Gene expression during the development of Mycobacterium smegmatis biofilms on hydroxyapatite surfaces / Expresión génica durante el desarrollo de biopelículas de Mycobacterium smegmatis en superficies de hidroxiapatita

Bacterial biofilms are a consortium of bacteria that are strongly bound to each other and the surface on which they developed irreversibly. Bacteria can survive adverse environmental conditions and undergo changes when transitioning from a planktonic form to community cells. The process of mycobacteria adhesion is complex, involving characteristics and properties of bacteria, surfaces, and environmental factors; therefore, the formation of different biofilms is possible. Cell wall-, lipid-, and lipid transporter-related genes (glycopeptidolipids, GroEL1, protein kinase) are important in mycobacterial biofilm development. We investigated gene expression during in vitro development of Mycobacterium smegmatis biofilms on a hydroxyapatite (HAP) surface. Biofilm formation by M. smegmatis cells was induced for 1, 2, 3, and 5 days on the HAP surface. Mycobacteria on polystyrene generated an air–liquid interface biofilm, and on the fifth day, it increased by 35% in the presence of HAP. Six genes with key roles in biofilm formation were analyzed by real-time RT‒qPCR during the biofilm formation of M. smegmatis on both abiotic surfaces. The expression of groEL1, lsr2, mmpL11, mps, pknF, and rpoZ genes during biofilm formation on the HAP surface did not exhibit significant changes compared to the polystyrene surface. These genes involved in biofilm formation are not affected by HAP.(AU)

Gene expression during the development of Mycobacterium smegmatis biofilms on hydroxyapatite surfaces.

Bacterial biofilms are a consortium of bacteria that are strongly bound to each other and the surface on which they developed irreversibly. Bacteria can survive adverse environmental conditions and undergo changes when transitioning from a planktonic form to community cells. The process of mycobacteria adhesion is complex, involving characteristics and properties of bacteria, surfaces, and environmental factors; therefore, the formation of different biofilms is possible. Cell wall-, lipid-, and lipid transporter-related genes (glycopeptidolipids, GroEL1, protein kinase) are important in mycobacterial biofilm development. We investigated gene expression during in vitro development of Mycobacterium smegmatis biofilms on a hydroxyapatite (HAP) surface. Biofilm formation by M. smegmatis cells was induced for 1, 2, 3, and 5 days on the HAP surface. Mycobacteria on polystyrene generated an air-liquid interface biofilm, and on the fifth day, it increased by 35% in the presence of HAP. Six genes with key roles in biofilm formation were analyzed by real-time RT‒qPCR during the biofilm formation of M. smegmatis on both abiotic surfaces. The expression of groEL1, lsr2, mmpL11, mps, pknF, and rpoZ genes during biofilm formation on the HAP surface did not exhibit significant changes compared to the polystyrene surface. These genes involved in biofilm formation are not affected by HAP.

Low Magnesium Concentration Enforces Bone Calcium Deposition Irrespective of 1,25-Dihydroxyvitamin D3 Concentration.

Efficient coordination between Mg2+ and vitamin D maintains adequate Ca2+ levels during lactation. This study explored the possible interaction between Mg2+ (0.3, 0.8, and 3 mM) and 1,25-dihydroxyvitamin D3 (1,25D; 0.05 and 5 nM) during osteogenesis using bovine mesenchymal stem cells. After 21 days, differentiated osteocytes were subjected to OsteoImage analysis, alkaline phosphatase (ALP) activity measurements, and immunocytochemistry of NT5E, ENG (endoglin), SP7 (osterix), SPP1 (osteopontin), and the BGLAP gene product osteocalcin. The mRNA expression of NT5E, THY1, ENG, SP7, BGLAP, CYP24A1, VDR, SLC41A1, SLC41A2, SLC41A3, TRPM6, TRPM7, and NIPA1 was also assessed. Reducing the Mg2+ concentration in the medium increased the accumulation of mineral hydroxyapatite and ALP activity. There was no change in the immunocytochemical localization of stem cell markers. Expression of CYP24A1 was higher in all groups receiving 5 nM 1,25D. There were tendencies for higher mRNA abundance of THY1, BGLAP, and NIPA1 in cells receiving 0.3 mM Mg2+ and 5 nM 1,25D. In conclusion, low levels of Mg2+ greatly enhanced the deposition of bone hydroxyapatite matrix. The effect of Mg2+ was not modulated by 1,25D, although the expression of certain genes (including BGLAP) tended to be increased by the combination of low Mg2+ and high 1,25D concentrations.

Morphology-Tailored Hydroxyapatite Nanocarrier for Rhizosphere-Targeted Phosphorus Delivery.

High hydrophilicity and soil fixation collectively hamper the delivery of phosphorus (P) released from conventional chemical phosphorus fertilizers (CPFs) to plant rhizosphere for efficient uptake. Here, a phosphorus nutrient nanocarrier (PNC) based on morphology-tailored nanohydroxyapatite (HAP) is constructed. By virtue of kinetic control of building blocks with designed calcium phosphate intermediates, rod-like and hexagonal prism-like PNCs are synthesized, both having satisfactory hydrophobicity (water contact angle of 105.4- 132.9°) and zeta potential (-17.43 to -58.4 mV at pH range from 3 to 13). Greenhouse experiments demonstrate that the P contents increase by up to 183% in maize rhizosphere and up to 16% in maize biomass when compared to the CPF. Due to the water potential gradient driven by photosynthesis and transpiration, both PNCs are stably transported to maize rhizosphere, and they are capable to counteract soil fixation prior to uptake by plant roots. Within the synergies of the HAP morphological characteristics and triggered phosphate starvation response, root anatomy confirms that two pathways are elucidated to enhance plant P replenishment from the PNCs. Together with structure tunability and facile synthesis, our results offer a new nanodelivery prototype to accommodate plant physiological traits by tailoring the morphology of HAP.

Osteocytes and the pathogenesis of hypophosphatemic rickets.

Since phosphorus is a component of hydroxyapatite, its prolonged deprivation affects bone mineralization. Fibroblast growth factor 23 (FGF23) is essential for maintaining phosphate homeostasis and is mainly produced by osteocytes. FGF23 increases the excretion of inorganic phosphate (Pi) and decreases the production of 1,25-dihydroxyvitamin D in the kidneys. Osteocytes are cells of osteoblastic lineage that have undergone terminal differentiation and become embedded in mineralized bone matrix. Osteocytes express FGF23 and other multiple genes responsible for hereditary hypophosphatemic rickets, which include phosphate-regulating gene homologous to endopeptidase on X chromosome (PHEX), dentin matrix protein 1 (DMP1), and family with sequence similarity 20, member C (FAM20C). Since inactivating mutations in PHEX, DMP1, and FAM20C boost the production of FGF23, these molecules might be considered as local negative regulators of FGF23. Mouse studies have suggested that enhanced FGF receptor (FGFR) signaling is involved in the overproduction of FGF23 in PHEX-deficient X-linked hypophosphatemic rickets (XLH) and DMP1-deficient autosomal recessive hypophosphatemic rickets type 1. Since FGFR is involved in the transduction of signals evoked by extracellular Pi, Pi sensing in osteocytes may be abnormal in these diseases. Serum levels of sclerostin, an inhibitor Wnt/ß-catenin signaling secreted by osteocytes, are increased in XLH patients, and mouse studies have suggested the potential of inhibiting sclerostin as a new therapeutic option for the disease. The elucidation of complex abnormalities in the osteocytes of FGF23-related hypophosphatemic diseases will provide a more detailed understanding of their pathogenesis and more effective treatments.

Degeneration of biological heart valve grafts in a rat model of superoxide dismutase-3 deficiency.

While oxidative stress is known as key element in the pathogenesis of atherosclerosis and calcific aortic valve disease, its role in the degeneration of biological cardiovascular grafts has not been clarified yet. Therefore, the present study aimed to examine the impact of oxidative stress on the degeneration of biological cardiovascular allografts in a standardized chronic implantation model realized in rats exhibiting superoxide dismutase 3 deficiency (SOD3(-) ). Rats with SOD3 loss-of-function mutation (n = 24) underwent infrarenal implantation of cryopreserved valved aortic conduits, while SOD3-competent recipients served as controls (n = 28). After a follow-up period of 4 or 12 weeks, comparative analyses addressed degenerative processes, hemodynamics, and evaluation of the oxidative stress model. SOD3(-) rats presented decreased circulating SOD activity (p = .0079). After 12 weeks, 58% of the implant valves in SOD3(-) rats showed regurgitation (vs. 31% in controls, p = .2377). Intima hyperplasia and chondro-osteogenic transformation contributed to progressive graft calcification (p = .0024). At 12 weeks, hydroxyapatite deposition (p = .0198) and the gene expression of runt-related transcription factor-2 (Runx2) (p = .0093) were significantly enhanced in group SOD3(-) . This study provides the first in vivo evidence that impaired systemic antioxidant activity contributes to biological cardiovascular graft degeneration.

Short and long-term effects of nanobiomaterials in fish cell lines. Applicability of RTgill-W1.

Nanobiomaterials (NBMs) are nanostructured materials for biomedical applications that can reach aquatic organisms. The short and long-term effects of these emerging contaminants are unknown in fish. The RTgill-W1 cell line has been proposed as a model to predict the acute toxicity of chemicals to fish (OECD Test Guideline nº 249). We assessed the applicability of this cell line to study the short and long-term toxicity of 15 NBMs based on hydroxyapatites (HA), lipid (LSNP/LNP), gold, iron oxide, carbon, poly l-Lactide acid (PLLA) fibers with Ag and poly (lactide-co-glycolide) acid. Two more rainbow trout cell lines (RTL-W1, from liver, and RTS-11, from spleen) were exposed, to identify possible sensitivity differences among cells. Exposures to a range of concentrations (0.78-100 µg/mL) lasted for 24 h. Additionally, the RTgill-W1 was used to perform long-term (28 d exposure) and recovery (14 d exposure/14 d recovery) assays. Cells were exposed to the 24 h-IC20 and/or to 100 µg/mL. A triple cytotoxicity assay was conducted. After 24 h, only PLLA Fibers-Ag showed cytotoxicity (IC50 < 100 µg/mL). However, the NBMs in general provoked concentration-dependent effects after long-term exposures, except the LSNPs. A recovery of viability was only observed for AuNPs, AuNRods, Fe3O4PEG-PLGA, MgHA-Collag_Scaffolds, Ti-HA and TiHA-Alg NPs.These results evidenced the need to test the long-term toxicity of NBMs and showed differences in cytotoxicity probably associated to different mechanisms of toxic action. The RTgill-W1 was useful to screen short and long-term toxicities of NBMs and appears as a promiseful model to assess possible toxicity of NBMs in fish.

Two-faced Janus: the dual role of macrophages in atherosclerotic calcification.

Calcification is an independent predictor of atherosclerosis-related cardiovascular events. Microcalcification is linked to inflamed, unstable lesions, in comparison to the fibrotic stable plaque phenotype generally associated with advanced calcification. This paradox relates to recognition that calcification presents in a wide spectrum of manifestations that differentially impact plaque's fate. Macrophages, the main inflammatory cells in atherosclerotic plaque, have a multifaceted role in disease progression. They crucially control the mineralization process, from microcalcification to the osteoid metaplasia of bone-like tissue. It is a bilateral interaction that weighs heavily on the overall plaque fate but remains rather unexplored. This review highlights current knowledge about macrophage phenotypic changes in relation to and interaction with the calcifying environment. On the one hand, macrophage-led inflammation kickstarts microcalcification through a multitude of interlinked mechanisms, which in turn stimulates phenotypic changes in vascular cell types to drive microcalcification. Macrophages may also modulate the expression/activity of calcification inhibitors and inducers, or eliminate hydroxyapatite nucleation points. Contrarily, direct exposure of macrophages to an early calcifying milieu impacts macrophage phenotype, with repercussions for plaque progression and/or stability. Macrophages surrounding macrocalcification deposits show a more reparative phenotype, modulating extracellular matrix, and expressing osteoclast genes. This phenotypic shift favours gradual displacement of the pro-inflammatory hubs; the lipid necrotic core, by macrocalcification. Parallels to bone metabolism may explain many of these changes to macrophage phenotype, with advanced calcification able to show homeostatic osteoid metaplasia. As the targeted treatment of vascular calcification developing in atherosclerosis is thus far severely lacking, it is crucial to better understand its mechanisms of development.

Calcium carbonate particles: synthesis, temperature and time influence on the size, shape, phase, and their impact on cell hydroxyapatite formation.

To develop materials for drug delivery and tissue engineering and to study their efficiency with respect to ossification, it is necessary to apply physicochemical and biological analyses. The major challenge is labor-intensive data mining during synthesis and the reproducibility of the obtained data. In this work, we investigated the influence of time and temperature on the reaction yield, the reaction rate, and the size, shape, and phase of the obtained product in the completely controllable synthesis of calcium carbonate. We show that calcium carbonate particles can be synthesized in large quantities, i.e., in gram quantities, which is a substantial advantage over previously reported synthesis methods. We demonstrated that the presence of vaterite particles can dramatically stimulate hydroxyapatite (HA) production by providing the continued release of the main HA component - calcium ions - depending on the following particle parameters: size, shape, and phase. To understand the key parameters influencing the efficiency of HA production by cells, we created a predictive model by means of principal component analysis. We found that smaller particles in the vaterite state are best suited for HA growth (HA growth was 8 times greater than that in the control). We also found that the reported dependence of cell adhesion on colloidal particles can be extended to other types of particles that contain calcium ions.

Porous Biphasic Calcium Phosphate Granules from Oyster Shell Promote the Differentiation of Induced Pluripotent Stem Cells.

Oyster shells are rich in calcium, and thus, the potential use of waste shells is in the production of calcium phosphate (CaP) minerals for osteopathic biomedical applications, such as scaffolds for bone regeneration. Implanted scaffolds should stimulate the differentiation of induced pluripotent stem cells (iPSCs) into osteoblasts. In this study, oyster shells were used to produce nano-grade hydroxyapatite (HA) powder by the liquid-phase precipitation. Then, biphasic CaP (BCP) bioceramics with two different phase ratios were obtained by the foaming of HA nanopowders and sintering by two different two-stage heat treatment processes. The different sintering conditions yielded differences in structure and morphology of the BCPs, as determined by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Brunauer-Emmett-Teller (BET) surface area analysis. We then set out to determine which of these materials were most biocompatible, by co-culturing with iPSCs and examining the gene expression in molecular pathways involved in self-renewal and differentiation of iPSCs. We found that sintering for a shorter time at higher temperatures gave higher expression levels of markers for proliferation and (early) differentiation of the osteoblast. The differences in biocompatibility may be related to a more hierarchical pore structure (micropores within macropores) obtained with briefer, high-temperature sintering.

A Novel Strategy for Caries Management: Constructing an Antibiofouling and Mineralizing Dual-Bioactive Tooth Surface.

Existing single-functional agents against dental caries are inadequate in antibacterial performance or mineralization balance. This problem can be resolved through a novel strategy, namely, the construction of an antibiofouling and mineralizing dual-bioactive tooth surface by grafting a dentotropic moiety to an antimicrobial peptide. The constructed bioactive peptide can strongly adsorb onto the tooth surface and has beneficial functions in a myriad of ways. It inhibits cariogenic bacteria Streptococcus mutans adhesion, kills planktonic S. mutans, and destroys the S. mutans biofilm on the tooth surface. It also protects teeth from demineralization in acidic environments, and induces self-healing regeneration in the remineralization environment. Molecular dynamics simulations elucidate the main adsorption mechanism that the positively charged amino acid residues in the bioactive peptide bind to phosphate groups on the tooth surface, and the main mineralization mechanism that the negative charges on the outermost layer of the bioactive peptide repel acetic acid ions and attract calcium ions as nucleation sites for remineralization. This study suggests that this in-house synthesized dual-bioactive peptide is a promising functional agent to prevent dental caries, and is effective in inducing in situ self-healing remineralization for the treatment of decayed teeth.

Positive role of calcium phosphate ceramics regulated inflammation in the osteogenic differentiation of mesenchymal stem cells.

Recently, researches have confirmed the crucial role of inflammatory response in Ca-P ceramic-induced osteogenesis, however, the underlying mechanism has not yet been fully understood. In this study, BCP and ß-TCP ceramics were used as material models to investigate the effect of physicochemical properties on inflammatory response in vitro. The results showed that BCP and ß-TCP could support macrophages attachment, proliferation, and spreading favorably, as well as promote gene expressions of inflammatory related cytokines (IL-1, IL-6, MCP-1, and TNF-α) and growth factors (TGF-ß, FGF, PDGF, VEGF, IGF, and EGF). BCP showed a facilitating function on the gene expressions earlier than ß-TCP. Further coculture experiments performed in vitro demonstrated that the CMs containing various increased cytokines for macrophages pre-culture could significantly promote MSCs osteogenic differentiation, which was confirmed by the gene expressions of osteogenic specific markers and the intracellular OCN product accumulation under the stimulation of BCP and ß-TCP ceramics. Further evidence was found from the formation of mineralized nodules in BCM and TCM. In addition, this study showed a concise relationship between Ca-P ceramic induced inflammation and its osteoinductivity that the increased cytokines and growth factors from macrophages could promote MSCs osteogenic differentiation.

[Slowly progressive joint destruction in an older man as expression of hydroxyapatite disease. Case report and literature review]. / Langsam progrediente Gelenkzerstörung bei einem älteren Mann als Ausdruck einer Hydroxylapatiterkrankung. Fallbericht und Literaturübersicht.

This article reports the case of a 75-year-old male patient presenting with arthralgia of the large joints that had existed for 10 years. Clinically, bursitis of the right elbow joint was found. Laboratory tests showed elevated inflammatory markers and imaging revealed erosive joint destruction. A surgical bursectomy was performed. Histologically, hydroxyapatite crystals were detected in alizarin red S staining and a crystal arthropathy was diagnosed. The diagnostics are difficult since crystals can only be detected by electron microscopy or special staining methods.

Hydroxylapatite-collagen hybrid scaffold induces human adipose-derived mesenchymal stem cells to osteogenic differentiation in vitro and bone regrowth in patients.

Tissue engineering-based bone graft is an emerging viable treatment modality to repair and regenerate tissues damaged as a result of diseases or injuries. The structure and composition of scaffolds should modulate the classical osteogenic pathways in human stem cells. The osteoinductivity properties of the hydroxylapatite-collagen hybrid scaffold named Coll/Pro Osteon 200 were investigated in an in vitro model of human adipose mesenchymal stem cells (hASCs), whereas the clinical evaluation was carried out in maxillofacial patients. Differentially expressed genes (DEGs) induced by the scaffold were analyzed using the Osteogenesis RT2 PCR Array. The osteoinductivity potential of the scaffold was also investigated by studying the alkaline phosphatase (ALP) activity, matrix mineralization, osteocalcin (OCN), and CLEC3B expression protein. Fifty patients who underwent zygomatic augmentation and bimaxillary osteotomy were evaluated clinically, radiologically, and histologically during a 3-year follow-up. Among DEGs, osteogenesis-related genes, including BMP1/2, ALP, BGLAP, SP7, RUNX2, SPP1, and EGFR, which play important roles in osteogenesis, were found to be upregulated. The genes to cartilage condensation SOX9, BMPR1B, and osteoclast cells TNFSF11 were detected upregulated at every time point of the investigation. This scaffold has a high osteoinductivity revealed by the matrix mineralization, ALP activity, OCN, and CLEC3B expression proteins. Clinical evaluation evidences that the biomaterial promotes bone regrowth. Histological results of biopsy specimens from patients showed prominent ossification. Experimental data using the Coll/Pro Osteon 200 indicate that clinical evaluation of bone regrowth in patients, after scaffold implantation, was supported by DEGs implicated in skeletal development as shown in "in vitro" experiments with hASCs.

The role of antimiR-26a-5p/biphasic calcium phosphate in repairing rat femoral defects.

Although miRNAs have been implicated in the osteogenic differentiation of stem cells, their role in bone repair and reconstruction in tissue­engineered bone grafts remains unclear. We previously reported that microRNA (miR)­26a­5p inhibited the osteogenic differentiation of adipose­derived mesenchymal stem cells (ADSCs), and that antimiR­26a­5p exerted the opposite effect. In the present study, the role of miR­26a­5p­ and antimiR­26a­5p­modified ADSCs combined with biphasic calcium phosphate (BCP) scaffolds was evaluated in a rat femur defect model. The aim of the present study was to improve the understanding of the role of miR­26a­5p in bone regeneration in vivo, as well as to provide a new method to optimize the osteogenic ability of BCPs. ADSCs were infected with Lv­miR­26a­5p, Lv­miR­NC, Lv­antimiR­26a­5p or Lv­antimiR­NC respectively, and then combined with BCP scaffolds to repair rat femoral defects. Using X­rays, micro­computed tomography and histology at 2, 4, and 8 weeks postoperatively, the quantity and rate of bone regeneration were analyzed, revealing that they were the highest in animals treated with antimiR­26a­5p and the lowest in the miR­26a­5p treatment group. The expression levels of osteocalcin, collagen I, Runt­related transcription factor 2, Wnt family member 5A and calmodulin­dependent protein kinase II proteins were positively correlated with the bone formation rate. Taken together, the present results demonstrated that miR­26a­5p inhibited bone formation while antimiR­26a­5p accelerated bone formation via the Wnt/Ca2+ signaling pathway. Therefore, antimiR­26a­5p­modified ADSCs combined with BCP scaffolds may be used to construct an effective tissue­engineering bone graft for bone repair and reconstruction.

Bone Regeneration Using Adipose-Derived Stem Cells in Injectable Thermo-Gelling Hydrogel Scaffold Containing Platelet-Rich Plasma and Biphasic Calcium Phosphate.

For bone regeneration, a biocompatible thermo-gelling hydrogel, hyaluronic acid-g-chitosan-g-poly(N-isopropylacrylamide) (HA-CPN) was used as a three-dimensional organic gel matrix for entrapping rabbit adipose-derived stem cells (rASCs). Biphasic calcium phosphate (BCP) ceramic microparticles were embedded within the gel matrix as a mineralized bone matrix, which was further fortified with platelet-rich plasma (PRP) with osteo-inductive properties. In vitro culture of rASCs in HA-CPN and HA-CPN/PRP/BCP was compared for cell proliferation and osteogenic differentiation. Overall, HA-CPN/PRP/BCP was a better injectable cell carrier for osteogenesis of rASCs with increased cell proliferation rate and alkaline phosphatase activity, enhanced calcium deposition and mineralization of extracellular matrix, and up-regulated expression of genetic markers of osteogenesis. By implanting HA-CPN/PRP/BCP/rASCs constructs in rabbit critical size calvarial bone defects, new bone formation at the defect site was successfully demonstrated from computed tomography, and histological and immunohistochemical analysis. Taken together, by combining PRP and BCP as the osteo-inductive and osteo-conductive factor with HA-CPN, we successfully demonstrated the thermo-gelling composite hydrogel scaffold could promote the osteogenesis of rASCs for bone tissue engineering applications.

Protein disorder-order interplay to guide the growth of hierarchical mineralized structures.

A major goal in materials science is to develop bioinspired functional materials based on the precise control of molecular building blocks across length scales. Here we report a protein-mediated mineralization process that takes advantage of disorder-order interplay using elastin-like recombinamers to program organic-inorganic interactions into hierarchically ordered mineralized structures. The materials comprise elongated apatite nanocrystals that are aligned and organized into microscopic prisms, which grow together into spherulite-like structures hundreds of micrometers in diameter that come together to fill macroscopic areas. The structures can be grown over large uneven surfaces and native tissues as acid-resistant membranes or coatings with tuneable hierarchy, stiffness, and hardness. Our study represents a potential strategy for complex materials design that may open opportunities for hard tissue repair and provide insights into the role of molecular disorder in human physiology and pathology.

In vivo biocompatibility and degradation of novel Polycaprolactone-Biphasic Calcium phosphate scaffolds used as a bone substitute.

BACKGROUND: Biocompatibility and degradation of poly ε-caprolactone (PCL)-Biphasic Calcium Phosphate (BCP) scaffolds fabricated by the "Melt Stretching and Compression Molding (MSCM)" technique were evaluated in rat models. OBJECTIVES: Degradation behaviors and histological biocompatibility of the PCL-20% BCP MSCM scaffolds and compare with those of PCL-20% ß-tricalcium phosphate (TCP) scaffolds commercially fabricated by Fused Deposition Modeling (FDM) were evaluated. METHODS: The study groups included Group A: PCL-20% BCP MSCM scaffolds and Group B: PCL-20% TCP FDM scaffolds, which were implanted subcutaneously in twelve male Wistar rats. On day 14, 30, 60 and 90, dimensional changes of the scaffolds and their surrounding histological features were assessed using Micro-Computed Tomography (µ-CT) and histological analysis. Changes of their molecular weight were assessed using Gel Permeation Chromatography (GPC). RESULTS: Formation of collagen and new blood vessels throughout the scaffolds of both groups increased with time with low degrees of inflammation. The µ-CT and GPC analysis demonstrated that the scaffolds of both groups degraded with time, but, their molecular weight slightly changed over the observation periods. All results of both groups were not significantly different. CONCLUSIONS: The PCL-20% BCP MSCM scaffolds were biocompatible and biodegradable in vivo. Their properties were comparable to those of the commercial PCL-20% TCP scaffolds.

Genotoxicity assessment of biphasic calcium phosphate of modified porosity on human dental pulp cells using Ames and Comet assays.

Biphasic Calcium Phosphate (BCP) with a ratio of 20/80 Hydroxyapatite (HA)/Beta-tricalcium phosphate (ß-TCP) promotes the differentiation of human dental pulp cells (HDPCs). In the current study, the genotoxicity of locally produced BCP of modified porosity (65%) with a mean pore size of 300micrometer (µm) was assessed using Comet and Ames assays. HDPCs were treated with BCP extract at three different inhibitory concentrations which were obtained based on cytotoxicity test conducted with concurrent negative and positive controls. The tail moment of HDPCs treated with BCP extract at all three concentrations showed no significant difference compared to negative control (p>0.05), indicating that BCP did not induce DNA damage to HDPCs. The BCP was evaluated using five tester strains of Salmonella typhimurium TA98, TA100, TA102, TA1537 and TA1538. Each strain was incubated with BCP extract with five different concentrations in the presence and absence of metabolic activation system (S9) mix. Concurrently, negative and positive controls were included. The average number of revertant colonies per plate treated with the BCP extract was less than double as compared to the number of revertant colonies in negative control plate and no dose-related increase was observed. Results from both assays suggested that the BCP of modified porosity did not exhibit any genotoxic effect under the present test conditions.