Bone bank service in Odense, Denmark. Audit of the first ten years with bone banking at the Department of Orthopaedics, Odense University Hospital

There has been an increase in the demand for allograft bone in recentyears. The Odense University Hospital bone bank has been in function since1990,and this paper outlines our results during the 10 year period 1990–1999.Potential donors were screened by contemporary banking techniques which includea social history, donor serum tests for HIV, hepatitis B and C, and graftmicrobiology. The bones were stored at –80 °C. No typeofsecondary sterilisation was made. 423 femoral heads were approved and donatedto300 patients,1–6 heads/operation. The allografts have been used mainly toreconstruct defects at revision hip arthroplasty (34%), and for fracturesurgery(24%). 7 % of all transplanted patients were reoperated because of infection.Inthe hip revision group the infection rate was 4 %. There were no cases ofdisease transmission. During the 10 year period there was a change in theclinical use of the allografts. In the first years the allografts were mainlyused for spinal fusion surgery, but today the majority are used in hip revisionand fracture surgery. The clinical results correspond to those reported inlarger international series.     

Validation of Radiation Dose Received by Frozen Unprocessed and Processed Bone during Terminal Sterilisation

Fresh frozen femoral heads (FH) and frozen processed bone (FP) are widely used as a source of allograft bone. The FP bone and some of the FH are terminally sterilised by the National Blood Service Tissue Services (NSBTS), via application of a minimum 25 kGy gamma radiation dose. To comply with the Guidelines for the Blood Transfusion Services in the United Kingdom (2002), frozen musculoskeletal tissue must be maintained below −40 °C during storage and transit. In practice, NBSTS stores bone long-term in −80 °C freezers. During transport for irradiation, a temperature of circa −79 °C is maintained by packing the bone in dry ice. An evaluation of the radiation dose received by bone has previously been made via dosimeters located within the tissue and dry ice, however, some evidence suggests that low temperature can influence the accuracy of the dosimeter readings. The aim of this study was to determine the actual radiation dose received by FH and FP bone during the irradiation process. This was accomplished by comparing radiation dose readings from dosimeters placed in dry ice with dosimeters placed in a dry ice substitute of similar dimensions and density i.e., polytetrafluoroethylene (PTFE) at ambient temperature. New packing formats were developed for both FH and FP bone such that 15 FH or 3 kg of FP bone could be irradiated in one transport box at any given time in a standardised fashion. The data show that low temperature consistently increased dosimeter readings 10–27%, and that radiation dose always fell within the range of 25–40 kGy (FH = 25.1–35.7 kGy; FP bone = 25.2–32.4 kGy).     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8–12 h at –50 to –40°C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45°C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4°C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.     

A simple and efficient procedure for cryopreservation of embryogenic cells of aromatic Indica rice varieties

Embryogenic suspension cells of two commercially cultivated aromatic Indica rice varieties, Basmati 385 and Pusa Basmati 1, were cryopreserved using a simple one-step freezing procedure that does not require a controlled-rate freezer. The procedure involves osmotic pre-conditioning of cells with mannitol, addition of a cryoprotectant solution consisting of sucrose, dimethyl sulfoxide, glycerol, proline, and modified R₂ medium, cooling to –25°C for 2 h in a freezer, and then storage in liquid nitrogen. After rapid thawing at 45°C, these cultures showed post-thaw cell viability of 5.6 to 10.5% and formed actively dividing, readyto-use cell suspensions in 20–35 d when cultured directly into liquid medium. Plants were regenerated from cell clumps as well as from colonies formed by protoplasts that were isolated from suspension cells re-established from cryopreserved cells, with frequencies higher (54–98%) than, or comparable to, those obtained from three to four-month-old original non-frozen cell cultures. Cell viability and regeneration frequencies of post-thawed Pusa Basmati 1 cultures were similar to those obtained from the suspension cells cryopreserved using the conventional slow-freezing procedure which involves pre-freezing cells to –40°C at the rate of –0.2°C per min prior to immersion in liquid nitrogen. In Basmati 385, however, cells frozen at ––25°C showed lower post-thaw cell viability than those preserved using the slow-freezing procedure, but these cells produced cell suspensions that had greater shoot morphogenetic potential. The study indicates the beneficial effect of this simple freezing procedure, not only for preserving desirable cultured cells but also for an enrichment of embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulfoxide - LN liquid nitrogen - MS Murashige and Skoog (1962) medium - NAA -napthaleneacetic acid - pcv packed cell volume - TTC 2,3,5-triphenyltetrazolium chloride     

Intracellular serine proteinase behaves as a heat-stress protein in nongrowing but as a cold-stress protein in growing populations of Bacillus megaterium

A temperature increase from 35° to 40–42°C enhances the rise of cytoplasmic serine proteinase (ISP1) activity in Bacillus megaterium incubated in a sporulation medium. A temperature shift from 27°C in the growth medium to 35°C in the sporulation medium has the same effect. Elevated temperature stimulates the increase of ISP1 level when applied immediately after the transfer of cells from the growth to the sporulation medium (at T₀) or at T₃, when sporulation becomes irreversible. The cytoplasmic PMSF-resistant activity or the proteolytic activity associated with the membrane fraction is stimulated only slightly or not at all. A temperature increase to 45–47°C suppresses the rise of proteolytic activities in all cell fractions. In addition to the elevation of the ISP1 activity by an upward temperature shift, the rise of this enzyme in nongrowing cells is also stimulated by osmotic stress. In growing populations, in contrast to the rise of the ISP1 activity caused by elevated temperature in nongrowing cells, this proteinase is induced by low temperatures (24–27°C). The ISP1 activity roughly correlates with the enzyme protein concentration determined by immunoblotting.     

A simple method forin situ freezing of anchorage-dependent cells including rat liver parenchymal cells

We developed a simple method for freezing anchorage-dependent cells, including primary cultured rat liver parenchymal cells, without detaching the cells from the culture dish. The method consists of preculture of the cells to confluence, changing the growth medium to a conventional freezing medium, packaging in a container, and storage at –80°C. After thawing and changing the freezing medium to regular growth medium, cell growth was nearly identical to that of cells freshly seeded into a new dish.     

Temperature and Abscisic Acid Can Be Used to Regulate Survival,Growth, and Differentiation of Cultured Guard Cell Protoplasts of Tree Tobacco

Guard cell protoplasts isolated from leaves of Nicotiana glauca (Graham) were cultured. Conditions were sought that would maximize survival and maintain cells in their differentiated state. Temperature was an important determinant of survival, growth, and differentiation. As temperatures were increased from 24 to 32[deg]C, survival for 1 week in culture increased from approximately 20% to approximately 80% of cells used to initiate cultures. At all of these temperatures, approximately 90% of surviving cells divided to form callus tissue. "Footprint" areas of cells cultured for 1 week at 32[deg]C increased almost 30-fold. Cells cultured for 1 week at 34 to 40[deg]C also survived in high percentages (approximately 80%), but they retained a morphology similar to that of guard cells and they did not divide. Footprint areas of cells cultured for 1 week at 38[deg]C increased 6-fold. Cells cultured at 36 to 40[deg]C in media containing 0.1 or 1.0 [mu]M abscisic acid survived in high percentages and did not divide. At 38[deg]C their footprint areas did not increase, but cells so cultured increased in diameter when treated with fusicoccin. Morphologies and electrophoretic profiles of total sodium dodecyl sulfate-extractable proteins suggest that cells cultured at 38[deg]C in media containing abscisic acid remain differentiated. L-[alpha]-(2-Aminoethoxyvinyl)-glycine reduced survival to <1% at 26 or 32[deg]C but had no effect at 38[deg]C. At lower temperatures, cell growth and survival appear to be ethylene dependent.     

Preservation of microplate-attached human hepatoma cells and their use in cytotoxicity tests

We investigated the feasibility of hypothermic- orcryogenically-preserved human hepatoma Hep G2 cell preculturedin 96-well plates in cytotoxicity testings. First, we observedthat microplates precoated with both collagen (CN) and pronectin (PN) showed significantly improved living cell adhesion (71.0 ± 5.5%) after 48 hr of cryopreservation with 10%-DMSO containing culture medium, whereas non-coated surfaces gave very low living cell adhesion (33.5 ± 2.1%). Hypothermic preservation was most suitable for short-term storage, and cryogenic preservation at –20 °C allowed cells to be used within a week of the storage period. Only cryopreservation in a deep freezer (–85 °C) gave satisfactory results in much longer period of storage. Second, we evaluated the cytotoxicity of ten chemicals during 48 hr of exposure using hypothermically – (4 °C for 2 days) or cryogenically – (–85 °C for 7 days) preserved cells cultured inCN/PN-precoated microplates in comparison with results fromfreshly inoculated cells. Although almost the same LD₅₀values were obtained, LD₁₀ values of relatively hydrophilic chemicals obtained with cryopreserved cell were significantly lowered. These results shown that CN/PN-precoating is effective in keeping cells attached even in recultivation of preserved cells and that the toxicities of relatively hydrophilic chemicals tend to be overestimated when we use preserved cells in that manner.     

Freeze-preservation of buds from Scots pine trees

A procedure has been developed for freeze-preservation of buds of the Scots pine (Pinus sylvestris L.). Instead of liquid nitrogen, cold storage in –80°C was used. The partly dormant material used in the experiments was obtained directly from a natural stand in Northern Finland and no prefreezing or cryoprotectants for preconditioning were used. Cooling velocity was 1°C/min up to a terminal freezing temperature of –39°C, after which the buds were immersed in liquid nitrogen at –196°C for 10 minutes. The material was then transferred to a deepfreezer at –80°C and stored up to 6 months. After rapid thawing, the buds were sterilized and their viability was tested by FDA staining and by culturing meristems on 1/2 MS medium for at least two weeks. All the freezing experiments were performed during March and April. The best survival of buds (90–100%) was achieved at the beginning of April, after which a pronounced decline in survival occurred obviously due to a rise in the water content of the buds.     

Vorläufige Ergebnisse von Untersuchungen an eingefrorenen und bei tiefen Temperaturen bestrahlten Lymphozyten

Zusammenfassung Lymphozytenkulturen werden nach Zugabe von Glyzerin als Gefrierschutzsubstanz zunächst gemeinsam mit ungefähr 1 °C/min Abkühlgeschwindigkeit bis zum vollständigen Erstarren eingefroren und dann mit verschiedener Geschwindigkeit ( 200 °C pro min und 3 bis 5 °C/min) auf verschieden tiefe Temperaturen (–22, –80 und –196 °C) gebracht. Alle eingefrorenen Proben weisen nach 24 h Kulturdauer eine kleinere, nach 48 h Kulturdauer eine wesentlich höhere Mitoserate als die Kontrollgruppe (unbehandelte, nicht eingefrorene Plasmaproben) auf. In den schnell auf –196 °C abgekühlten Proben wurden in beiden Fällen (24 h und 48 h Kulturdauer) keine Metaphaseplatten gefunden. Die Zellkonzentrationen waren nur bei den schnell auf –196 °C abgekühlten Kulturen stark verringert. Die Chromosomenaberrationsrate der eingefrorenen Kulturen ist nicht signifikant erhöht.

---

Preliminary results of investigations of lymphocytes, frozen and irradiated at low temperaturesI. The effects of cooling rate, low temperature, and incubation time on frozen lymphocytes

Summary Peripheral human lymphocytes were cooled to the temperature of solidification at a rate of less than 1 °C/min using glycerol as a protective agent against the effects of freezing. After solidification at temperatures of –10 to –15 °C and cooling to about –22 °C one group of the samples was thawn and the others were cooled rapidly to –80 or –196 °C by immersion into solid carbon dioxide or liquid nitrogen. Other samples of the frozen cell suspension were cooled down to the same temperatures much slower at a cooling rate of 3 to 5 °C/min. After rapid thawing in a 25 °C water bath the cell suspensions were removed from glycerol and cultured for 24 or 48 h before stopping mitoses by adding colchicine. The samples frozen at –22 °C, the samples cooled both rapidly and slowly to –79 °C, and the ones cooled slowly to –196 °C showed a lower rate of mitosis when colchicine was added after 24 h and a significantly higher rate of mitosis after 48 h of incubation before adding colchicine as compared to the controls (untreated, unfrozen plasma). In the culture frozen rapidly to –196 °C no metaphases could be found. The cell concentrations before and after freezing showed no significant differences except those of the culture frozen rapidly to –196 °C. The chromosome aberration rate is not significantly increased.

---

---

Herrn Prof. Dr. H. A. Künkel zum 60. Geburtstag gewidmet.     

Cheese whey: an alternative growth and protective medium for<Emphasis Type="Italic"> Rhizobium loti</Emphasis> cells

Cheese whey (CW)-based growth medium efficiently protects Rhizobium loti cells during freezing and desiccation and can maintain their growth in a manner similar to that of traditional mannitol-based medium (YEM). The cheese-whey-based medium (CW) improved viability when used to re-suspend cell pellets kept at –20 °C and –80 °C and resulted in the survival of over 90% of the cells. Moreover, bacterial pellets obtained from cells grown in CW withstand desiccation better than cells grown in YEM. Survival was over 60% after 30 days at 4 °C. No differences were observed in nodulation efficiency between YEM-grown and CW-grown cells. Fast protein liquid chromatography (FPLC) protocols are presented for total protein profile analyses of sweet and acid cheese whey.In memoriam of Sylvio Cortina Vicepresident of Fundación COREPRO     

Oligosaccharides extracted from cell walls of Kluyveromyces marxianus grown on whey

After whey fermentation by Kluyveromyces marxianus var. marxianus (30°C, pH 4.5, 24 h) and autolysis of the cells (50°C, pH 6.5, 12 h), the subsequent extracts were centrifuged (10,000 × g, 4°C, 15 min), and the cell walls were separated from the autolysates. Cell walls were then treated with: (i) 0.75M NaOH (75°C, 20 h) ; or (ii) lytic enzymes, 0.0025–5.0% (w/v), in 5 mM phosphate buffer (pH 6.5–7.0) (40°C, 24 h). The lytic enzymes were denaturated (80°C, 15 min), and the alkali solutions were neutralized with 0.5M acetic acid, before centrifugation. The supernatants were concentrated by a Speed-Vac concentrator, and analyzed by HPLC, equipped with a TSK-Amide 80 column (1.0 ml/ min of water/acetonitrile, 35/65 ratio, 60°C, 40 min). Tetrasaccharides were detected. Gels were formed when cell walls were treated with NaOH. © Rapid Science Ltd. 1998     

The biocompatibility of calcium phosphate cements containing alendronate-loaded PLGA microparticles in?vitro

The composite of poly-lactic-co-glycolic acid (PLGA) and calcium phosphate cements (CPC) are currently widely used in bone tissue engineering. However, the properties and biocompatibility of the alendronate-loaded PLGA/CPC (APC) porous scaffolds have not been characterized. APC scaffolds were prepared by a solid/oil/water emulsion solvent evaporation method. The morphology, porosity, and mechanical strength of the scaffolds were characterized. Bone marrow mesenchymal stem cells (BMSCs) from rabbit were cultured, expanded and seeded on the scaffolds, and the cell morphology, adhesion, proliferation, cell cycle and osteogenic differentiation of BMSCs were determined. The results showed that the APC scaffolds had a porosity of 67.43 ± 4.2% and pore size of 213 ± 95 µm. The compressive strength for APC was 5.79 ± 1.21 MPa, which was close to human cancellous bone. The scanning electron microscopy, cell counting kit-8 assay, flow cytometry and ALP activity revealed that the APC scaffolds had osteogenic potential on the BMSCs in vitro and exhibited excellent biocompatibility with engineered bone tissue. APC scaffolds exhibited excellent biocompatibility and osteogenesis potential and can potentially be used for bone tissue engineering.     

Isolated microspore culture of Chinese flowering cabbage (Brassica campestris ssp. parachinensis)

Microspores of several genotypes of Brassica campestris ssp. parachinensis have been cultured in vitro and induced to undergo embryogenesis and plant formation. Conditions favourable for embryogenesis in this species include a bud size of 2–2.9 mm, NLN-13 culture medium (Nitsch and Nitsch 1967; Lichter 1981, 1982; Swanson 1990), and an induction through exposure to 32°C for a period of 48 h. Longer periods of an elevated temperature for induction of embryogenesis resulted in embryo abortion at early developmental stages. With the protocol developed here, microspores of 60–80% of donor plants could be induced to produce embryos, although embryo yields were low, i.e. 2–5 embryos per 10 buds. Some genotypes responded to culture conditions with high numbers of embryo formation (100–150 embryos per 10 buds) but most of these subsequently failed to mature. The pattern of cell division and morphological changes of the microspores in culture were studied using various microscopic techniques.     

Brief History of the Tissue Bank, Charles University Hospital, Hradec Králové, Czech Republic

A brief history of the Tissue Bank (TB) of the University Hospital Hradec Králové, Czech Republic, established by Dr. R. Klen in 1952 is presented. In Dr. Klen's original concept the TB was defined as a department specialised in the harvesting, processing, preservation, storage and distribution of various kinds of tissue for clinical and experimental practice. The first kinds of tissue collected in cadaveric donors were corneas, bone and skin. Xenogeneic cartilage and bone grafts were prepared at the same time. Later, preparation of soft connective tissues and chorion–amnion was introduced. During the first 15 years of activity a total of 11,443 grafts preserved by hypothermy at +4°C or freezing in absence of cryoprotectants (–20°C) were prepared. In the 60's freeze-drying of tissue grafts was introduced and the bank of cryopreserved cell lines was established. In the 80's cryopreservation of haematopoietic progenitor cells for clinical transplantations was started and the spectrum of tissue grafts was enlarged (xenogeneic pericardium and allogeneic specially treated dura mater for neurosurgical operations, pigskin for burn treatment, demineralised bone for parodontology and implantology). In the 90's human keratinocyte culture for treatment of burns and chronic skin defects was started. The human milk bank and organ bank co-operating with the Regional Transplantation Centre are component parts of the TB as well. The TB is an institutional member of the European Association of Tissue Banks and annually delivers approximately 1000 grafts that are used in University and county hospitals as well as in surgeons' private practices. Health insurance companies reimburse all grafts on a non-profit and tax-free basis.     

Outwardly rectifying chloride channels in lymphocytes

Summary Outwardly rectifying Cl^– channels in cultured human Jurkat T-lymphocytes were activated by excising a patch of membrane using the inside-out (i/o) patch-clamp configuration and holding at depolarized voltages for prolonged periods of time (1–6 min at +80 mV, 20°C). The single-channel current at +80 mV was 4.5 ± 0.3 pA and at –80 mV, it was 1.0 ± 0.4 pA. After activation, the probability of being open (P ₀)for the lymphocyte channel was voltage independent. Activation of the Cl^– channel in lymphocytes was temperature dependent. Nineteen percent of i/o recordings from lymphocytes made at 20°C exhibited Cl^– channel activity. In contrast, 49% of recordings made at 30°C showed channel activity. The number of channels in an active patch was not significantly different at the two temperatures. Channel activation in excised, depolarized patches also occurred 20-fold faster at 30°C than at 20°C. There was no marked change in the single-channel conductance at 30°C. Open-channel conductance was blocked by 200 m indanyloxyacetic acid (IAA) or 1 mm SITS when applied to the intracellular side of the patch. The characteristics of this channel are similar to epithelial outwardly rectifying Cl^– channels thought to be involved in fluid secretion     

Expansion of Endothelial Progenitor Cells in High Density Dot Culture of Rat Bone Marrow Cells

In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×10⁵ cells/cm²) by seeding total 9×10⁵ cells into six high density dots or cultured in regular density (1.6×10⁴ cells/cm²) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells.     

An efficient protocol for plant regeneration from protoplasts of the moss <Emphasis Type="Italic">Atrichum undulatum P. Beauv in vitro</Emphasis>

A system for plant regeneration from protoplasts of the moss, Atrichum undulatum (Hedw.) P. Beauv. in vitro, is first reported. Viable protoplasts were isolated at about 9 × 10⁵ protoplasts g⁻¹ fresh weight from 10 to 18 days protonemata. For regeneration of protoplasts, viable protoplasts were cultured in liquid–solid medium containing surface liquid medium MS (0.4 M mannitol) and subnatant solid medium Benecke (0.3 M mannitol) at 20 °C under a 16-h photoperiod white light after 12 h preculture in darkness at 20 °C. The great majority of protoplasts follow a regenerative sequence: formation of asymmetric cells in 2–3 days; division of the asymmetric cells to 2–3 cells in 4–5 days, and further develop to produce a new chloronemal filament in 15 days. Juvenile gametophyte can be visible in 20 days. The plating ratio of cell cluster regenerated from protoplasts reaches up to 45%. Transient expression experiments indicate the electroporation uptake of DNA is possible.     

Optimal conditions for heart cell cryopreservation for transplantation

Cultured myocyte transplantation into an infarcted myocardium has been shown to improve contractile function. Cryopreservation of cultured muscle cells or heart tissue will be important for the technology to be practical. This study, using fetal cardiomyocytes, evaluated the optimal conditions for muscle cell cryopreservation. Study 1: Fetal rat cardiomyocytes were isolated and cultured. The freshly isolated and passage 1, 2, 3 and 4 cells were cryopreserved in a solution containing 70% IMDM, 20% FBS and 10% DMSO and stored in –196°C for 1, 2, 4, 8, 12 and 24 weeks. The cells were thawed and cultured. Cell number and contractility were evaluated at 0, 2, 4, 6, 8 and 10 days of culture. Study 2: Rat myocardium was cryopreserved in sizes of 0.2, 2 and 6 mm³ for 1 week. The tissue was thawed and cells were isolated. Cell growth and contractility were evaluated. (1) Cardiomyocytes grew and contracted after cryopreservation. Storage time did not affect cell survival rate, beating cell numbers and beating rates. Increasing cell passage prior to cryopreservation decreased the percentage of beating cells. (2) Cells isolated from cryopreserved tissue grew in vitro and contracted normally. Cell yield decreased with increased cryopreserved tissue size. Fetal rat cardiomyocytes survived and functioned after in vitro cryopreservation. Viable cells can be isolated from cryopreserved myocardium and cultured. Cryopreservation of small pieces of myocardium is preferred for maximal cell yields.     

Effect of β-alanyl-L-histidinato zinc on differentiation of osteoblastic MC3T3-El cells: Increases in alkaline phosphatase activity and protein concentration

The effect of -alany-L-histidinato zinc (AHZ) on bone cell function was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus AHZ (10^–7–10^–5 M) or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5 M) produced a remarkable increase of alkaline phosphatase activity and protein concentration in osteoblastic cells. Thus increases were seen with the prolonged cultivation (12–21 days). With the culture of 1, 3 and 12 days, the effect of AHZ (10^–6 M) to increase alkaline phosphatase activity and protein concentration was more intensive than the effect of zinc sulfate, (10^–6 M). The AHZ effects were completely abolished by the presence of cycloheximide (10^–6 M), indicating that AHZ stimulates protein synthesis in the cells. The present study suggests that AHZ has a stimulatory effect on cell differentiation, and that this effect is partly involved on protein synthesis in osteoblastic cells.