In vitro inflammatory modulation of bioceramic endodontic sealer in macrophages stimulated by bacterial lipopolysaccharide.

AIM: To evaluate the effects of AH Plus (Dentsply), Sealer 26 (Dentsply), and Sealer Plus BC (Produtos Médicos e Odontológicos) on cytotoxicity and inflammation in macrophage cultures exposed to bacterial lipopolysaccharide (LPS). METHODOLOGY: After initial setting, the sealers were conditioned with serum-free culture medium for 24 h (1 ml/cm2 ). Macrophages from the RAW 264.7 strain were exposed to sealer extracts in a 1:16 ratio in a culture medium with or without LPS. Cell morphology, viability, mitochondrial activity, oxidative stress and gene expression of interleukin 1ß (IL-1ß) and tumour necrosis factor-alpha (TNF-α) were evaluated. Data on mitochondrial activity, oxidative stress and TNF-α were analysed using a two-way analysis of variance (anova) test, followed by the Student-Newman-Keuls post-test. IL-1ß data were analysed using one-way anova, followed by SNK, and the t-test was used for intragroup comparison. The significance level was set at 5%. RESULTS: In the absence of LPS, only AH Plus and Sealer 26 showed a reduction in cell density, while in the presence of LPS, Sealer 26 had the lowest density compared to the other groups. In terms of mitochondrial activity, at 24 and 48 h, Sealer Plus BC had significantly higher mean values than Sealer 26 and AH Plus (p < .05). Sealer 26 exhibited the lowest levels of oxidative stress and IL-1ß and TNF-α expression, regardless of the presence of LPS (p < .05). CONCLUSIONS: Although all sealers interfere with the response of macrophages to LPS, contact with epoxy resin-based sealers can impair cell activity in vitro, while bioceramic sealer seems to favour the inflammatory functions of these cells.

Histologic and Histometric Analysis of Bone Repair at the Site of Mandibular Body Osteotomy and at the Bone-Screw Interface After Using a Biodegradable 2.0-mm Internal Fixation System.

The aim of the study was to evaluate histologically and histometrically the bone repair at the mandibular body osteotomy and at the bone-screw interface after using a biodegradable 2.0-mm internal fixation system. Six dogs were subjected to an osteotomy in the mandibular body, which was stabilized by applying a fixation device manufactured with poly-L-DL-lactic acid (70:30). The dogs were euthanized at 2 and 18 weeks. Each screw was sectioned along its long axis, and the osteotomy sites were divided into 3 parts: the upper part was labeled the tension third (TT); the lower part, compression third (CT); and the part between the TT and CT, intermediary third (IT). Histologic analysis showed areas of direct contact between the screw surface and the parent lamellar bone at 2 weeks. At 18 weeks, 3 microscopically distinct layers at the bone-screw interface were noted. At the osteotomy sites, union between the bone fragments was observed at 18 weeks. Statistically significant differences in the newly formed bone among TT, IT, and CT (P = 0.019) were observed. In conclusion, the biomechanical environment created by the biodegradable IF system used in this study facilitated bone repair at the osteotomy site.

Antibacterial, cytotoxic and mechanical properties of a orthodontic cement with phosphate nano-sized and phosphorylated chitosan: An in vitro study.

OBJECTIVES: Evaluate, in vitro, the effect of incorporating nano-sized sodium trimetaphosphate (TMPnano) and phosphorylated chitosan (Chi-Ph) into resin-modified glass ionomer cement (RMGIC) used for orthodontic bracket cementation, on mechanical, fluoride release, antimicrobial and cytotoxic properties. METHODS: RMGIC was combined with Chi-Ph (0.25%/0.5%) and/or TMPnano (14%). The diametral compressive/tensile strength (DCS/TS), surface hardness (SH) and degree of conversion (%DC) were determined. For fluoride (F) release, samples were immersed in des/remineralizing solutions. Antimicrobial/antibiofilm activity was evaluated by the agar diffusion test and biofilm metabolism (XTT). Cytotoxicity in fibroblasts was assessed with the resazurin method. RESULTS: After 24 h, the RMGIC-14%TMPnano group showed a lower TS value (p < 0.001); after 7 days the RMGIC-14%TMPnano-0.25%Chi-Ph group showed the highest value (p < 0.001). For DCS, the RMGIC group (24 h) showed the highest value (p < 0.001); after 7 days, the highest value was observed for the RMGIC-14%TMPnano-0.25%Chi-Ph (p < 0.001). RMGIC-14%TMPnano, RMGIC-14%TMPnano-0.25%Chi-Ph, RMGIC-14%TMPnano-0.5%Chi-Ph showed higher and similar release of F (p > 0.001). In the SH, the RMGIC-0.25%Chi-Ph; RMGIC-0.5%Chi-Ph; RMGIC-14%TMPnano-0.5%Chi-Ph groups showed similar results after 7 days (p > 0.001). The RMGIC-14%TMPnano-0.25%Chi-Ph group showed a better effect on microbial/antibiofilm growth, and the highest efficacy on cell viability (p < 0.001). After 72 h, only the RMGIC-14%TMPnano-0.25%Chi-Ph group showed cell viability (p < 0.001). CONCLUSION: The RMGIC-14%TMPnano-0.25%Chi-Ph did not alter the physical-mechanical properties, was not toxic to fibroblasts and reduced the viability and metabolism of S. mutans. CLINICAL RELEVANCE: The addition of phosphorylated chitosan and organic phosphate to RMGIC could provide an antibiofilm and remineralizing effect on the tooth enamel of orthodontic patients, who are prone to a high cariogenic challenge due to fluctuations in oral pH and progression of carious lesions.

Cytotoxicity testing of methyl and ethyl 2-cyanoacrylate using direct contact assay on osteoblast cell cultures.

PURPOSE: Cyanoacrylate has been used as a commercial tissue adhesive. Recently, ethyl 2-cyanoacrylate has been suggested for the fixation of onlay autogenous bone graft. However, ethyl 2-cyanoacrylate must be biocompatible with bone tissue. This study evaluated the cytotoxicity of cyanoacrylate adhesives using a direct contact assay on human oral osteoblast cells. MATERIALS AND METHODS: Osteoblastic cells derived from human alveolar bone of the mandible were cultured with or without cyanoacrylate. The CA1 group contained methyl 2-cyanoacrylate, the CA2 group contained ethyl 2-cyanoacrylate, and the CA3 group did not contain cyanoacrylate (control). This study investigated cell morphology, which included the inhibition zone, and cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which was measured as optical density. Data from the MTT assay were tested statistically using SigmaStat 3.5. RESULTS: Dead cells found around the CA1- and CA2-treated cells constituted inhibitory zones that varied from 200 to 500 µm. There was no inhibitory zone in the CA3 group. Cell viability evaluated by the MTT assay showed that the CA2 and CA3 optical densities were not significantly different. The CA1 optical densities differed significantly from the CA3 optical densities. CONCLUSIONS: Within the limits of this study, the MTT method supported the conclusion that ethyl 2-cyanoacrylate is biocompatible according to a direct contact assay on human osteoblast cell cultures and suggests its usefulness in bone graft fixation.

Characterization and in vitro evaluation of bacterial cellulose membranes functionalized with osteogenic growth peptide for bone tissue engineering.

The aim of this study was to characterize the physicochemical properties of bacterial cellulose (BC) membranes functionalized with osteogenic growth peptide (OGP) and its C-terminal pentapeptide OGP[10-14], and to evaluate in vitro osteoinductive potential in early osteogenesis, besides, to evaluate cytotoxic, genotoxic and/or mutagenic effects. Peptide incorporation into the BC membranes did not change the morphology of BC nanofibers and BC crystallinity pattern. The characterization was complemented by Raman scattering, swelling ratio and mechanical tests. In vitro assays demonstrated no cytotoxic, genotoxic or mutagenic effects for any of the studied BC membranes. Culture with osteogenic cells revealed no difference in cell morphology among all the membranes tested. Cell viability/proliferation, total protein content, alkaline phosphatase activity and mineralization assays indicated that BC-OGP membranes enabled the highest development of the osteoblastic phenotype in vitro. In conclusion, the negative results of cytotoxicity, genotoxicity and mutagenicity indicated that all the membranes can be employed for medical supplies, mainly in bone tissue engineering/regeneration, due to their osteoinductive properties.

Mandibular bisphosphonate-related osteonecrosis after dental implant rehabilitation: a case report.

It has been a matter of debate as to whether dental implant therapies are suitable for patients subjected to long-term use of bisphosphonates (BPs). This report presents a case of a 76-year-old woman who developed BPs-related osteonecrosis of the jaw (BRONJ) in the left hemimandible after dental implant exposure. The implants and the necrotic crestal bone were removed, and postoperatively, a delay in tissue healing with bone exposure was noticed. The histologic analysis of the block biopsies revealed a lamellar bone tissue exhibiting necrotic areas and bacterial colonies associated with the bone outer surface. The bone-implant interface showed viable lamellar bone with enlarged vascular spaces in the areas between the implant threads. The possible mechanisms for the loss of implants in BRONJ patients are discussed, and the potential protocols for dental implant rehabilitation for patients under BP therapies are presented.

Human alveolar bone cell proliferation, expression of osteoblastic phenotype, and matrix mineralization on porous titanium produced by powder metallurgy.

OBJECTIVE: This study aimed at investigating the influence of the porous titanium (Ti) structure on the osteogenic cell behaviour. MATERIALS AND METHODS: Porous Ti discs were fabricated by the powder metallurgy process with the pore size typically between 50 and 400 microm and a porosity of 60%. Osteogenic cells obtained from human alveolar bone were cultured until subconfluence and subcultured on dense Ti (control) and porous Ti for periods of up to 17 days. RESULTS: Cultures grown on porous Ti exhibited increased cell proliferation and total protein content, and lower levels of alkaline phosphatase (ALP) activity than on dense Ti. In general, gene expression of osteoblastic markers-runt-related transcription factor 2, collagen type I, alkaline phosphatase, bone morphogenetic protein-7, and osteocalcin was lower at day 7 and higher at day 17 in cultures grown on porous Ti compared with dense Ti, a finding consistent with the enhanced growth rate for such cultures. The amount of mineralized matrix was greater on porous Ti compared with the dense one. CONCLUSION: These results indicate that the porous Ti is an appropriate substrate for osteogenic cell adhesion, proliferation, and production of a mineralized matrix. Because of the three-dimensional environment it provides, porous Ti should be considered an advantageous substrate for promoting desirable implant surface-bone interactions.

Development of the osteoblastic phenotype in human alveolar bone-derived cells grown on a collagen type I-coated titanium surface.

OBJECTIVE: The aim of this study was to evaluate the development of the osteoblastic phenotype in human alveolar bone-derived cells grown on collagen type I-coated titanium (Ti) surface (Col-Ti) obtained by plasma deposition acrylic acid grafting compared with machined Ti (M-Ti). MATERIAL AND METHODS: Osteoblastic cells were cultured until subconfluence and subcultured on Col-Ti and M-Ti for periods of up to 21 days. RESULTS: Cultures grown on Col-Ti and M-Ti exhibited similar cell morphology. Cell adhesion, total protein content, and alkaline phosphatase (ALP) activity were not affected by Ti surface modification in all evaluated periods. Growth analyses indicated that there were significantly more cells in cultures grown on Col-Ti at day 3. Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG) mRNA expression of cells subcultured on Col-Ti was higher, whereas collagen type I (COL) was lower compared with M-Ti. Ti surface modification neither affected the osteocalcin (OC), ALP and receptor activator of NF-kappaB ligand (RANKL) mRNA expression nor the calcium content extracted from mineralized matrix. CONCLUSIONS: These results demonstrated that Col-Ti favours cell growth during the proliferative phase (day 3) and osteoblastic differentiation, as demonstrated by changes in mRNA expression profile during the matrix mineralization phase (day 14), suggesting that this Ti surface modification may affect the processes of bone healing and remodelling.

Bone repair in mandibular body osteotomy after using 2.0 miniplate system--histological and histometric analysis in dogs.

The objective of this study was to evaluate the bone repair along a mandibular body osteotomy after using a 2.0 miniplate system. Nine adult mongrel dogs were subjected to unilateral continuous defect through an osteotomy between the mandibular 3rd and 4th premolars. Two four-hole miniplates were placed in accordance with the Arbeitgeimeinschaft für Osteosynthesefragen Manual. Miniplates adapted to the alveolar processes were fixed monocortically with 6.0-mm-length titanium alloy self-tapping screws, whereas miniplates placed near the mandible bases were fixed bicortically. At 2, 6 and 12 weeks, three dogs were sacrificed per period, and the osteotomy sites were removed, divided into three thirds (Tension Third, TT; Intermediary Third, IT; Compression Third, CT) and prepared for conventional and polarized light microscopy. At 6 weeks, while the CT repaired faster and showed bone union by woven bone formation, the TT and IT exhibited a ligament-like fibrous connective tissue inserted in, and connecting, newly formed woven bone overlying the parent lamellar bone edges. At 12 weeks, bone repair took place at all thirds. Histometrically, proportions of newly formed bone did not alter at TT, IT and CT, whereas significantly enhanced bone formation was observed for the 12-week group, irrespective of the third. The results demonstrated that although the method used to stabilize the mandibular osteotomy allowed bone repair to occur, differences in the dynamics of bone healing may take place along the osteotomy site, depending on the action of tension and compression forces generated by masticatory muscles.

Bone cell responses to the composite of Ricinus communis polyurethane and alkaline phosphatase.

The aim of this study was to evaluate the response of osteoblastic cells to the composite of Ricinus communis polyurethane (RCP) and alkaline phosphatase (ALP) incubated in synthetic body fluid (SBF). RCP pure (RCPp) and RCP blended with ALP 6 mg/mL polymer (RCP+ALP) were incubated in SBF for 17 days. Four groups of RCP were tested: RCPp, RCP+ALP, and RCPp and RCP+ALP incubated in SBF (RCPp/SBF and RCP+ALP/SBF). Stem cells from rat bone marrow were cultured in conditions that allowed osteoblastic differentiation on RCP discs and were evaluated: cell adhesion, culture growth, cell viability, total protein content, ALP activity, and bone-like nodule formation. Data were compared by ANOVA or Kruskal-Wallis test. The group RCP+ALP was highly cytotoxic and, therefore, was not considered here. Cell adhesion (p = 0.14), culture growth (p = 0.39), viability (p = 0.46) and total protein content (p = 0.12) were not affected by either RCP composition or incubation in SBF. ALP activity was affected (p = 0.0001) as follows: RCPp < RCPp/SBF < RCP+ALP/SBF. Bone-like nodule formation was not observed on all evaluated groups. The composite RCP+ALP prior to SBF incubation is cytotoxic and must not be considered as biomaterial, but the incorporation of ALP to the RCP followed by SBF incubation could be a useful alternative to improve the biological properties of the RCP.

Macroporous scaffolds associated with cells to construct a hybrid biomaterial for bone tissue engineering.

Bone tissue has the ability to heal without a scar and to remodel, which promotes three basic functions: locomotion, protection of internal organs and mineral homeostasis. Although bone regeneration is highly efficient, some clinical situations - such as large bone defects - require specific treatments in order to promote bone healing. Allogenic or autologous bone grafts have been used in these procedures with limited success and, based on this, bone tissue-engineering approaches have been investigated extensively. Tissue engineering has been defined as the application of principles and techniques of the life sciences and engineering to the design, modification and growth of living tissues using biomaterials, cells and growth factors, alone or in combination. The association of cells with porous scaffolds to produce 3D hybrid osteogenic constructs is a common subject in bone tissue-engineering research and will be the focus of this review. We will present some aspects of bone biology, the cells and scaffolds used to engineer bone, and techniques to fabricate the hybrid biomaterial.

Partial replacement of the dentin-pulp complex by periodontal supporting tissues in a traumatically intruded primary maxillary incisor.

This paper describes a case of a 9-year-old patient who presented a completely intruded primary maxillary incisor because of a traumatic injury sustained at the age of 3 years. After tooth extraction, histological analysis revealed that the dentin-pulp complex was partially replaced by cementum, periodontal ligament and alveolar bone. No signs of ankylosis were noticed. It is suggested that the lack of spontaneous re-eruption of the traumatized primary tooth after 6 years could be due to the development of functional periodontal supporting tissues in the pulp chamber secondary to the traumatism.

Enhancement of in vitro osteogenesis on titanium by chemically produced nanotopography.

The surface characteristics of biomaterials can influence protein adsorption, cellular functions, and ultimately tissue formation. Controlled chemical oxidation of titanium-based surfaces with a mixture of H(2)SO(4)/H(2)O(2) creates a nanopatterned surface that has been shown to affect early osteogenic events. The objective of this study was to evaluate the effect over time of this nanopattern on various key parameters of osteogenesis, and determine whether these effects ultimately translate into more mineralized matrix production. Osteogenic cells were obtained by enzymatic digestion of newborn rat calvaria and grown on treated and untreated titanium discs for periods of up to 14 days. Alkaline phosphatase activity peaked earlier and cell number was higher as of day 7 on the nanopatterned discs. Immunofluorescence showed that the treated surface favored early bone sialoprotein and osteopontin secretion, and fibronectin accumulation. Alizarin red staining revealed that, at days 10 and 14, there were significantly more mineralized nodules on treated than on untreated discs. These results demonstrate that simple chemical treatment of titanium with H(2)SO(4)/H(2)O(2) accelerates the in vitro osteogenic potential of calvaria-derived cells. They also suggest that this treatment may represent an advantageous approach for producing "intelligent surfaces" that stimulate bone formation and enhance bone-implant contact.

In vitro osteogenesis on a highly bioactive glass-ceramic (Biosilicate).

One of the strategies to improve the mechanical performance of bioactive glasses for load-bearing implant devices has been the development of glass-ceramic materials. The present study aimed to evaluate the effect of a highly bioactive, fully-crystallized glass-ceramic (Biosilicate) of the system P(2)O(5)-Na(2)O-CaO-SiO(2) on various key parameters of in vitro osteogenesis. Surface characterization was carried out by scanning electron microscopy and Fourier transform infrared spectroscopy. Osteogenic cells were obtained by enzymatic digestion of newborn rat calvarial bone and by growing on Biosilicate discs and on control bioactive glass surfaces (Biosilicate) parent glass and Bioglass(R) 45S5) for periods of up to 17 days. All materials developed an apatite layer in simulated body fluid for 24h. Additionally, as early as 12 h under culture conditions and in the absence of cells, all surfaces developed a layer of silica-gel that was gradually covered by amorphous calcium phosphate deposits, which remained amorphous up to 72 h. During the proliferative phase of osteogenic cultures, the majority of cells exhibited disassembly of the actin cytoskeleton, whereas reassembly of actin stress fibers took place only in areas of cell multilayering by day 5. Although no significant differences were detected in terms of total protein content and alkaline phosphatase activity at days 11 and 17, Biosilicate supported significantly larger areas of calcified matrix at day 17. The results indicate that full crystallization of bioactive glasses in a range of compositions of the system P(2)O(5)-Na(2)O-CaO-SiO(2) may promote enhancement of in vitro bone-like tissue formation in an osteogenic cell culture system.

Bioactive-glass ceramic with two crystalline phases (BioS-2P) for bone tissue engineering.

We aimed to evaluate the in vitro osteogenic and osteoinductive potentials of BioS-2P and its ability to promote in vivo bone repair. To investigate osteogenic potential, UMR-106 osteoblastic cells were cultured on BioS-2P and Bioglass 45S5 discs in osteogenic medium. The osteoinductive potential was evaluated using mesenchymal stem cells (MSCs) cultured on BioS-2P, Bioglass 45S5 and polystyrene in non-osteogenic medium. Rat bone calvarial defects were implanted with BioS-2P scaffolds alone or seeded with MSCs. UMR-106 proliferation was similar for both materials, while alkaline phosphatase (ALP) activity and mineralization were higher for BioS-2P. Bone sialoprotein (BSP), RUNX2 and osteopontin (OPN) gene expression and BSP, OPN, ALP and RUNX2 protein expression were higher on BioS-2P. For MSCs, ALP activity was higher on Bioglass 45S5 than on BioS-2P and was lower on polystyrene. All genes were highly expressed on bioactive glasses compared to polystyrene. BioS-2P scaffolds promoted in vivo bone formation without differences in the morphometric parameters at 4, 8 and 12 weeks. After 8 weeks, the combination of BioS-2P with MSCs did not increase the quantity of new bone compared to the BioS-2P alone. To stimulate osteoblast activity, drive MSC differentiation and promote bone formation, BioS-2P is a good choice as a scaffold for bone tissue engineering.

Nanocellulose-collagen-apatite composite associated with osteogenic growth peptide for bone regeneration.

Despite advances in the field of biomaterials for bone repair/regeneration, some challenges for developing an ideal bone substitute need to be overcome. Herein, this study synthesized and evaluated in vitro a nanocomposite based on bacterial cellulose (BC), collagen (COL), apatite (Ap) and osteogenic growth peptide (OGP) or its C-terminal pentapeptide [OGP(10-14)] for bone regeneration purposes. The BC-COL nanocomposites were successfully obtained by carbodiimide-mediated coupling as demonstrated by spectroscopy analysis. SEM, FTIR and 31P NMR analyses revealed that in situ synthesis to apatite was an effective route for obtaining of bone-like apatite. The OGP-containing (BC-COL)-Ap stimulated the early development of the osteoblastic phenotype. Additionally, the association among collagen, apatite, and OGP peptides enhanced cell growth compared with OGP-containing BC-Ap. Furthermore, none of the nanocomposites showed cytotoxic, genotoxic or mutagenic effects. These promising results suggest that the (BC-COL)-Ap associated with OGP peptides might be considered a potential candidate for bone tissue engineering applications.

Nanotexturing of titanium-based surfaces upregulates expression of bone sialoprotein and osteopontin by cultured osteogenic cells.

Bone formation around implants is influenced by surface geometry. Since cell/matrix/substrate interactions associated with cell signaling occur in the nanoscale dimension, we have evaluated the influence of nanotexturing of titanium-based surfaces on the expression of matrix proteins by cultured osteogenic cells at initial time points. Cells were obtained by enzymatic digestion of newborn rat calvaria and grown on titanium and titanium alloy discs with nanotextured or machined surfaces, and on glass coverslips for periods of 6 h, 1 day, and 3 days, under standard culture conditions. Cultures were processed for single or dual immunolabeling with monoclonal and/or polyclonal antibodies against bone sialoprotein (BSP), fibronectin (FN), osteopontin (OPN), type-I pro-collagen, or tubulin, followed by corresponding fluorophore-conjugated secondary antibodies. Some samples were processed for scanning electron microscope analysis of morphology and immunogold labeling. After 6 h, nanotextured surfaces exhibited up to a nine-fold increase in the proportion of cells with peripheral OPN labeling. At day 3, the proportion of OPN and BSP labeled cells was higher, and the intensity of immunoreactivity dramatically increased. No significant differences were observed in the expression pattern and the proportion of cells immunoreactive for FN or type-I pro-collagen. Our results demonstrate that nanotexturing of titanium-based surfaces upregulates the early expression of BSP and OPN in osteogenic cell cultures.

Effects of type I collagen coating on titanium osseointegration: histomorphometric, cellular and molecular analyses.

The investigation of titanium (Ti) surface modifications aiming to increase implant osseointegration is one of the most active research areas in dental implantology. This study was carried out to evaluate the benefits of coating Ti with type I collagen on the osseointegration of dental implants. Acid etched Ti implants (AETi), either untreated or coated with type I collagen (ColTi), were placed in dog mandibles for three and eight weeks for histomorphometric, cellular and molecular evaluations of bone tissue response. While the histological aspects were essentially the same with both implants being surrounded by lamellar bone trabeculae, histomorphometric analysis showed more abundant bone formation in ColTi, mainly at three weeks. Cellular evaluation showed that cells harvested from bone fragments in close contact with ColTi display lower proliferative capacity and higher alkaline phosphatase activity, phenotypic features associated with more differentiated osteoblasts. Confirming these findings, molecular analyses showed that ColTi implants up-regulates the expression of a panel of genes well known as osteoblast markers. Our results present a set of evidences that coating AETi with collagen fastens the osseointegration by stimulating bone formation at the cellular and molecular levels, making this combination of morphological and biochemical modification a promising approach to treat Ti surfaces.

Pore size regulates cell and tissue interactions with PLGA-CaP scaffolds used for bone engineering.

A common subject in bone tissue engineering is the need for porous scaffolds to support cell and tissue interactions aiming at repairing bone tissue. As poly(lactide-co-glycolide)-calcium phosphate (PLGA-CaP) scaffolds can be manufactured with different pore sizes, the aim of this study was to evaluate the effect of pore diameter on osteoblastic cell responses and bone tissue formation. Scaffolds were prepared with 85% porosity, with pore diameters in the ranges 470-590, 590-850 and 850-1200 µm. Rat bone marrow stem cells differentiated into osteoblasts were cultured on the scaffolds for up to 10 days to evaluate cell growth, alkaline phosphatase (ALP) activity and the gene expression of the osteoblast markers RUNX2, OSX, COL, MSX2, ALP, OC and BSP by real-time PCR. Scaffolds were implanted in critical size rat calvarial defects for 2, 4, and 8 weeks for histomorphometric analysis. Cell growth and ALP activity were not affected by the pore size; however, there was an increase in the gene expression of osteoblastic markers with the increase in the pore sizes. At 2 weeks all scaffolds displayed a similar amount of bone and blood vessels formation. At 4 and 8 weeks much more bone formation and an increased number of blood vessels were observed in scaffolds with pores of 470-590 µm. These results show that PLGA-CaP is a promising biomaterial for bone engineering. However, ideally, combinations of larger (-1000 µm) and smaller (-500 µm) pores in a single scaffold would optimize cellular and tissue responses during bone healing.