Sequential delivery of BMP-2 and IGF-1 using a chitosan gel with gelatin microspheres enhances early osteoblastic differentiation

The purpose of this study was to develop and characterize a chitosan gel/gelatin microsphere (MSs) dual delivery system for sequential release of bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-1 (IGF-1) to enhance osteoblast differentiation in vitro. We made and characterized the delivery system based on its degree of cross-linking, degradation, and release kinetics. We also evaluated the cytotoxicity of the delivery system and the effect of growth factors on cell response using pre-osteoblast W-20-17 mouse bone marrow stromal cells. IGF-1 was first loaded into MSs, and then the IGF-1-containing MSs were encapsulated into the chitosan gel which contained BMP-2. Cross-linking of gelatin with glyoxal via Schiff bases significantly increased thermal stability and decreased the solubility of the MSs, leading to a significant decrease in the initial release of IGF-1. Encapsulation of the MSs into the chitosan gel generated polyelectrolyte complexes by intermolecular interactions, which further affected the release kinetics of IGF-1. This combinational delivery system provided an initial release of BMP-2 followed by a slow and sustained release of IGF-1. Significantly greater alkaline phosphatase activity was found in W-20-17 cells treated with the sequential delivery system compared with other treatments (P<0.05) after a week of culture.     

Retention of in vitro and in vivo BMP-2 bioactivities in sustained delivery vehicles for bone tissue engineering

In this study, we investigated the in vitro and in vivo biological activities of bone morphogenetic protein 2 (BMP-2) released from four sustained delivery vehicles for bone regeneration. BMP-2 was incorporated into (1) a gelatin hydrogel, (2) poly(lactic-co-glycolic acid) (PLGA) microspheres embedded in a gelatin hydrogel, (3) microspheres embedded in a poly(propylene fumarate) (PPF) scaffold and (4) microspheres embedded in a PPF scaffold surrounded by a gelatin hydrogel. A fraction of the incorporated BMP-2 was radiolabeled with (125)I to determine its in vitro and in vivo release profiles. The release and bioactivity of BMP-2 were tested weekly over a period of 12 weeks in preosteoblast W20-17 cell line culture and in a rat subcutaneous implantation model. Outcome parameters for in vitro and in vivo bioactivities of the released BMP-2 were alkaline phosphatase (AP) induction and bone formation, respectively. The four implant types showed different in vitro release profiles over the 12-week period, which changed significantly upon implantation. The AP induction by BMP-2 released from gelatin implants showed a loss in bioactivity after 6 weeks in culture, while the BMP-2 released from the other implants continued to show bioactivity over the full 12-week period. Micro-CT and histological analysis of the delivery vehicles after 6 weeks of implantation showed significantly more bone in the microsphere/PPF scaffold composites (Implant 3, p<0.02). After 12 weeks, the amount of newly formed bone in the microsphere/PPF scaffolds remained significantly higher than that in the gelatin and microsphere/gelatin hydrogels (p<0.001), however, there was no statistical difference compared to the microsphere/PPF/gelatin composite. Overall, the results from this study show that BMP-2 could be incorporated into various bone tissue engineering composites for sustained release over a prolonged period of time with retention of bioactivity.     

Sustained local delivery of siRNA from an injectable scaffold

Controlled gene silencing technologies have significant, unrealized potential for use in tissue regeneration applications. The design described herein provides a means to package and protect siRNA within pH-responsive, endosomolytic micellar nanoparticles (si-NPs) that can be incorporated into nontoxic, biodegradable, and injectable polyurethane (PUR) tissue scaffolds. The si-NPs were homogeneously incorporated throughout the porous PUR scaffolds, and they were shown to be released via a diffusion-based mechanism for over three weeks. The siRNA-loaded micelles were larger but retained nanoparticulate morphology of approximately 100 nm diameter following incorporation into and release from the scaffolds. PUR scaffold releasate collected in vitro in PBS at 37 °C for 1-4 days was able to achieve dose-dependent siRNA-mediated silencing with approximately 50% silencing achieved of the model gene GAPDH in NIH3T3 mouse fibroblasts. This promising platform technology provides both a research tool capable of probing the effects of local gene silencing and a potentially high-impact therapeutic approach for sustained, local silencing of deleterious genes within tissue defects.     

Design of an injectable system based on bioerodible polyanhydride microspheres for sustained drug delivery

The fabrication, morphological characterization, and drug release kinetics from microspheres of three bioerodible polyanhydrides, poly[1,6-bis(p-carboxyphenoxy)hexane] (poly(CPH)), poly(sebacic anhydride) (poly(SA)), and the copolymer poly(CPH-co-SA) 50:50 (CPH:SA 50:50) is reported. The fabrication technique yields microspheres with different morphologies for each of the three polymers studied, ranging from very smooth exterior surfaces for poly(CPH) to coarse surface roughness with large pores for poly(SA). Release profiles for the model drug, p-nitroaniline are also different for each polymer. The release profile from poly(CPH) has a large initial burst and shows little additional release after 2 days. The release from poly(SA) is nearly zero-order and lasts for about 8 days. The release profile from CPH:SA 50:50 shows a relatively small burst and then exhibits zero-order release for about I month. The different release profiles are attributed to both polymer erosion rates and drug distribution characteristics of the microspheres. Tailored release profiles of a burst followed by zero-order release are obtained by appropriately combining the microspheres. This technique enables independent modulation of both the burst and the zero-order release rate by varying the number of poly(CPH) and poly(SA) microspheres respectively. Additionally, the zero-order release can be extended from about a week to a month by including CPH:SA 50:50 microspheres.     

In vivo evaluation of an oral delivery system for P-gp substrates based on thiolated chitosan

Recently, thiolated polymers, so called thiomers, have been reported to modulate drug absorption by inhibition of intestinal P-glycoprotein (P-gp). The aim of the present study was to provide a proof-of-principle for a delivery system based on thiolated chitosan in vivo in rats, using rhodamine-123 (Rho-123) as representative P-gp substrate. In vitro, the permeation enhancing effect of unmodified chitosan, chitosan-4 thiobutylamidine (Ch-TBA) and the combination of Ch-TBA with reduced glutathione (GSH) was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to buffer only, Rho-123 transport in presence of 0.5% (w/v) chitosan, 0.5% (w/v) Ch-TBA and the combination of 0.5% (w/v) Ch-TBA/0.5% (w/v) GSH, was 1.8-fold, 2.6-fold, 3.8-fold improved, respectively. Furthermore, enteric-coated tablets based on unmodified chitosan or Ch-TBA/GSH, were investigated in vivo. In rats, the Ch-TBA/GSH tablets increased the area under the plasma concentration time curve (AUC0-12) of Rho-123 by 217% in comparison to buffer control and by 58% in comparison to unmodified chitosan. This in vivo study showed that a delivery system based on thiolated chitosan significantly increased the oral bioavailability of P-gp substrate Rho-123.     

Porous membrane with reverse gradients of PDGF-BB and BMP-2 for tendon-to-bone repair: In vitro evaluation on adipose-derived stem cell differentiation

Polycaprolactone (PCL)/Pluronic F127 membrane with reverse gradients of dual platelet-derived growth factor-β (PDGF-BB) and bone morphogenetic protein 2 (BMP-2) concentrations was fabricated using a diffusion method to investigate the effect of reverse gradients of dual growth factor concentrations on adipose-derived stem cell (ASC) differentiations, such as tenogenesis and osteogenesis. The PDGF-BB and BMP-2 were continuously released from the membrane for up to 35 days, with reversely increasing/decreasing growth factors along the membrane length. Human ASCs were seeded on the membrane with reverse PDGF-BB and BMP-2 gradients. The cells were confluent after 1 week of culture, regardless of growth factor types or concentrations on the membrane. Gene expression (real-time polymerase chain reaction), Western blot and immunohistological analyses after 1 and 2 weeks of ASC culture showed that the membrane sections with higher PDGF-BB and lower BMP-2 concentrations provided a better environment for ASC tenogenesis, while the membrane sections with higher BMP-2 and lower PDGF-BB concentrations were better for promoting osteogenesis. The results suggest that the membrane with reverse gradients of PDGF-BB and BMP-2 may be promising for tendon-to-bone repair, as most essential biological processes are mediated by gradients of biological molecules in the body.     

A hybrid thermo-sensitive chitosan gel for sustained release of Meloxicam

The purpose of this work was to develop a multi-phase gel system for sustained release drug delivery. A thermo-sensitive hydrogel composed of chitosan and glycerol was prepared, and then an o/w emulsion was introduced to the thermo-sensitive gel in order to modulate the gelation behavior. Meloxicam was chosen as a model drug in this study and its release profile was investigated. This study revealed that the factors such as pH, chitosan molecular mass and glycerol concentration could significantly influence the gel formation. Chitosan with a molecular mass of 950 kDa and glycerol proportion ranging from 30 to 60% can form a pH-dependent thermo-sensitive gel system. Both the chitosan–glycerol gel and chitosan–glycerol-emulsion gel systems were applied in delivering drugs. The drug release from the two gels was both in Higuchi mode. Higuchi moduli were 3.04 × 10^?3 mg · h^½ in the chitosan–glycerol-emulsion gel and 1.28 mg · h^½ in the chitosan– glycerol gel. The former was significantly slower in sustained release. The in vivo investigation on the chitosan–glycerol gel indicated that the gel may be useful in sustained drug release in situ. Thermosensitive hydrogels composed of chitosan and glycerol were well formed and could act as a sustained release drug carrier in the work, it showed that this hybrid thermo-sensitive hydrogel system may be a promising sustained release drug carrier.     

A hybrid thermo-sensitive chitosan gel for sustained release of Meloxicam

The purpose of this work was to develop a multi-phase gel system for sustained release drug delivery. A thermo-sensitive hydrogel composed of chitosan and glycerol was prepared, and then an o/w emulsion was introduced to the thermo-sensitive gel in order to modulate the gelation behavior. Meloxicam was chosen as a model drug in this study and its release profile was investigated. This study revealed that the factors such as pH, chitosan molecular mass and glycerol concentration could significantly influence the gel formation. Chitosan with a molecular mass of 950 kDa and glycerol proportion ranging from 30 to 60% can form a pH-dependent thermo-sensitive gel system. Both the chitosan-glycerol gel and chitosan-glycerol-emulsion gel systems were applied in delivering drugs. The drug release from the two gels was both in Higuchi mode. Higuchi moduli were 3.04 x 10(-3) mg x h((1/2)) in the chitosan-glycerol-emulsion gel and 1.28 mg x h((1/2)) in the chitosan- glycerol gel. The former was significantly slower in sustained release. The in vivo investigation on the chitosan-glycerol gel indicated that the gel may be useful in sustained drug release in situ. Thermosensitive hydrogels composed of chitosan and glycerol were well formed and could act as a sustained release drug carrier in the work, it showed that this hybrid thermo-sensitive hydrogel system may be a promising sustained release drug carrier.     

In vitro evaluation of electrospun silk fibroin/nano-hydroxyapatite/BMP-2 scaffolds for bone regeneration

Bone tissue engineering by using osteoinductive scaffolds seeded with stem cells to promote bone extracellular matrix (ECM) production and remodeling has evolved into a promising approach for bone repair and regeneration. In order to mimic the ECM of bone tissue structurally and compositionally, nanofibrous silk fibroin (SF) scaffolds containing hydroxyapatite (HAP) nanoparticles and bone morphogenetic protein 2 (BMP-2) were fabricated in this study using electrospinning technique. The microstructure, mechanical property, biocompatibility, and osteogenic characteristics were examined. It was found that the HAP nanoparticles were successfully incorporated in the SF nanofibers (diameter, 200–500 nm). The mechanical properties of SF/HAP/BMP-2 composite scaffolds increased with HAP content when it was less than 20 wt%, after which the mechanical properties dropped as HAP content increased. Cell culture tests using bone marrow mesenchymal stem cells (BMSCs) showed that the scaffolds had good biocompatibility and promoted the osteogenic differentiation of BMSCs. Therefore, the electrospun SF/HAP/BMP-2 scaffolds may serve as a promising biomaterial for bone tissue engineering.     

A novel polymer gel for the delivery of local therapies to intracranial tumors: In vivo safety evaluation

The treatment of intracranial malignancies is limited by the ability of systemically administered therapies to cross the blood-brain barrier. Royer resorbable matrix, or R-Gel, is a dextran polymer administered in liquid form via needle injection. Within minutes of preparation, the polymer forms a gel and subsequently solidifies, thereby conforming to the dimensions of the injection cavity. R-Gel can accommodate a wide variety of therapeutic agents that may provide new options for local treatment delivery. This preclinical study evaluates the neurotoxicity of R-Gel implanted in the rat brain. Fifteen rats underwent intracranial administration of R-Gel (N = 9) or saline (N = 6) were monitored for systemic and neurotoxicity, and sacrificed at pre-determined time points. Animals that received the R-Gel injection demonstrated no behavioral changes or weight loss. Histopathologic analysis revealed an inflammatory response in both groups on day 3 and day 7 after implantation, which resolved by day 42. These results suggest that intracranial R-Gel is well tolerated. Therapeutic studies of chemotherapy-complexed R-Gel are underway.     

In vitro evaluation of a chitosan membrane cross-linked with genipin

The study was to evaluate the characteristics of a chitosan membrane cross-linked with a naturally-occurring cross-linking reagent, genipin. This newly-developed genipin-cross-linked chitosan membrane may be used as an implantable drug-delivery system. The chitosan membrane without cross-linking (fresh) and the glutaraldehyde-cross-linked chitosan membrane were used as controls. The characteristics of test chitosan membranes evaluated were their cross-linking degree, swelling ratio, mechanical properties, antimicrobial activity, cytotoxicity, and degradability. It was found that cross-linking of chitosan membrane using genipin increased its ultimate tensile strength but significantly reduced its strain-at-fracture and swelling ratio. There was no significant difference in antimicrobial activity between the genipin-cross-linked chitosan membrane and its fresh counterpart. Additionally, the results showed that the genipin-cross-linked chitosan membrane had a significantly less cytotoxicity and a slower degradation rate compared to the glutaraldehyde-cross-linked membrane. These results suggested that the genipin-cross-linked chitosan membrane may be a promising carrier for fabricating an implantable drug-delivery system. The drug-release characteristics of the genipin-cross-linked chitosan membrane are currently under investigation.     

In vitro evaluation of a chitosan membrane cross-linked with genipin.

The study was to evaluate the characteristics of a chitosan membrane cross-linked with a naturally-occurring cross-linking reagent, genipin. This newly-developed genipin-cross-linked chitosan membrane may be used as an implantable drug-delivery system. The chitosan membrane without cross-linking (fresh) and the glutaraldehyde-cross-linked chitosan membrane were used as controls. The characteristics of test chitosan membranes evaluated were their cross-linking degree, swelling ratio, mechanical properties. antimicrobial activity, cytotoxicity, and degradability. It was found that cross-linking of chitosan membrane using genipin increased its ultimate tensile strength but significantly reduced its strain-at-fracture and swelling ratio. There was no significant difference in antimicrobial activity between the genipin-cross-linked chitosan membrane and its fresh counterpart. Additionally, the results showed that the genipin-cross-linked chitosan membrane had a significantly less cytotoxicity and a slower degradation rate compared to the glutaraldehyde-cross-linked membrane. These results suggested that the genipin-cross-linked chitosan membrane may be a promising carrier for fabricating an implantable drug-delivery system. The drug-release characteristics of the genipin-cross-linked chitosan membrane are currently under investigation.     

pH-triggered injectable hydrogels prepared from aqueous N-palmitoyl chitosan: In vitro characteristics and in vivo biocompatibility

In-situ forming hydrogels triggered by environmental stimuli have emerged as a promising injectable strategy targeted for various biomedical applications. However, several drawbacks associated with temperature-stimulated hydrogels have been reported. Employing a hydrophobically-modified chitosan (N-palmitoyl chitosan, NPCS), we developed a pH-triggered hydrogel system which showed a rapid nanostructure transformation within a narrow pH range (pH 6.5–7.0). NPCS in an aqueous environment was found to be a shear-thinning fluid and exhibited an instant recovery of its elastic properties after shear thinning, thereby making it an injectable material. Additionally, aqueous NPCS, an associating polyelectrolyte, can be rapidly transformed into hydrogel triggered simply by its environmental pH through a proper balance between charge repulsion and hydrophobic interaction. This in-situ hydrogel system was shown to be nontoxic. Subcutaneous injection of aqueous NPCS (pH 6.5) into a rat model resulted in rapid formation of a massive hydrogel at the location of injection. The implanted hydrogel was found to be degradable and was associated with an initial macrophage response which decreased with time as the degradation proceeded. These results suggested that the developed NPCS hydrogel may be used as an injectable drug/cell delivery system.     

Bioactive porous beads as an injectable urethral bulking agent: their in vitro evaluation on smooth muscle cell differentiation

Growth factor (basic fibroblast growth factor or vascular endothelial growth factor)-immobilized polycaprolactone (PCL)/Pluronic F127 porous beads were prepared as an injectable bulking agent for effective treatment of urinary incontinence. The growth factor-immobilized porous beads may stimulate smooth muscle cell (SMC) differentiation of muscle-derived stem cells or defect tissues around urethra to improve the sphincter function (bioactive therapy) as well as to provide a bulking effect (passive therapy). The porous PCL/F127 beads were fabricated by an isolated particle-melting/melt-molding particulate-leaching method. The growth factors were easily immobilized onto the surfaces of the PCL/F127 porous beads via heparin binding and were continuously released for up to 28 days. Both growth factor-immobilized porous beads had a positive effect for the SMC differentiation of muscle-derived stem cells, as were demonstrated by the analyses of quantitative polymerase chain reactions, Western blot using SMC-specific markers, and immunohistochemical staining. In particular, the basic fibroblast growth factor-immobilized porous beads showed desirable SMC differentiation behavior that can be applied as an injectable bulking agent for the treatment of urinary incontinence.     

BMP-2和FGF-2对小鼠骨髓间充质干细胞向成骨细胞分化的影响

目的:观察骨形成蛋白(BMP-2)和成纤维细胞生长因子2(FGF-2)对小鼠骨髓间充质干细胞(MSCs)向成骨细胞分化的影响.方法:取3～18月雄性C57BL/6J小鼠(共50只)的骨髓细胞, 分离贴壁培养后, 用免疫磁珠法纯化, 并鉴定为MSCs后, 再进行贴壁培养24 h后, 分别在成骨细胞诱导培养液中加入100 μg/L BMP-2和0.5 nmol/L FGF-2持续诱导7、 14、 21 d后, 进行碱性磷酸酶染色、碱性磷酸酶活性检测, Vonkossa染色以及茜素红染色, 并用递转录荧光定量PCR法检测向成骨细胞分化的标志性基因(Runx2/cbfa1、 Alp、 collagen-1、 osteocalcin)的表达情况.结果:BMP-2刺激组的ALP活性以及钙化结节明显高于对照组, Runx2/cbfa1, ALP, collage-1, osteocalcin的mRNA呈高表达; FGF-2刺激组的ALP活性以及钙化结节也高于对照组, Runx2/cbfa1、 Alp、 collagen-1、 osteocalcin的mRNA表达量也高于对照组, 但不如BMP-2明显. 结论:BMP-2和 FGF-2在不同程度上促进体外培养的小鼠MSCs向成骨细胞方向分化.     

The use of injectable sonication-induced silk hydrogel for VEGF(165) and BMP-2 delivery for elevation of the maxillary sinus floor

Sonication-induced silk hydrogels were previously prepared as an injectable bone replacement biomaterial, with a need to improve osteogenic features. Vascular endothelial growth factor (VEGF(165)) and bone morphogenic protein-2 (BMP-2) are key regulators of angiogenesis and osteogenesis, respectively, during bone regeneration. Therefore, the present study aimed at evaluating in situ forming silk hydrogels as a vehicle to encapsulate dual factors for rabbit maxillary sinus floor augmentation. Sonication-induced silk hydrogels were prepared in?vitro and the slow release of VEGF(165) and BMP-2 from these silk gels was evaluated by ELISA. For in?vivo studies for each time point (4 and 12 weeks), 24 sinus floors elevation surgeries were made bilaterally in 12 rabbits for the following four treatment groups: silk gel (group Silk gel), silk gel/VEGF(165) (group VEGF), silk gel/BMP-2 (group BMP-2), silk gel/VEGF(165)/BMP-2 (group V?+?B) (n?=?6 per group). Sequential florescent labeling and radiographic observations were used to record new bone formation and mineralization, along with histological and histomorphometric analysis. At week 4, VEGF(165) promoted more tissue infiltration into the gel and accelerated the degradation of the gel material. At this time point, the bone area in group V?+?B was significantly larger than those in the other three groups. At week 12, elevated sinus floor heights of groups BMP-2 and V?+?B were larger than those of the Silk gel and VEGF groups, and the V?+?B group had the largest new bone area among all groups. In addition, a larger blood vessel area formed in the remaining gel areas in groups VEGF and V?+?B. In conclusion, VEGF(165) and BMP-2 released from injectable and biodegradable silk gels promoted angiogenesis and new bone formation, with the two factors demonstrating an additive effect on bone regeneration. These results indicate that silk hydrogels can be used as an injectable vehicle to deliver multiple growth factors in a minimally invasive approach to regenerate irregular bony cavities.     

Effect of local sequential VEGF and BMP-2 delivery on ectopic and orthotopic bone regeneration

Bone regeneration is a coordinated cascade of events regulated by several cytokines and growth factors. Angiogenic growth factors are predominantly expressed during the early phases for re-establishment of the vascularity, whereas osteogenic growth factors are continuously expressed during bone formation and remodeling. Since vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are key regulators of angiogenesis and osteogenesis during bone regeneration, the aim of this study was to investigate if their sequential release could enhance BMP-2-induced bone formation. A composite consisting of poly(lactic-co-glycolic acid) microspheres loaded with BMP-2 embedded in a poly(propylene) scaffold surrounded by a gelatin hydrogel loaded with VEGF was used for the sequential release of the growth factors. Empty composites or composites loaded with VEGF and/or BMP-2 were implanted ectopically and orthotopically in Sprague–Dawley rats (n = 9). Following implantation, the local release profiles were determined by measuring the activity of ¹²⁵I-labeled growth factors using scintillation probes. After 8 weeks blood vessel and bone formation were analyzed using microangiography, μCT and histology. The scaffolds exhibited a large initial burst release of VEGF within the first 3 days and a sustained release of BMP-2 over the full 56-day implantation period. Although VEGF did not induce bone formation, it did increase the formation of the supportive vascular network (p = 0.03) in ectopic implants. In combination with local sustained BMP-2 release, VEGF significantly enhanced ectopic bone formation compared to BMP-2 alone (p = 0.008). In the orthotopic defects, no effect of VEGF on vascularisation was found, nor was bone formation higher by the combination of growth factors, compared to BMP-2 alone. This study demonstrates that a sequential angiogenic and osteogenic growth factor release may be beneficial for the enhancement of bone regeneration.     

An injectable calcium phosphate cement for the local delivery of paclitaxel to bone

Bone metastases are usually treated by surgical removal, fixation and chemotherapeutic treatment. Bone cement is used to fill the resection voids. The aim of this study was to develop a local drug delivery system using a calcium phosphate cement (CPC) as carrier for chemotherapeutic agents. CPC consisted of alpha-tricalcium phosphate, calcium phosphate dibasic and precipitated hydroxyapatite powders and a 2% Na(2)HPO(4) hardening solution. Scanning electron microscopy (SEM) was used to observe CPC morphology. X-ray diffraction (XRD) was used to follow CPC transformation. The loading/release capacity of the CPC was studied by a bovine serum albumin-loading model. Release/retention was measured by high performance liquid chromatography and X-ray photoelectron spectrometry. For chemotherapeutic loading, paclitaxel (PX) was loaded onto the CPC discs by absorption. Viability of osteosarcoma U2OS and metastatic breast cancer MDA-MB-231 cells was measured by an AlamarBlue assay. Results of SEM and XRD showed changes in CPC due to its transformation. The loading model indicated a high retention behavior by the CPC composition. Cell viability tests indicated a PX minimal lethal dose of 90?μg/ml. PX released from CPC remained active to influence cell viability. In conclusion, this study demonstrated that CPC is a feasible delivery vector for chemotherapeutic agents.     

In vitro osteoblastic differentiation of human bone marrow cells in the presence of metal ions.

For periods up to 21 days human bone marrow was cultured in control conditions that favor the proliferation and differentiation of osteoblastic cells. The effect of AISI 316L corrosion products and the corresponding major separate metal ions (Fe, Cr, and Ni) were studied in three different phases of the culture period in order to investigate the effects of metal ions in cell populations representative of osteoblastic cells in different stages of differentiation. Toxicity consequences of the presence of metal ions in bone marrow cultures were evaluated by biochemical parameters (enzymatic reduction of MTT, alkaline phosphatase activity, and total protein content), histochemical assays (identification of ALP-positive cells and Ca and phosphates deposits), and observation of the cultures by light and scanning electron microscopy. Culture media were analyzed for total and ionized Ca and P and also for metal ions (Fe, Cr, and Ni). The presence of AISI 316L corrosion products and Ni salt in bone marrow cultures during the first and second weeks of culture significantly disturbs the normal behavior of these cultures, interfering in the lag phase and exponential phase of cell growth and ALP expression. However, the presence of these species during the third week of culture, when expression of osteoblastic functions occurs (mineralization process), did not result in any detectable effect. Fe salt also disturbs the behavior of bone marrow cell cultures when present during the lag phase and proliferation phase, and a somewhat compromised response between the normal pattern (control cultures) and intense inhibition (AISI 316L corrosion products and Ni salt-added cultures) was observed. Fe did not affect the progression of the mineralization phase. Osteogenic cultures exposed to Cr salt (Cr3+) presented a pattern similar to the controls, indicating that this element does not interfere, in the concentration studied, in the osteoblastic differentiation of bone marrow cells. Quantification of metal ions in the culture media showed that Cr (originated from AISI 316L corrosion products but from not Cr3+ salt) and Ni (originated from AISI 316L corrosion products and Ni salt) appear to be retained by the bone marrow cultures.