10 research outputs found
Tetracycline-Loaded Electrospun Poly(L-lactide-co-Δ-caprolactone) Membranes for One-Step Periodontal Treatment
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 31â56% 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
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
verÀnderter 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
ungefÀhr 70% der FÀllen 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
TrÀger rezessiver Mutationen haben keinen PhÀnotyp. Da es gegenwÀrtig
ungeklÀrt ist, ab welchem Grad von Funktionsverlust von ClC-7 die
Osteoklastenfunktion eingeschrÀnkt 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
(unabhÀngiger 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 PhÀnotyp, 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 annÀhernd normale Knochendichte, wÀhrend F1 MÀuse eine milde
Osteopetrose darboten, die in etwa dem PhÀnotyp 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, wÀhrend die Expression in F1 nur
2,51±0,89 % betrug. Schlussfolgerung: Die Clcn7-/- Tg+ Tiere der Linie F1
zeigten einen milden Osteopetrose-PhÀnotyp, der einer autosomal dominanten
Osteopetrose nahekommt, wÀhrend in Linie F3 die Knochendichte durch die
transgene Expression von ClC-7 im Clcn7-/- Hintergrund annÀhernd 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 mutationsâ 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
Abstract Background The Thai traditional herbal formulaâMathurameha, 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 15Â 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 2Â weeks. Two-hour postprandial plasma glucose (2hâPPG) 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 2Â weeks of daily oral administration, the aqueous extract showed a dose-dependent glucose lowering effect. At doses of 12.5, 25, and 50Â mg/kg, the 2hâPPG 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 (5Â 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
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
Abstract Osseointegration is vital to success in orthopedic and dental reconstructions with implanted materials. The bone matrix or cellsâparticularly osteoblastsâare 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 (Tiâ6Alâ4V) 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 10â100 ”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