The evaluation of a biphasic osteochondral implant coupled with an electrospun membrane in a large animal model

Conventional clinical therapies are unable to resolve osteochondral defects adequately, hence tissue engineering solutions are sought to address the challenge. A biphasic implant which was seeded with Mesenchymal Stem Cells (MSC) and coupled with an electrospun membrane was evaluated as an alternative. This dual phase construct comprised of a Polycaprolactone (PCL) cartilage scaffold and a Polycaprolactone - Tri Calcium Phosphate (PCL - TCP) osseous matrix. Autologous MSC was seeded into the entire implant via fibrin and the construct was inserted into critically sized osteochondral defects located at the medial condyle and patellar groove of pigs. The defect was resurfaced with a PCL - collagen electrospun mesh that served as a substitute for periosteal flap in preventing cell leakage. Controls either without implanted MSC or resurfacing membrane were included. After 6 months, cartilaginous repair was observed with a low occurrence of fibrocartilage at the medial condyle. Osteochondral repair was promoted and host cartilage degeneration was arrested as shown by the superior Glycosaminoglycan (GAG) maintenance. This positive morphological outcome was supported by a higher relative Young's modulus which indicated functional cartilage restoration. Bone in growth and remodeling occurred in all groups with a higher degree of mineralization in the experimental group. Tissue repair was compromised in the absence of the implanted cells or the resurfacing membrane. Moreover healing was inferior at the patellar groove as compared to the medial condyle and this was attributed to the native biomechanical features.

A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex

This study describes the design of a biphasic scaffold composed of a Fused Deposition Modeling scaffold (bone compartment) and an electrospun membrane (periodontal compartment) for periodontal regeneration. In order to achieve simultaneous alveolar bone and periodontal ligament regeneration a cell-based strategy was carried out by combining osteoblast culture in the bone compartment and placement of multiple periodontal ligament (PDL) cell sheets on the electrospun membrane. In vitro data showed that the osteoblasts formed mineralized matrix in the bone compartment after 21 days in culture and that the PDL cell sheet harvesting did not induce significant cell death. The cell-seeded biphasic scaffolds were placed onto a dentin block and implanted for 8 weeks in an athymic rat subcutaneous model. The scaffolds were analyzed by μCT, immunohistochemistry and histology. In the bone compartment, a more intense ALP staining was obtained following seeding with osteoblasts, confirming the μCT results which showed higher mineralization density for these scaffolds. A thin mineralized cementum-like tissue was deposited on the dentin surface for the scaffolds incorporating the multiple PDL cell sheets, as observed by H&E and Azan staining. These scaffolds also demonstrated better attachment onto the dentin surface compared to no attachment when no cell sheets were used. In addition, immunohistochemistry revealed the presence of CEMP1 protein at the interface with the dentine. These results demonstrated that the combination of multiple PDL cell sheets and a biphasic scaffold allows the simultaneous delivery of the cells necessary for in vivo regeneration of alveolar bone, periodontal ligament and cementum. © 2012 Elsevier Ltd.

A biphasic response to adenosine in the coronary vasculature of the K+-arrested perfused rat heart

Biphasic vasodilatory responses to adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) were observed in the coronary vasculature of K(+)-arrested perfused rat hearts. Dose-response data for both agonists were best represented by two-site models. For adenosine, two sites with negative log ED50 (pED50) values of 8.1 +/- 0.1 (mean +/- S.E.M) and 5.2 +/- 0.1 were obtained, mediating 31 +/- 2% and 69 +/- 2% of the total response. In the presence of 8-phenyltheophylline, the vasodilatory response to adenosine remained best fitted to a two-site model with pED50 values of 7.0 +/- 0.2 and 5.4 +/- 0.2. The relative contribution of each site to the total response remained unchanged. For NECA, pED50 values of 9.6 +/- 0.1 and 6.8 +/- 0.2 were obtained, representing 48 +/- 3% and 52 +/- 3% of the sites, respectively. In contrast, ATP produced a monophasic response with a pED50 value of 8.8 +/- 0.1. These results provide evidence of adenosine receptor and response heterogeneity in the in situ coronary vasculature.

High-throughput bone and cartilage micropellet manufacture, followed by assembly of micropellets into biphasic osteochondral tissue

Engineered biphasic osteochondral tissues may have utility in cartilage defect repair. As bone-marrow-derived mesenchymal stem/stromal cells (MSC) have the capacity to make both bone-like and cartilage-like tissues, they are an ideal cell population for use in the manufacture of osteochondral tissues. Effective differentiation of MSC to bone-like and cartilage-like tissues requires two unique medium formulations and this presents a challenge both in achieving initial MSC differentiation and in maintaining tissue stability when the unified osteochondral tissue is subsequently cultured in a single medium formulation. In this proof-of-principle study, we used an in-house fabricated microwell platform to manufacture thousands of micropellets formed from 166 MSC each. We then characterized the development of bone-like and cartilage-like tissue formation in the micropellets maintained for 8–14 days in sequential combinations of osteogenic or chondrogenic induction medium. When bone-like or cartilage-like micropellets were induced for only 8 days, they displayed significant phenotypic changes when the osteogenic or chondrogenic induction medium, respectively, was swapped. Based on these data, we developed an extended 14-day protocol for the pre-culture of bone-like and cartilage-like micropellets in their respective induction medium. Unified osteochondral tissues were formed by layering 12,000 osteogenic micropellets and 12,000 chondrogenic micropellets into a biphasic structure and then further culture in chondrogenic induction medium. The assembled tissue was cultured for a further 8 days and characterized via histology. The micropellets had amalgamated into a continuous structure with distinctive bone-like and cartilage-like regions. This proof-of-concept study demonstrates the feasibility of micropellet assembly for the formation of osteochondral-like tissues for possible use in osteochondral defect repair.

The Use of Liquid-Liquid Interface (Biphasic) for the Preparation of Benzenetricarboxylate Complexes of Cobalt and Nickel

New metal-organic frameworks (MOFs) [Ni(C12N2H10)(H2O)][C6H3(COO)2(COOH)] (I), [Co2(H2O)6][C6H3(COO)3]2·(C4N2H12)(H2O)2 (II), [Ni2(H2O)6][C6H3(COO)3]2·(C4N2H12)(H2O)2 (III), [Ni(C13N2H14)(H2O)][C6H3(COO)2(COOH)] (IV), [Ni3(H2O)8][C6H3(COO)3] (V) and [Co(C4N2H4)(H2O)][C6H3(COO)3] (VI) {C6H3(COOH)3 = trimesic acid, C12N2H10 = 1,10-phenanthroline, C4N2H12 = piperazine dication, C13N2H14 = 1,3-bis(4-pyridyl)propane and C4N2H4 = pyrazine} have been synthesized by using an interface between two immiscible solvents, water and cyclohexanol. The compounds are constructed from the connectivity between the octahedral M2+ (M = Ni, Co) ions coordinated by oxygen atoms of carboxylate groups and water molecules and/or by nitrogen atoms of the ligand amines and the carboxylate units to form a variety of structures of different dimensionality. Strong hydrogen bonds of the type O-H···O are present in all the compounds, which give rise to supramolecularly organized higher-dimensional structures. In some cases ··· interactions are also observed. Magnetic studies indicate weak ferromagnetic interactions in I, IV and V and weak antiferromagnetic interactions in the other compounds (II, III and VI). All the compounds have been characterized by a variety of techniques.

Liquid-Liquid Interphase (Biphasic) as the Reaction Medium in the Assembly of a Hierarchy of Structures of 4,4 `-Azodibenzoic Acid with Zinc and Cadmium

The compounds Zn(C12H8N2)](2)C12N2H8(COO)(2)](2)center dot(C6H12O)center dot(H2O), I, Zn(C12H8N2)]C12N2H8(COO)(2)], II, Cd(C12H8N2)(H2O)]C12N2H8(COO)(2)]center dot(H2O), III, Zn(C10N2H8)]C12N2H8(COO)(2)]center dot 0.5(C10N2H8), IV, Cd(C12N2H8(COO)(2)center dot H2O], V, and Zn-3(mu(2)-O)(mu(3)-O)(3)]C12N2H8(COO)(2)], VI, have been synthesized by using a biphasic approach (I, III, V, VI) or regular hydrothermal method (II, IV). The compounds exhibit one (I and II), two (In), and three dimensionally (IV, V, VI) extended structures. The flexible azodibenzoate ligand gives rise to a 3-fold interpenetration (IV) when the synthesis was carried out using normal hydrothermal methods. The biphasic approach forms structures without any interpenetrations, especially in the three-dimensional structures of V and VI. Formation of Cd2O2 dimers in V and extended M-O(H)-M two-dimensional layers in VI suggests the subtle structural control achieved by the biphasic method. Transformation studies indicate that it is possible to transform I to II. Lewis acid catalytic studies have been performed to evaluate the role of the coordination environment in such reactions. All the compounds have been characterized by a variety of techniques that includes powder X-ray diffraction, infrared, thermogravitric analysis, UV-vis, photoluminescence studies.

Polar and nonpolar solvation dynamics, ion diffusion, and vibrational relaxation: Role of biphasic solvent response in chemical dynamics

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Biphasic composite of Tricalcium phosphate reinforced with Hydroxyapatite Whiskers

Biphasic calcium phosphates have received considerable attention due to their optimum dissolution rate in the human body after implantation. These materials are composed of hydroxyapatite (HA) and resorbable tricalcium phosphate (TCP). In the present investigation, HA whiskers are reinforced into TCP to enhance the mechanical properties of this biphasic composite. Various amounts (30-50 wt%) HA whiskers are reinforced in TCP matrix. Microstructural characterization has been carried out using field-emission scanning electron microscope. Mechanical properties have been investigated by microindentation in a universal testing machine (UTM). As TCP is resorbable, it will dissolve in body fluid and there is a strong possibility for the faceted HA whiskers to interact with functional groups present in the body fluid surroundings.

Biphasic regulation of H2O2 on angiogenesis implicated NADPH oxidase

ROS (reactive oxygen species) take an important signalling role in angiogenesis. Although there are several ways to produce ROS in cells, multicomponent non-phagocytic NADPH oxidase is an important source of ROS that contribute to angiogenesis. In the present work, we examined the effects of H2O2 on angiogenesis including proliferation and migration in HUVECs (human umbilical vein endothelial cells), new vessel formation in chicken embryo CAM (chorioallantoic membrane) and endothelial cell apoptosis, which is closely related to anti-angiogenesis. Our results showed that H2O2 dose-dependently increased the generation of O-2(-) (superoxide anion) in HUVECs, which was suppressed by DPI (diphenylene iodonium) and APO (apocynin), two inhibitors of NADPH oxidase. H2O2 at low concentrations (10 mu M) stimulated cell proliferation and migration, but at higher concentrations, inhibited both. Similarly, H2O2 at 4 nmol/cm(2) strongly induced new vessel formation in CAM, while it suppressed at high concentrations (higher than 4 nmol/cm(2)). Also, H2O2 (200 similar to 500 mu M) could stimulate apoptosis in HUVECs. All the effects of H2O2 on angiogenesis could be suppressed by NADPH oxidase inhibitors, which suggests that NADPH oxidase acts downstream of H2O2 to produce O-2(-) and then to regulate angiogenesis. In summary, our results suggest that H2O2 as well as O-2(-) mediated by NADPH oxidase have biphasic effects on angiogenesis in vitro and in vivo.

Selective hydrogenation of unsaturated aldehydes in a poly(ethylene glycol)/compressed carbon dioxide biphasic system

Hydrogenation of alpha,beta-unsaturated aldehydes (citral, 3-methyl-2-butenal, cinnamaldehyde) has been studied with tetrakis(triphenylphosphine) ruthenium dihydride (H2Ru(TPP)(4)) catalyst in a poly(ethylene glycol) (PEG)/ compressed carbon dioxide biphasic system. The hydrogenation reaction was slow under PEG/ H-2 biphasic conditions at H-2 4 MPa in the absence of CO2. When the reaction mixture was pressurized by a non-reactant of CO2, however, the reaction was significantly accelerated.

Phase Diagram of [Amim]Cl plus Salt Aqueous Biphasic Systems and Its Application for [Amim]Cl Recovery

In this study, binodal curves and tie line data of [Amim]Cl + salt (K3PO4, K2HPO4, K2CO3) + water aqueous biphasic systems (ABS) were measured and correlated satisfactorily with the Merchuk equation and Othmer-Tobias and Bancroft equations, respectively. [Amim]Cl could be recovered from aqueous solutions using the ABS, and the recovery efficiency could reach 96.80%. The recovery efficiency was influenced by the concentrations of the salts and their Homeister series: K3PO4 > K2HPO4 > K2CO3. Our method provides a new and effective route for the recovery of hydrophilic IL using [Amim]Cl + salt + water ABS from aqueous solutions.

Phase diagram data for several salt plus salt aqueous biphasic systems at 298.15 K

Phase diagrams corresponding to aqueous biphasic systems of salt (the organic ionic liquid of salts [C(4)mim]Cl, [C(6)mim]Cl, and [C(8)mim]Cl) + salt (K3PO4, K2CO3) + water were determined at 298.15 K. The binodal curve was fitted to the Merchuk equation. Tie lines assigned from mass phase ratios according to the lever arm rule were satisfactorily described using the Othmer-Tobias and Bancroft equations.

Characteristics of the biphasic action of androgens and of the potent antiproliferative effects of the new pure antiestrogen EM-139 on cell cycle kinetic parameters in LNCaP human prostatic cancer cells

The most potent steroid in human prostatic carcinoma LNCaP cells, i.e. dihydrotestosterone (DHT), has a biphasic stimulatory effect on cell proliferation. At the maximal stimulatory concentration of 0.1 nM DHT, analysis of cell kinetic parameters shows a decrease of the G0-G1 fraction with a corresponding increase of the S and G2 + M fractions. In contrast, concentrations of 1 nM DHT or higher induce a return of cell proliferation to control levels, reflected by an increase in the G0-G1 fraction at the expense of the S and especially the G2 + M fractions. Continuous labeling for 144 h with the nucleotide analogue 5'-bromodeoxyuridine shows that the percentage of cycling LNCaP cells rises more than 90% after treatment with stimulatory concentrations of DHT, whereas in control cells as well as in cells treated with high concentrations of the androgen, this value remains below 50%. Although LNCaP cells do not contain detectable estrogen receptors, the new pure steroidal antiestrogen EM-139 not only reversed the stimulation of cell proliferation and cell kinetics induced by stimulatory doses of DHT but also inhibited basal cell proliferation.