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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-ε-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 μg/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.

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--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 increasing 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 1,500 µg/ml of TC showed a release concentration of 30 ppm over a certain time period to promote greater levels of human oral fibroblast (HOF) and human oral keratinocyte (HOK) 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 non-fibrous FPLCL loaded with TC at the same concentration.

Osteopetrosis is an inherited disorder of impaired bone resorption, with the most commonly affected genes being CLCN7 and TCIRG1, encoding the Cl(-) /H(+) exchanger CLC-7 and the a3 subunit of the vacuolar H(+) -ATPase, respectively. We and others have previously shown that the disease is frequently accompanied by osteomalacia, and that this additional pathology is also found in Tcirg1-deficient oc/oc mice. The remaining question was whether osteoid enrichment is specifically associated with TCIRG1 inactivation, or whether CLCN7 mutations would also cause skeletal mineralization defects. Here we describe a complete osteologic assessment of one family carrying a novel mutation in CLCN7 (D145G), which impairs the activation and relaxation kinetics of the CLC-7 ion transporter. The two siblings carrying the mutation in the homozygous state displayed high bone mass, increased serum levels of bone formation markers, but no impairment of calcium homeostasis when compared to the other family members. Most importantly, however, undecalcified processing of an iliac crest biopsy from one of the affected children clearly demonstrated a pathological increase of trabecular bone mass, but no signs of osteomalacia. Given the potential relevance of these findings we additionally performed undecalcified histology of iliac crest biopsies from seven additional cases with osteopetrosis caused by a mutation in TNFRSF11A (n=1), CLCN7 (n=3), or TCIRG1 (n=3). Here we observed that all cases with TCIRG1-dependent osteopetrosis displayed severe osteoid accumulation and decreased calcium content within the mineralized matrix. In contrast, there was no detectable bone mineralization defect in the cases with TNFRSF11A-dependent or CLCN7-dependent osteopetrosis. Taken together, our analysis demonstrates that CLCN7 and TCIRG1 mutations differentially affect bone matrix mineralization, and that there is a need to modify the current classification of osteopetrosis.

Periodontal disease is caused by microorganisms and host-derived inflammation involving increased cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production. We previously demonstrated that human β-defensin-3 induces COX-2 and PGE(2) in human gingival fibroblasts (HGFs). We, therefore, aimed to examine the inducible effects of LL-37, the only cathelicidin expressed in humans, on COX-2 expression and PGE(2) synthesis in HGFs and to elucidate the relevant signaling pathways. The COX-2 expression was upregulated by LL-37 in dose- and time-dependent manners. Accordingly, the synthesis of PGE(2) in cell-free culture supernatants was raised by LL-37 (p < 0.01) and blocked by NS-398, a specific COX-2 inhibitor (p < 0.01). P2X inhibitors and a neutralizing antibody against P2X(7) purinergic receptor significantly abrogated COX-2 induction and PGE(2) production by LL-37 (p < 0.01). LL-37 upregulated COX-2 expression and PGE(2) synthesis via activation of extracellular signal-regulated kinase (ERK) and p46 c-Jun N-terminal kinase (JNK), while interleukin-1β did so via nuclear factor-ĸB and all three mitogen-activated protein kinases. In summary, LL-37 can control arachidonic acid metabolism by induction of COX-2 expression and PGE(2) synthesis via the P2X(7) receptor, ERK, and p46 JNK. The pro-inflammatory effects of LL-37 may be essential for initiating oral mucosal inflammation in periodontal disease.

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 veränderter 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 Säuresekretion der Osteoklasten, welche für die Resorptionstätigkeit essentiell ist. Es ist daher folgerichtig, dass ein völliger Verlust des Proteins die infantil maligne Osteopetrose in Mensch (ARO) bzw. einen letalen Osteopetrose-Phänotyp in der Maus zur Folge hat. Darüber hinaus werden in ungefähr 70% der Fällen von dominanter Osteopetrose (ADOII) heterozygote Mutationen im Gen für ClC-7 identifiziert, die statistisch die Menge an funktionellen ClC-7 Dimeren maximal auf 25% reduzieren sollten. Heterozygote Träger rezessiver Mutationen haben keinen Phänotyp. Da es gegenwärtig ungeklärt ist, ab welchem Grad von Funktionsverlust von ClC-7 die Osteoklastenfunktion eingeschränkt 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+/- Mäusen 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 (unabhängiger 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-/- Mäuse und Clcn7-/- Tg+ Tiere der Linie F7 hatten einen gleich schweren osteopetrotischen Phänotyp, 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 für eine teilweise Wiederherstellung der Osteoklastenfunktion spricht. F3 Mäuse zeigten eine annähernd normale Knochendichte, während F1 Mäuse eine milde Osteopetrose darboten, die in etwa dem Phänotyp 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 überraschend niedrig. In F3 wurde die Proteinexpression auf 5,70±1,4 % gemindert, während die Expression in F1 nur 2,51±0,89 % betrug. Schlussfolgerung: Die Clcn7-/- Tg+ Tiere der Linie F1 zeigten einen milden Osteopetrose-Phänotyp, der einer autosomal dominanten Osteopetrose nahekommt, während in Linie F3 die Knochendichte durch die transgene Expression von ClC-7 im Clcn7-/- Hintergrund annähernd normalisiert wurde. Damit korreliert die Proteinexpression, die jedoch unter den erwarteten Werten von 25-50% liegt. Dies könnte durch einen Speziesunterschied begründet sein. Schlüsselwörte: Knochendichte, Knochenumbau, Chloridkanal, Histomorphometrie, Osteoklast, Ostepetrose, Transgen, Mausmodel, Bone 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