In vitro delivery of dexamethasone using hydroxyapatite reservoirs.

Dexamethasone has been shown to stimulate bone nodule formation in vitro. A hydroxyapatite (HA) reservoir drug release device was designed to release dexamethasone in vitro. Two HA particle sizes (< 38 microns or 63-75 microns) were used to fabricate the reservoirs. Each HA reservoir was loaded with 2 mg of dexamethasone and suspended in 100 ml of 50% aqueous ethanol at 37 degrees C for a period of 28 days. The positive controls indicated a limited solubility of dexamethasone of 1.18 mg per 100 ml of 50% aqueous ethanol. Dexamethasone was not released from any of the HA reservoirs for the first 24 hours. The largest amount of dexamethasone (0.0137 microgram/microliter) was released from the 63-75 microns particle HA reservoirs. A significantly lesser amount (0.00855 microgram/microliter) of dexamethasone was released from the < 38 microns particle HA reservoirs. The results of this study suggest that a HA ceramic reservoir loaded with dexamethasone can be used to deliver dexamethasone over long periods of time.

Establishment of a shear stress protocol to study the mechanosensitivity of human primary osteogenic cells in vitro.

We report here the establishment of a protocol to study the influence of mechanical stress on the behavior of primary human osteogenic cells. We first developed a method for the specific isolation of human bone marrow osteoprogenitors and studied their potential of differentiation into osteoblasts in vitro. Then, using NIH-3T3 cells as a model for cells of mesenchymal origin, we employed a parallel plate flow chamber apparatus to apply shear stress to cells in culture. This technique will be useful to determine the influence of shear stress on the rate of proliferation and differentiation of human osteoprogenitors and osteoblasts. Ultimately, this line of research will extend our knowledge on osteogenesis and bone remodeling.

Transcriptional pathways induced by fluid shear stress in mouse preosteoblast cells.

Recent findings support the idea that fluid movement in the bone is a key factor in bone formation and related cellular responses. However, the precise molecular mechanisms are not known. Our data demonstrates that fluid shear stress is a key factor in the activation of specific transcriptional pathways in murine preosteoblast (MC3T3E1) cells. MC3T3E1 cells have increased expression of the egr-1 and p57kip2 genes after being subjected to 0.3 dynes/cm2 of fluid shear stress. The MC3T3E1 cells are already known to express egr-1, a multipotent transcription factor, after high levels of fluid shear stress, but this is the first demonstration of egr-1 expression after low levels of fluid shear stress. Moreover, this is the first study showing expression of p57kip2 by the MC3T3E1 cells after fluid shear stress. The expression of p57kip2, a cyclin dependent kinase inhibitor, is a strong indicator that the cells are exiting the cell cycle and are beginning to differentiate. Our data shows decreased 3H-thymidine incorporation up to 18 hours post stress, which agrees with the upregulation of p57kip2 as a result of fluid shear stress.

Immunomagnetic isolation of osteoprogenitors from human bone marrow stroma.

We report here the efficient and specific isolation of human bone marrow osteoprogenitors using magnetic beads coated with a mouse mAb (STRO-1) that recognizes a stromal cell surface protein. According to their pattern of differentiation, osteoprogenitors accounted for 100% of the STRO-1-positive cell population. Upon long term culture, osteoprogenitors differentiated down the osteoblastic lineage as evidenced by their in vitro morphology, their increased expression of alkaline phosphatase, their production of osteocalcin, and the deposition of a mineralized matrix. Upon differentiation, the cells first expressed alkaline phosphatase on their surface before they began proliferating as phenotypically recognizable osteoblasts. This observation conflicts with previous studies that have characterized human osteoprogenitors as highly proliferative cells that form colonies before differentiating into osteoblasts. The ability to isolate and cultivate pure populations of primary human osteoprogenitors will substantially advance investigations on osteogenesis and bone remodeling.

Preparation and characterization of ZCAP blends.

Six different blends of zinc oxide, calcium oxide, phosphorous pentoxide (ZCAP) were prepared by mixing zinc oxide (ZnO), calcium oxide (CaO), and phosphorous pentoxide (P2O5) powders. The blends were 50:30:20, 48:32:20, 44:26:30, 40:40:20, 30:40:30, and 30:30:40, ZnO:CaO:P2O5 by weight, respectively. The mixed powders were calcined at 800 degrees C for 12 hours. Each blend was then characterized using X-ray diffraction. The X-ray diffraction patterns indicated that in some cases the reaction between oxides may not have gone to completion. Compositions of beta-3CaO.P2O5 and alpha-CaZn2(PO4)2 were found in many of the blends.

A ceramic system for delivering interleukin-2.

Interleukin-2 (IL-2) is a potentially effective cytokine to be used for cancer treatment. Since keeping IL-2 doses at a low, continuous level is important to avoid side effects which accompany high IL-2 doses, a ceramic which release IL-2 could prove to be a beneficial method of drug delivery for cancer patients [1]. In vitro studies were performed to determine the optimal hydroxyapatite (HA) to IL-2 ratio for continuous release of IL-2 from HA using both a matrix and insert ceramic pellet. In the matrix pellet experiment, HA to IL-2 ratios of 25:1, 50:1 and 100:1 were tested by compressing a homogenous mixture of each variable in triplicate and placing each ceramic in 2.5 mL simulated body fluid (SBF) at 37 degrees C. The fluid was collected and replaced for each ceramic every two hours for 12 hours and at 24 hours. The collected fluid was then assayed for IL-2 content. Pellets consisting of 25:1 ceramic to drug ratio delivered almost the entire amount of IL-2 at the fastest rate. Composites of 100:1 IL-2 to HA delivered the least amount of IL-2 at the slowest rate. Release of IL-2 from the 50:1 ratio pellets was intermediate with respect to speed and amount of delivery. In the insert pellet experiment, similar laboratory procedures were used to show that 100:1 ratio insert pellets delivered IL-2 at a slower rate and in lesser amounts than 50:1 ratio insert pellets. Results of this study show that HA can deliver IL-2 at different rates by varying the ratio of HA to IL-2 in both matrix and insert ceramic pellets.

Parameters of protein delivery using hydroxyapatite in simulated body fluid.

Various studies have been conducted using hydroxyapatite (HA) to deliver therapeutic drugs over a long period of time. However, the rate of drug release from ceramics varies tremendously. Thus a study was designed to observe the effect of particle size, pressure, drug ratio, and the addition of a zinc stearate binder on the release of BSA from ceramics. Samples were collected every two hours for a 12 hour period. Three particle sizes were used in the study (< 38, 45-63, and 63-75 microns). Variations in particle size did not influence the release of BSA. Ceramics compressed at a pressure of 150 Mpa delivered more protein than pressures of 300 MPa, 450 MPa, and 900 MPa. Drug to ceramic ratio had the most significant effect. A ratio of 1:25 BSA to HA delivered the protein quickly whereas the 1:100 BSA to HA delivered BSA to HA delivered BSA slowly and in zero order kinetics. The addition of the zinc binder improved the quality of the composite and decreased the release rate of protein delivery when present in 5% or less of the total ceramic weight. HA ceramics can be used to deliver proteins at different rates by varying compression pressure and drug to HA ratio.

In vitro characterization of a zinc based bioceramic.

Six different blends of zinc calcium phosphorous oxide ceramics (ZCAP) were prepared by mixing and calcining powders (ZnO:CaO:P2O5) of weight percent ratio: 50:30:20, 48:32:20, 44:20:26, 40:40:20, 30:40:30, and 30:30:40. ZCAP is a resorbable bioceramic and has been used to repair bone defects and deliver drugs in a continuous manner. The chemical composition, porosity, and elements released on exposure to buffered Tris HCl were measured for each blend of ZCAP. The products of mixing and thermal reaction were beta-Ca3(PO4)2, alpha-CaZn2(PO4)2, and 2CaO.P2O5. Free calcium and/or zinc oxide was present in several blends. The components of ZCAP and their volume percentages influenced the interconnected porosity of ZCAP bioceramics. The interconnected porosity for all blends of ZCAP ranged from 35 to 38%. Pore sizes from these six blends of ZCAP ranged from less 1 micron to greater than 100 microns. Results of the 12 hour dissolution study showed that more calcium was released than zinc or phosphorous from all blends of ZCAP. Zinc was released in trace amounts from all blends of ZCAP. Release of phosphorous from the different blends of ZCAP was not detected by the procedures used to detect phosphorous in this investigation. These blends of ZCAP have the potential to be used as bone substitutes and probably long term treatment of zinc deficiency in humans.

Hydroxyapatite-oil composites for delivering AZT in simulated body fluid.

Hydroxyapatite (HA) has been used to deliver therapeutic drugs both in vitro and in vivo at a constant rate showing zero order kinetics. This study was designed specifically to analyze the effects of wheat germ (WG) incorporation with HA on the rate of delivery of AZT from an insert system over a one month period in vitro. Insert systems which were saturated with wheat germ oil delivered AZT at a slower rate over the one month period than did half-saturated or unsaturated insert systems. All systems containing 50 mg of AZT in the outer shell delivered 80% of the 100 mg AZT dosage over the first eight days. The systems which had a 100 mg AZT insert surrounded by an oily HA shell lacking AZT delivered AZT in a linear manner over the course of one month. The amounts and rates of AZT release from composites was indirectly proportional to the amount of wheat germ oil used. The results of this study show that the lipids incorporated in the ceramic composites can be tailored to deliver a 100 mg AZT dosage for a period of one month in vitro.