Al). The MG-63 cells from each group were collected from 3 different wells. Membrane materials were also collected to further digest the cells adhered against the porous wall (A2) using the equation as follows: Cell adhesion rate= [(A0—A1—A2)/A0]× 100%(A0 represents the number of cells during inoculation).2. A 100 μL cell suspension containing 5 × 105 MG-63 cells were added to a 24-well plate containing 3 membrane material groups of the pre-wet membranes. Cell suspensions from each group was incubated for 1,3, and 6 h per well. The media was then removed, plates washed with PBS followed by 4% paraformaldehyde fixation for 10 min and repeated PBS washing. A 300 μL aliquot of Hoechst 33342 dye (10 μg/mL) was added to the membrane and for 30 min at room temperature followed by PBS washing and blotting with filter paper. We observed cell adhesion on the membrane under fluorescence microscopy.3. A 100 μL cell suspension containing 5 × 105 MG-63 cells were inoculated into a 24-well plate containing 3 membrane material groups of the pre-wet membranes. Cell suspension from each group was incubated for 1,3, and 12 days. Material membranes were then removed from the well followed by PBS washing. These were placed into a new 24-well plate with 300 μL fresh media and 60 uL MTS in the dark for 3 h at 37℃. A 100 μL of solution from each well was extracted twice and placed into a 96-well plate to measure the optical density (OD) from each well at 490 nm with a microplate reader.4. Pre-wet oxygen plasma treated PLLA/BG composite membranes were placed in a 24-well plate and inoculated with 100 μL cell suspension containing 1 × 104 MG-63 cells in each well. The cell suspension was then incubated for 3 and 7 days, followed by removing the culture medium and fixing in 2.5% pentanediol at 4℃ overnight. The membranes were than dehydrated in gradient ethanol and coated with colloidal gold before observing cell-adhesion, cell proliferation, and calcium nodule formation with SEM.5. A 100 μL cell suspension containing 1 × 105 MG-63 cells was inoculated into a 24-well plate containing 3 membrane material groups of the pre-wet membranes. A cell suspension from each group was incubated for 3,7, and 14 days. The media was then removed, plates rinsed 3 times with PBS followed by 200 μL 1% Triton X-100 solution to lyse for 10-15 min at 4℃. A 30 μL solution from each well was removed and added into a new 96-well plate for a ALP assay according to the manufacturer’s instruction. The absorbance of each well (OD) was measured at 520 nm.Results1. The longer the cell inoculation, the higher the cell adhesion rate in the 3 membrane material groups. Either the PLLA membrane or the untreated PLLA/BG membrane showed no significant differences in the cell adhesion rate at 3 time points (P> 0.05). After oxygen plasma treatment on the PLLA/BG composite membrane, the cell adhesion rates were significantly higher than the other 2 membrane material groups (P< 0.001).2. We evaluated cell adhesion with Hoechst 33342. Because the PLLA membrane surface is hydrophobic, the cell-membrane material surface adhesion was relatively weak. After repeatedly washing with PBS, the number of cells adhered on the PLLA membrane or PLLA/BG membrane was reduced at the 1 and 3 h time points. Oxygen plasma treatment improved the cell-membrane material surface adhesion in the PLLA/BG membrane especially at the early time points of cell culture. At 100x magnification, the cell nuclei began to show a relatively uniform dispersion under blue fluorescence at 1 h. The fluorescent intensity increased at 3 h, and the MG-63 cell adhesion rate increased at 6 h. Oxygen plasma treatment on the composite membrane showed a significant increase in fluorescent intensity. Furthermore, the oxygen plasma treatment significantly improved the cell adhesion at early time points versus the other two samples.3. The longer the cell inoculation, the higher the cell proliferation rate. Oxygen plasma treatment on PLLA/BG composite membranes significantly increased the cell proliferation at all time points versus PLLA membrane or untreated PLLA/BG composite membrane (P< 0.01), respectively. No significant differences in cell proliferation were seen between the untreated PLLA/BG membrane and PLLA membrane (P> 0.05).4. SEM data shows that the day 3 MG-63 cell culture sample had spindle-shaped cells adhering to the surface of the oxygen plasma treated PLLA/BG composite membrane. These cells began to extend pseudopodia in all directions to cross-link with the composite membrane. After 7 days of cell culture, the number of cells increased, and the cells had grown into a sheet with a high number of calcified nodules. When the cell became confluent and turned into a monolayer, the cell proliferation reduced and the cell body increased. This was followed by the secretion of an extracellular matrix. The osteoblast secretion of spherical matrix was observed, and these spherical matrices were merged together into aggregates. The osteoblasts grew into colonies after aggregating on multiple layers. At the center of the colony, the overlapping cells blurred the cell boundaries and projections of the peripheral cells were intertwined. These colonies occurred on three-dimensional clumps of the scaffold, while the matrix secreted from the osteoblasts mineralized to form calcified nodules. Some cells grew inside the membrane through the enlarged pore due to gradual degradation of the membrane surface. These cells were tightly cross-linked to secrete matrix and form calcium nodules.5. The overall ALP activity of the MG-63 cells in the 3 groups increased over time and reached a peak at day 7 after cell inoculation. In comparing day 3 cell culture on the 3 groups, we see that oxygen plasma treatment on the PLLA/BG composite membrane had a significantly higher ALP activity than PLLA membrane and untreated PLLA/BG membrane (P< 0.01). However, no significant differences were seen in ALP activity among the 3 groups on day 7 and day 14 (P> 0.05).ConclusionsThe PLLA/BG composite membrane prepared in this study had good biocompatibility, and osteogenic properties. |
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