10 research outputs found

    Tetracycline-Loaded Electrospun Poly(L-lactide-co-Δ-caprolactone) Membranes for One-Step Periodontal Treatment

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    In this research, a one-step periodontal membrane, with the required function and properties, has been designed as an alternative method of tissue regenerative treatments. Designed nanoporous prototypes from poly(l-lactide-co-Δ-caprolactone) (PLCL, 70:30 mol %) were fabricated by electrospinning, denoted as S-PLCL. They were subsequently loaded with tetracycline (TC) in order to enhance periodontal regeneration and deliver an anti-inflammatory and antibiotic drug. It was found that TC loading did not have any significant effect on the fiber diameter but did increase hydrophilicity. With the increase in TC loading, the water vapor permeability (WVP) of the S-PLCL membrane decreased within the range of 31–56% when compared with neat S-PLCL membranes, while in the solvent-cast film (F-PLCL), no significant change in WVP was observed. Moreover, S-PLCL demonstrated a controllable slow release rate of TC. S-PLCL loaded with 1500 ÎŒg/mL of TC showed a release concentration of 30 ppm over a certain time period to promote greater levels of human oral fibroblast and human oral keratinocyte cell proliferation and plaque inhibition. In conclusion, a TC-loaded S-PLCL fibrous membrane has been designed and fabricated to provide the ideal conditions for cell proliferation and antibiotic activity during treatment, outperforming nonfibrous F-PLCL loaded with TC at the same concentration

    Charakterisierung der osteopetrotischen Clcn7-/- Mausmutante mit Osteoklasten- spezifischer Expression von ClC-7

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    Die Struktur des Knochens wird bestimmt durch ein komplexes Zusammenspiel von Osteoblasten, die Knochen aufbauen, und Osteoklasten, die Knochen abbauen. Durch ein Ungleichgewicht dieses Prozesses resultieren Erkrankungen mit verĂ€nderter Knochendichte. Die Osteopetrose ist eine generalisierte osteosklerotische Erkrankung, die durch Verlust der Osteoklastenfunktion bzw. Defekte der Osteoklastendifferenzierung entstehen. Das Chloridkanal-Protein ClC-7, das in Osteoklasten stark exprimiert ist, hat eine wichtige Funktion bei der SĂ€uresekretion der Osteoklasten, welche fĂŒr die ResorptionstĂ€tigkeit essentiell ist. Es ist daher folgerichtig, dass ein völliger Verlust des Proteins die infantil maligne Osteopetrose in Mensch (ARO) bzw. einen letalen Osteopetrose-PhĂ€notyp in der Maus zur Folge hat. DarĂŒber hinaus werden in ungefĂ€hr 70% der FĂ€llen von dominanter Osteopetrose (ADOII) heterozygote Mutationen im Gen fĂŒr ClC-7 identifiziert, die statistisch die Menge an funktionellen ClC-7 Dimeren maximal auf 25% reduzieren sollten. Heterozygote TrĂ€ger rezessiver Mutationen haben keinen PhĂ€notyp. Da es gegenwĂ€rtig ungeklĂ€rt ist, ab welchem Grad von Funktionsverlust von ClC-7 die Osteoklastenfunktion eingeschrĂ€nkt wird, wurden in dieser Studie verschiedene Mauslinien mit unterschiedlich starkem ClC-7 Expressionsniveau untersucht. Methoden: Drei Mauslinien (F1, F3, und F7), die ein transgenes Konstrukt, das aus dem Osteoklasten-spezifischen Acp5 (Trap)-Promoter und der ClC-7 cDNA besteht, stabil im Genom integriert haben, wurden mit Clcn7+/- MĂ€usen verpaart, so dass in der zweiten Generation Clcn7-/- Tg+ Tiere entstanden, bei denen der Funktionsverlust des Clcn7-Gens durch transgene Expression teilweise ausgeglichen wurde. Die Clcn7-/- Tg+ Tiere wurden histomorphometrisch untersucht. Die Strukturparameter und Zellzahlen wurden statistisch (unabhĂ€ngiger gepaarter t-Test) zwischen den Genotypen und den Linien verglichen. ClC-7 wurde in situ im Vergleich mit anderen Osteoklastenproteinen durch Immunofluoreszenz nachgewiesen. Weiterhin wurden Osteoklasten der verschiedenen Mauslinien in vitro kultiviert. mRNA und Proteine wurden extrahiert und durch quantitative PCR und Western-Blot quantifiziert. Ergebnisse: Clcn7-/- MĂ€use und Clcn7-/- Tg+ Tiere der Linie F7 hatten einen gleich schweren osteopetrotischen PhĂ€notyp, so dass keine transgene Expression vorhanden sein konnte. Diese Linie wurde nicht weiter untersucht. Dagegen wurde die Osteopetrose bei den Linien F1 und F3 deutlich abgemildert, was fĂŒr eine teilweise Wiederherstellung der Osteoklastenfunktion spricht. F3 MĂ€use zeigten eine annĂ€hernd normale Knochendichte, wĂ€hrend F1 MĂ€use eine milde Osteopetrose darboten, die in etwa dem PhĂ€notyp der dominanten Osteopetrose entspricht. Die Anzahl der Osteoblasten und die Anzahl der Osteoklasten korrelierte umgekehrt mit der Knochendichte. Die Expression von Clcn7 mRNA und Protein war in beiden Linien ĂŒberraschend niedrig. In F3 wurde die Proteinexpression auf 5,70±1,4 % gemindert, wĂ€hrend die Expression in F1 nur 2,51±0,89 % betrug. Schlussfolgerung: Die Clcn7-/- Tg+ Tiere der Linie F1 zeigten einen milden Osteopetrose-PhĂ€notyp, der einer autosomal dominanten Osteopetrose nahekommt, wĂ€hrend in Linie F3 die Knochendichte durch die transgene Expression von ClC-7 im Clcn7-/- Hintergrund annĂ€hernd normalisiert wurde. Damit korreliert die Proteinexpression, die jedoch unter den erwarteten Werten von 25-50% liegt. Dies könnte durch einen Speziesunterschied begrĂŒndet sein. SchlĂŒsselwörte: Knochendichte, Knochenumbau, Chloridkanal, Histomorphometrie, Osteoklast, Ostepetrose, Transgen, MausmodelBone quality is a consequence of simultaneous functions of bone formation cells or osteoblasts and bone resorption cells or osteoclasts. Imbalance function of these cells disturbs bone remodeling and consequently leads to pathological bone diseases. Complete loss of function or insufficient differentiation of osteoclasts causes excessive bone density or osteopetrosis. Intracellular transmembrane protein chloride channel-7 (ClC-7), which is highly expressed in osteoclasts is required for electroneutralization during bone degradation. Complete depletion of the protein gives rise to autosomal recessive osteopetrosis (ARO) in humans and osteopetrosis phenotype in mice. Moreover, in approximately 70% of autosomal dominant osteopetrosis type II (ADO II) patients, heterozygous mutations of CLCN7 are identified. Because ClC-7 structure is dimmers, in which each of two subunits has its own pore, the heterozygous mutations should be encoded for <25% of the entire protein, statistically. However, the mutations’ carriers do not express phenotype. The pathogenesis of the disease remains unclear. Hence, the present study was aimed to explain the mechanism of the diseases relevant to ClC-7 expression. Methods: To answer the question, three transgenic mouse lines were constructed by genome-integration of ClC-7 cDNA which is under control of osteoclast- specific Acp5 (Trap) promoter. Heterozygous mice (Clcn7+/- Tg+) were mated. Then, Clcn7-/- Tg+ mice could be generated in the second generation. Accordingly, ClC-7 could be expressed partially. Bone phenotypes of the transgenic mice with different Clcn7 genotypes were investigated histomorphologically. Structural and cellular parameters were compared between the genotypes, and groups statistically (independent paired t-test). ClC-7 and other osteoclast specific proteins were also determined in situ using the immunofluorescent technique. mRNA and protein extracted from osteoclasts in vitro were examined by quantitative PCR and Western blot analysis, respectively. Results: The Clcn7 knockout mice and F7 showed severe osteopetrosis phenotype, whereas F1 and F3 had different degrees of bone density. F1 rescue mice had mild osteopetrosis phenotype, while F3 rescue mice and heterozygous mice displayed insignificant bone phenotype. Osteoclast and osteoblast numbers were reversely associated with the degree of bone density. Interestingly, Clcn7 mRNA and ClC-7 expressed very low in F1 and F3. Moreover, ClC-7 expression in F3 was 5.70±1.4%, while the average value in F1 was decreased to 2.51±0.89% of control signals. Conclusion: The expression of ClC-7 is associated with the degrees of bone density. Low degree expression of ClC-7 can rescue the osteopetrosis phenotype. The reduced bone resorption function due to impaired ClC-7 prolongs the bone remodeling process and results in mild osteopetrosis. Recessive pattern of the inheritance of the mutated gene leads to severe osteopetrosis in humans and mice, whereas heterozygous mutations cannot affect osteopetrosis phenotype in mice. Thus, the pattern of genotype-phenotype relationship would be different between species. The study would be meaningful for genetic diagnostics and the prediction of severity of the disease. Furthermore, F1 mice displayed a phenotype very similar to ADO II, thus the F1 mice would be a beneficial model for treatment trials of the disease as well as for the study of osteoporosis. Keywords: bone density, bone remodeling, chloride channel, histomorphometry, osteoclast, osteopetrosis, transgene, mice mode

    Hypoglycaemic activity of Mathurameha, a Thai traditional herbal formula aqueous extract, and its effect on biochemical profiles of streptozotocin-nicotinamide-induced diabetic rats

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    Abstract Background The Thai traditional herbal formula–Mathurameha, consisting of 26 medicinal plants, has been used as an alternative and complementary medicine for diabetes treatment in Wangnamyen Hospital, Thailand. To provide scientific evidences on the efficacy and safety of this herbal formula, in vivo hypoglycaemic activity, effect on serum biochemical profiles and acute toxicity were investigated. Methods Experimental type 2 diabetes was induced in male Sprague-Dawley rats by intraperitoneal injection of nicotinamide 15 min prior to intravenous injection of streptozotocin. The most effective extract from the oral glucose tolerant test (OGTT) was administered daily via the oral route to diabetic rats for 2 weeks. Two-hour postprandial plasma glucose (2h–PPG) levels were measured on days 0, 7, and 14. Biochemical data were measured at the end of daily oral administration experiment. Results Aqueous extract of the herbal formula was the most potent extract for improving glucose tolerance of streptozotocin-nicotinamide-induced diabetic rats after single oral administration. After 2 weeks of daily oral administration, the aqueous extract showed a dose-dependent glucose lowering effect. At doses of 12.5, 25, and 50 mg/kg, the 2h–PPG level of diabetic rats decreased by 3.32%, 15.78%, and 17.94%, respectively. Most of the biochemical profiles of diabetic rats were improved, including the total cholesterol (TC), alkaline phosphatase (ALP), total protein, albumin, globulin, creatinine, and uric acid levels. The significantly increased triglyceride (TG) level observed in treated diabetic rats indicated a lack of a beneficial effect of the extract on lipid homeostasis. Nevertheless, there were no signs or symptoms of acute toxicity observed after oral administration of aqueous extract (5 g/kg) to both male and female rats. Conclusions The results revealed that the herbal formula aqueous extract has hypoglycaemic activity, beneficial effects on biochemical profiles and a lack of acute toxicity. This study confirms the efficacy and safety of the Mathurameha herbal formula used for treating type 2 diabetes mellitus

    Preparation and Characterization of PLG Microparticles by the Multiple Emulsion Method for the Sustained Release of Proteins

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    Rapid release and diminished stability are two of the limitations associated with the growth factors that are essentially used in dental applications. These growth factors are employed to enhance the quality and quantity of tissue or bone matter during regeneration. Therefore, drug delivery devices and systems have been developed to address these limitations. In this study, bovine serum albumin (BSA), as a representative growth factor, was successfully sustained by encapsulation with the medium-absorbable copolymer, poly(L-lactide-co-glycolide) (PLG) 70:30% mol, via the multiple emulsion method. Different PLG, PVA, and BSA concentrations were used to investigate their effects on the BSA encapsulation efficiency. The suitable ratios leading to a better characterization of microparticles and a higher encapsulation efficiency in producing encapsulated PLG microparticles were 8% (w/v) of PLG, 0.25% (w/v) of PVA, and 8% (w/v) of BSA. Furthermore, an in vitro release study revealed a bursting release of BSA from the encapsulated PLG microsphere in the early phase of development. Subsequently, a gradual release was observed over a period of eight weeks. Furthermore, to encapsulate LL-37, different proteins were used in conjunction with PLG under identical conditions with regard to the loading efficiency and morphology, thereby indicating high variations and poor reproducibility. In conclusion, the encapsulated PLG microparticles could effectively protect the protein during encapsulation and could facilitate sustainable protein release over a period of 60 days. Importantly, an optimal method must be employed in order to achieve a high degree of encapsulation efficiency for all of the protein or growth factors. Accordingly, the outcomes of this study will be useful in the manufacture of drug delivery devices that require medium-sustained release growth factors, particularly in dental treatments

    Recombinant human secretory leukocyte protease inhibitor (rhSLPI) coated titanium enhanced human osteoblast adhesion and differentiation

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    Abstract Osseointegration is vital to success in orthopedic and dental reconstructions with implanted materials. The bone matrix or cells—particularly osteoblasts—are required to achieve functional contact on the implant surface. Osteoblast induction is therefore essential for osteogenesis to occur. Enhancement of osteoblast adhesion, proliferation, and differentiation, particularly by implant surface modifications, have been found challenging to develop. Secretory Leukocyte Protease Inhibitor (SLPI), a cation ionic protein with anti-inflammatory and anti-bacterial activities, showed activation in osteoblast proliferation and differentiation. However, the effects of coating recombinant human (rh) SLPI on a titanium alloy surface on human osteoblast adhesion, proliferation, and differentiation has never been investigated. In this study, titanium alloys (Ti–6Al–4V) were coated with rhSLPI, while human osteoblast adhesion, proliferation, differentiation, actin cytoskeletal organization, and gene expressions involved in cell adhesion and differentiation were investigated. The results indicate that coating titanium with 10–100 ”g/ml rhSLPI enhanced the physical properties of the Ti surface and enhanced human osteoblast (hFOB 1.19) cell adhesion, activated actin dynamic, enhanced adhesive forces, upregulated integrins α1, α2, and α5, enhanced cell proliferation, mineralization, alkaline phosphatase activity, and upregulated ALP, OCN, and Runx2. This is the first study to demonstrate that coating SLPI on titanium surfaces enhances osseointegration and could be a candidate molecule for surface modification in medical implants

    Medicinal plants utilized in Thai Traditional Medicine for diabetes treatment: Ethnobotanical surveys, scientific evidence and phytochemicals

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