Production of three-dimensional tissue-engineered cartilage through mutual fusion of chondrocyte pellets.

In this study, the mutual fusion of chondrocyte pellets was promoted in order to produce large-sized tissue-engineered cartilage with a three-dimensional (3D) shape. Five pellets of human auricular chondrocytes were first prepared, which were then incubated in an agarose mold. After 3 weeks of culture in matrix production-promoting medium under 5.78g/cm(2) compression, the tissue-engineered cartilage showed a sufficient mechanical strength. To confirm the usefulness of these methods, a transplantation experiment was performed using beagles. Tissue-engineered cartilage prepared with 50 pellets of beagle chondrocytes was transplanted subcutaneously into the cell-donor dog for 2 months. The tissue-engineered cartilage of the beagles maintained a rod-like shape, even after harvest. Histology showed fair cartilage regeneration. Furthermore, 20 pellets were made and placed on a beta-tricalcium phosphate prism, and this was then incubated within the agarose mold for 3 weeks. The construct was transplanted into a bone/cartilage defect in the cell-donor beagle. After 2 months, bone and cartilage regeneration was identified on micro-computed tomography and magnetic resonance imaging. This approach involving the fusion of small pellets into a large structure enabled the production of 3D tissue-engineered cartilage that was close to physiological cartilage tissue in property, without conventional polyper scaffolds.

Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up-regulates inflammatory cytokines, prostaglandin E2 synthesis and osteoclast formation in interleukin-1 receptor antagonist-deficient mice.

BACKGROUND AND OBJECTIVE: The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS). MATERIAL AND METHODS: Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10 µg/mL of Aggregatibacter actinomycetemcomitans LPS for 24 h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9 d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA. RESULTS: The levels of IL-1α, IL-1ß, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A. actinomycetemcomitans LPS were significantly increased by approximately 4- (p < 0.05), 5- (p < 0.05), 1.3- (p < 0.05) and 6- (p < 0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p < 0.05), 1.6- (p < 0.05), 2.5- (p < 0.05), 1.6- (p < 0.05) and 1.9- (p < 0.05) fold, respectively, in IL-1Ra KO mice stimulated with A. actinomycetemcomitans LPS compared with WT mice. CONCLUSION: IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption.

Selection of highly osteogenic and chondrogenic cells from bone marrow stromal cells in biocompatible polymer-coated plates.

To enrich the subpopulation that preserves self-renewal and multipotentiality from conventionally prepared bone marrow stromal cells (MSCs), we attempted to use 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer-coated plates that selected the MSCs with strong adhesion ability and evaluated the proliferation ability or osteogenic/chondrogenic potential of the MPC polymer-selected MSCs. The number of MSCs that were attached to the MPC polymer-coated plates decreased with an increase in the density of MPC unit (0-10%), whereas no significant difference in the proliferation ability was seen among these cells. The surface epitopes of CD29, CD44, CD105, and CD166, and not CD34 or CD45, were detectable in the cells of all MPC polymer-coated plates, implying that they belong to the MSC category. In the osteogenic and chondrogenic induction, the MSCs selected by the 2-5% MPC unit composition showed higher expression levels of osteoblastic and chondrocytic markers (COL1A1/ALP, or COL2A1/COL10A1/Sox9) at passage 2, compared with those of 0-1% or even 10% MPC unit composition, while the enhanced effects continued by passage 5. The selection based on the adequate cell adhesiveness by the MPC polymer-coated plates could improve the osteogenic and chondrogenic potential of MSCs, which would provide cell sources that can be used to treat the more severe and various bone/cartilage diseases.

Gene transfer of bFGF to recipient bed improves survival of ischemic skin flap.

BACKGROUND: The recipient bed is a promising target of angiogenic therapy to treat ischemic skin flaps. We delivered basic fibroblast growth factor (bFGF) gene to the recipient bed by a plasmid-based method with electroporation, and assessed the effects on flap viability in a rat dorsal skin flap model. METHODS: A 25 x 90 mm(2) axial skin flap was elevated on the back of male Sprague-Dawley rats. Two days before flap elevation, an expression plasmid vector containing the bFGF gene with the signal sequence was injected into the dorsal muscles beneath the skin flap, and then electroporation was delivered (FGF-E(+) group). As control, rats were injected with a plasmid vector containing LacZ gene (LacZ-E(+) group), instead of bFGF gene. Other groups of animals received plasmid vector containing bFGF (FGF-E(-) group) or LacZ (LacZ-E(-) group) gene without electroporation. Seven days later, the area of necrosis and neovascularisation of the skin flap were evaluated. RESULTS: The bFGF gene was successfully transferred to the dorsal muscles, and bFGF was expressed in muscle tissue. The area of flap necrosis (%) in the FGF-E(+) group (21.7+/-5.3%) was significantly smaller than that in the LacZ-E(+) (28.3+/-4.1%), FGF-E(-) (29.7+/-3.3%), and LacZ-E(-) (28.1+/-2.5%) groups. Postmortem angiograms and histological analyses showed that vascularisation in the distal part of the skin flap was significantly increased in the FGF-E(+) group compared with the other groups. CONCLUSION: These findings suggested that gene delivery of bFGF to the recipient bed muscles enhanced vascularity and viability of an ischemic skin flap, and that plasmid-based gene delivery with electroporation was a suitable delivery method.

Evidence for lipopolysaccharide as the predominant proinflammatory mediator in supernatants of antibiotic-treated bacteria.

Lipopolysaccharide (LPS), purified from gram-negative bacteria, is well known to induce proinflammatory responses in monocytes and macrophages, and release of LPS from the microbial surface has been suggested to be an important initiating event in the sepsis syndrome. However, numerous studies have documented that a variety of constituents present in the outer cell membrane of gram-negative bacteria have the capacity to activate cells of the immune system. Given that the majority of immunotherapeutic approaches designed to intervene in gram-negative sepsis have to date targeted the LPS molecule, it would be of value to assess the relative proinflammatory properties of LPS and other gram-negative structures. Experiments were therefore undertaken to assess stimulation of human monocytes by components released from Escherichia coli following bacteriolysis by the cell wall-active antibiotic ceftazidime. As assessed by both induction of procoagulant activity and release of tumor necrosis factor, bacterial culture supernatants contain significant proinflammatory activity. When culture supernatants are fractionated via either velocity sedimentation in sucrose gradients or isopycnic density gradient ultracentrifugation in cesium chloride, the predominant monocyte-stimulating activity is identified in LPS-containing fractions. Further, such activity can be readily abrogated by the addition of polymyxin B. These results provide support for the hypothesis that LPS may be responsible for the majority of the proinflammatory activity released from E. coli following bacteriolysis in vitro.

A protective effect of glutathione-dextran macromolecular conjugates on acetaminophen-induced hepatotoxicity dependent on molecular size.

Glutathion (GSH) was covalently attached to dextrans with various molecular weights of 2, 5, 10, 40, and 70 kDa by the cyanogen bromide activation method. The conjugates obtained synthetically were white or pale yellowish powders containing 6-10% (w/w) of GSH. The average molecular weights of the conjugates were estimated to be larger and the molecular weight distribution was a little broader than that of each original dextran. The conjugates significantly stabilized GSH and liberated it gradually under physiological conditions (t1/2 = 0.99-1.6h). Mice depleted of GSH by treatment with buthionine sulfoximine, a potent inhibitor of gamma-glutamylcysteine synthetase, exhibited a significant increase in hepatic GSH level after intravenous injection of the conjugates. In mice given a hepatotoxic dose of acetaminophen, the survival rate increased progressively with coadministration of the conjugates, whereas a small improvement was found when free GSH was given. The conjugate of GSH attached to dextran with the molecular weight of 40 kDa exhibited the highest prophylactic effect on acetaminophen-induced hepatotoxicity in mice. The prolonged retention of the conjugates of larger molecular weight in the circulation would cause a higher hepatic accumulation. These results suggested that molecular size would be the most critical factor in the delivery of GSH, as a dextran conjugate, into the liver.

[Clinical evaluation of stent placement for tracheal and bronchial stenosis].

Expandable metallic stents were successfully introduced in 7 patients, including 4 with left main bronchial stenosis caused by bronchopulmonary tuberculosis, 2 with main bronchial stenosis caused by lung cancer and one with tracheal stenosis caused by adenoid cystic carcinoma. The length of stenosis was 1.5-5 cm. The stents were 1.5-2.5 cm long with barbs, and their full expanded diameter was 1.5 cm. Balloon dilatation was performed before stenting in all cases. The stents were inserted by using a 10-12 Fr catheter. In all patients except the one with tracheal stenosis, stents were introduced under local anesthesia without any difficulties. No migration of stents occurred. After stent placement, there were no respiratory difficulties, and radionuclide lung perfusion scan and chest radiographic findings such as lung atelectasis showed marked improvement in three cases. Combined therapy of stent placement and bronchial arterial infusion chemotherapy showed marked effectiveness in one case with lung cancer. Expandable metallic stents were very useful in eliminating tracheobronchial stenosis symptoms.

Intrahepatic delivery of glutathione by conjugation to dextran.

Glutathione was covalently attached to dextran (T-40) by the CNBr activation method. The compound obtained was a water-soluble powder containing 10 (w/w%) glutathione, which was gradually released from the conjugate in aqueous media. Mice depleted of glutathione by treatment with buthionine sulfoximine, a potent inhibitor of gamma-glutamylcysteine synthetase, exhibited a significant increase in hepatic glutathione level after intravenous injection of the conjugate. In mice given a lethal dose of acetaminophen, the survival rate increased progressively with coadministration of the conjugate, whereas little improvement was found when free glutathione was given. The conjugate maintained the serum transaminase activities at lower level after acetaminophen administration. These findings suggest that the dextran conjugate of glutathione is transported into hepatic cells and is intracellularly hydrolyzed to free form, which protects mice from hepatotoxicity due to acetaminophen.

Dose escalation study of recombinant human granulocyte-colony-stimulating factor (KRN8601) in patients with advanced malignancy.

To evaluate the toxicity and efficacy of recombinant human granulocyte-colony-stimulating factor (rh G-CSF) administered with intensive chemotherapy, 39 patients with advanced pulmonary cancers were enrolled in a dose escalation trial of rh G-CSF. Three days after initiation of chemotherapy rh G-CSF was administered i.v. for 14 consecutive days at five dose levels (50-800 micrograms/m2). Absolute neutrophil counts showed a dose-dependent increase with an increasing dose of rh G-CSF and the durations of neutropenia (less than 1000/mm3) shortened significantly at doses of 200, 400, and 800 micrograms/m2 compared to those at 50 micrograms/m2 (P less than 0.01). The duration of neutropenia was shortened significantly at all five dose levels following treatment with rh G-CSF compared to treatment without rh G-CSF (P less than 0.05). Adverse side effects associated with rh G-CSF administration were fever higher than 38 degrees C (21%), chest pain, and low back pain (13%). No intolerable side effects were experienced. It can be concluded that rh G-CSF is effective in shortening the duration of neutropenia following intensive chemotherapy at a dose level of 100 to 200 micrograms/m2 i.v. a 400-micrograms/m2 dose of rh G-CSF is recommended in patients with prior treatment because of the possibility of a lower bone marrow response.

Effects of glutathione, as the dextran conjugate, on acetaminophen-induced hepatotoxicity.

Glutathione was covalently attached to dextran (T-40) by the CNBr activation method. In mice given a lethal dose of acetaminophen, the 30-d survival rate increased progressively with coadministration of the conjugate, whereas little improvement was found when free glutathione was given. The dextran conjugate of glutathione maintained the serum transaminase activities at lower levels after acetaminophen administration, giving effective protection against acetaminophen hepatotoxicity.