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Autophagy, a conserved cellular degradation and recycling process, can be enhanced by nutrient depletion, oxidative stress or other harmful conditions to maintain cell survival. 6-Hydroxydopamine/ascorbic acid (6-OHDA/AA) is commonly used to induce experimental Parkinson’s disease (PD) lesions by causing oxidative damage to dopaminergic neurons. Activation of autophagy has been observed in the 6-OHDA-induced PD models. However, the mechanism and exact role of autophagy activation in 6-OHDA PD model remain inconclusive. In this study, we report that autophagy was triggered via mucolipin 1/calcium/calcineurin/TFEB (transcription factor EB) pathway upon oxidative stress induced by 6-OHDA/AA. Interestingly, overexpression of TFEB alleviated 6-OHDA/AA toxicity. Moreover, autophagy enhancers, Torin1 (an mTOR-dependent TFEB/autophagy enhancer) and curcumin analog C1 (a TFEB-dependent and mTOR-independent autophagy enhancer), significantly rescued 6-OHDA/AA-induced cell death in SH-SY5Y cells, iPSC-derived DA neurons and mice nigral DA neurons. The behavioral abnormality of 6-OHDA/AA-treated mice can also be rescued by Torin 1 or C1 administration. The protective effects of Torin 1 and C1 can be blocked by autophagy inhibitors like chloroquine (CQ) or by knocking down autophagy-related genes TFEB and ATG5. Taken together, this study supports that TFEB-mediated autophagy is a survival mechanism during oxidative stress and pharmacological enhancement of this process is a neuroprotective strategy against oxidative stress-associated PD lesions.

Alzheimer’s disease (AD) is a neurodegenerative disease with a high incidence in the elderly. Many preclinical studies show that a natural product, ferulic acid (FA), displays neuroprotective effects in AD models. This review aims to systematically review and meta-analyze published pre-clinical researches about the effects, mechanism, and clinical prospects of FA in the treatment of AD. According to the pre-determined search strategy and inclusion criteria, a total of 344 animals in 12 papers were included in the meta-analysis. We used the fixed effects model to analyze data and I2 and p values to indicate heterogeneity. Results show that FA treatment can effectively improve rodents’ spatial memory ability in MWM and Y maze experiments (I2 ≥ 70, p < 0.005), and reduce the deposition of Aβ in the brains of various model animals (I2 ≥ 50, p < 0.005). The potential mechanisms include anti-amyloidogenesis, anti-inflammation, anti-oxidation, mitochondrial protection, and inhibition of apoptosis. In conclusion, we systematically review and meta-analyze the literature reporting the effects of FA treatment on AD rodent models and solidify the benefits of FA in reducing Aβ deposition and improving memory in preclinical experiments. We also point out the limitations in the current research design and provide a strategy for the production research of FA in the future.

Copper nanoparticles encapsulated by carbon (Cu-NPs@C) were synthesized by a two-step method by mixing cupric chloride and glucose in a solution, followed by carbonization. The microstructures and antibacterial activities of the Cu-NPs@C samples were characterized by TEM, XRD, XPS, nitrogen adsorption and antibacterial activity tests. Results indicated that the as-synthesized Cu-NPs had a FCC crystal structure and a spherical morphology of diameter 4-46 nm and were well dispersed in the porous graphitic carbon layers. When the mass ratio of cupric chloride to glucose was less than 1:5, the Cu-NPs@C were of pure metallic copper without any oxides, and were stable against ambient air oxidation. The Cu-NPs@C synthesized with a mass ratio of cupric chloride to glucose of 1:5 had a specific surface area of 418 m2/g, which showed much higher antibacterial activities against three different common bacteria (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa) than pure nano-copper powder. It was believed that the carbon layers are responsible for capturing the bacteria while the copper ions released from the copper nanoparticles kill them. The outer carbon layers effectively protected the metallic copper inside from oxidation. These findings indicate that Cu-NPs@C can be used as a stable antibacterial agent in biomedical applications.

Background Berberine is an isoquinoline alkaloid extracted from various Berberis species which is widely used in East Asia for a wide range of symptoms. Recently, neuroprotective effects of berberine in Alzheimer’s disease (AD) animal models are being extensively reported. So far, no clinical trial has been carried out on the neuroprotective effects of berberine. However, a review of the experimental data is needed before choosing berberine as a candidate drug for clinical experiments. We conducted a systematic review on AD rodent models to analyze the drug effects with minimal selection bias. Methods Five online literature databases were searched to find publications reporting studies of the effect of berberine treatment on animal models of AD. Up to March 2018, 15 papers were identified to describe the efficacy of berberine. Results The included 15 articles met our inclusion criteria with different quality ranging from 3 to 5. We analyzed data extracted from full texts with regard to pharmacological effects and potential anti-Alzheimer’s properties. Our analysis revealed that in multiple memory defects animal models, berberine showed significant memory-improving activities with multiple mechanisms, such as anti-inflammation, anti-oxidative stress, cholinesterase (ChE) inhibition and anti-amyloid effects. Conclusion AD is likely to be a complex disease driven by multiple factors. Yet, many therapeutic strategies based on lowering β-amyloid have failed in clinical trials. This suggest that the threapy should not base on a single cause of Alzheimer’s disease but rather a number of different pathways that lead to the disease. Overall we think that berberine can be a promising multipotent agent to combat Alzheimer’s disease.

NRBF2, a regulatory subunit of the ATG14-BECN1/Beclin 1-PIK3C3/VPS34 complex, positively regulates macroautophagy/autophagy. In this study, we report that NRBF2 is required for the clearance of apoptotic cells and alleviation of inflammation during colitis in mice. NRBF2-deficient mice displayed much more severe colitis symptoms after the administration of ulcerative colitis inducer, dextran sulfate sodium salt (DSS), accompanied by prominent intestinal inflammation and apoptotic cell accumulation. Interestingly, we found that nrbf2(−/-) mice and macrophages displayed impaired apoptotic cell clearance capability, while adoptive transfer of nrbf2(+/+) macrophages to nrbf2(−/-) mice alleviated DSS-induced colitis lesions. Mechanistically, NRBF2 is required for the generation of the active form of RAB7 to promote the fusion between phagosomes containing engulfed apoptotic cells and lysosomes via interacting with the MON1-CCZ1 complex and regulating the guanine nucleotide exchange factor (GEF) activity of the complex. Evidence from clinical samples further reveals the physiological role of NRBF2 in maintaining intestinal homeostasis. In biopsies of UC patient colon, we observed upregulated NRBF2 in the colon macrophages and the engulfment of apoptotic cells by NRBF2-positive cells, suggesting a potential protective role for NRBF2 in UC. To confirm the relationship between apoptotic cell clearance and IBD development, we compared TUNEL-stained cell counts in the UC with UC severity (Mayo Score) and observed a strong correlation between the two indexes, indicating that apoptotic cell population in colon tissue correlates with UC severity. The findings of our study reveal a novel role for NRBF2 in regulating apoptotic cell clearance to restrict intestinal inflammation. Abbreviation: ANOVA: analysis of variance; ATG14: autophagy related 14; ATG16L1: autophagy related 16-like 1 (S. cerevisiae); BMDM: bone marrow-derived macrophage; BSA: bovine serum albumin; CD: Crohn disease; CD68: CD68 molecule; CFP: cyan fluorescent protein; CMFDA: 5-chloromethylfluorescein diacetate; Co-IP, co-immunoprecipitation; CPR: C-reactive protein; Cy7: cyanine 7 maleimide; DAB: diaminobezidine 3; DAI: disease activity indexes; DAPI: 4ʹ6-diamidino-2-phenylindole; DMEM: dulbecco’s modified eagle’s medium; DMSO: dimethyl sulfoxide; DOC: sodium deoxycholate; DSS: dextran sulfate sodium; EDTA: ethylenediaminetetraacetic acid; EGTA: ethylenebis (oxyethylenenitrilo) tetraacetic acid; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; FRET: Förster resonance energy transfer; GDP: guanine dinucleotide phosphate; GEF: guanine nucleotide exchange factor; GFP: green fluorescent protein; GTP: guanine trinucleotide phosphate; GWAS: genome-wide association studies; HEK293: human embryonic kidney 293 cells; HRP: horseradish peroxidase; IBD: inflammatory bowel disease; IgG: immunoglobin G; IL1B/IL-1β: interleukin 1 beta; IL6: interleukin 6; IRGM: immunity related GTPase M; ITGAM/CD11b: integrin subunit alpha M; KO: knockout; LRRK2: leucine rich repeat kinase 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MPO: myeloperoxidase; NaCl: sodium chloride; NEU: neutrophil; NOD2: nucleotide binding oligomerization domain containing 2; NP40: nonidet-P40; NRBF2: nuclear receptor binding factor 2; PBS: phosphate buffer saline; PCR: polymerase chain reaction; PE: P-phycoerythrin; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PTPRC/CD45: protein tyrosine phosphatase receptor type C; SDS-PAGE: sodium dodecylsulphate-polyacrylamide gel electrophoresis; TBST: tris-buffered saline Tween-20; Tris-HCl: trihydroxymethyl aminomethane hydrochloride; TUNEL: TdT-mediated dUTP nick-end labeling; UC: ulcerative colitis; ULK1: unc-51 like autophagy activating kinase 1; WB: western blotting; WT: wild type; YFP: yellow fluorescent protein.

NRBF2 is a component of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex. Our previous study has revealed its role in regulating ATG14-associated PtdIns3K activity for autophagosome initiation. In this study, we revealed an unknown mechanism by which NRBF2 modulates autophagosome maturation and APP-C-terminal fragment (CTF) degradation. Our data showed that NRBF2 localized at autolysosomes, and loss of NRBF2 impaired autophagosome maturation. Mechanistically, NRBF2 colocalizes with RAB7 and is required for generation of GTP-bound RAB7 by interacting with RAB7 GEF CCZ1-MON1A and maintaining the GEF activity. Specifically, NRBF2 regulates CCZ1-MON1A interaction with PI3KC3/VPS34 and CCZ1-associated PI3KC3 kinase activity, which are required for CCZ1-MON1A GEF activity. Finally, we showed that NRBF2 is involved in APP-CTF degradation and amyloid beta peptide production by maintaining the interaction between APP and the CCZ1-MON1A-RAB7 module to facilitate the maturation of APP-containing vesicles. Overall, our study revealed a pivotal role of NRBF2 as a new RAB7 effector in modulating autophagosome maturation, providing insight into the molecular mechanism of NRBF2-PtdIns3K in regulating RAB7 activity for macroautophagy/autophagy maturation and Alzheimer disease-associated protein degradation.. Abbreviations: 3xTg AD, triple transgenic mouse for Alzheimer disease; Aβ, amyloid beta peptide; Aβ1-40, amyloid beta peptide 1–40; Aβ1-42, amyloid beta peptide 1–42; AD, Alzheimer disease; APP, amyloid beta precursor protein; APP-CTFs, APP C-terminal fragments; ATG, autophagy related; ATG5, autophagy related 5; ATG7, autophagy related 7; ATG14, autophagy related 14; CCD, coiled-coil domain; CCZ1, CCZ1 homolog, vacuolar protein trafficking and biogenesis associated; CHX, cycloheximide; CQ, chloroquine; DAPI, 4ʹ,6-diamidino-2-phenylindole; dCCD, delete CCD; dMIT, delete MIT; FYCO1, FYVE and coiled-coil domain autophagy adaptor 1; FYVE, Fab1, YGL023, Vps27, and EEA1; GAP, GTPase-activating protein; GDP, guanine diphosphate; GEF, guanine nucleotide exchange factor; GTP, guanine triphosphate; GTPase, guanosine triphosphatase; HOPS, homotypic fusion and vacuole protein sorting; ILVs, endosomal intralumenal vesicles; KD, knockdown; KO, knockout; LAMP1, lysosomal associated membrane protein 1; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MLVs, multilamellar vesicles; MON1A, MON1 homolog A, secretory trafficking associated; NRBF2, nuclear receptor binding factor 2; PtdIns3K, class III phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; RILP, Rab interacting lysosomal protein; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62, sequestosome 1; UVRAG, UV radiation resistance associated; VPS, vacuolar protein sorting; WT, wild type.

Alzheimer disease (AD) is the most common neurodegenerative disease characterized by the deposition of amyloid plaque in the brain. The autophagy-associated PIK3C3-containing phosphatidylinositol 3-kinase (PtdIns3K) complex has been shown to interfere with APP metabolism and amyloid beta peptide (Aβ) homeostasis via poorly understood mechanisms. Here we report that NRBF2 (nuclear receptor binding factor 2), a key component and regulator of the PtdIns3K, is involved in APP-CTFs homeostasis in AD cell models. We found that NRBF2 interacts with APP in vivo and its expression levels are reduced in hippocampus of 5XFAD AD mice; we further demonstrated that NRBF2 overexpression promotes degradation of APP C-terminal fragments (APP-CTFs), and reduces Aβ1–40 and Aβ1-42 levels in human mutant APP-overexpressing cells. Conversely, APP-CTFs, Aβ1–40 and Aβ1-42 levels were increased in Nrbf2 knockdown or nrbf2 knockout cells. Furthermore, NRBF2 positively regulates autophagy in neuronal cells and NRBF2-mediated reduction of APP-CTFs levels is autophagy dependent. Importantly, nrbf2 knockout attenuates the recruitment of APP and APP-CTFs into phagophores and the sorting of APP and APP-CTFs into endosomal intralumenal vesicles, which is accompanied by the accumulation of the APP and APP-CTFs into RAB5-positive early endosomes. Collectively, our results reveal the potential connection between NRBF2 and the AD-associated protein APP by showing that NRBF2 plays an important role in regulating degradation of APP-CTFs through modulating autophagy.

// Ming-Yue Wu 1, * , Sheng-Fang Wang 1, * , Cui-Zan Cai 1 , Jie-Qiong Tan 2 , Min Li 3 , Jin-Jian Lu 1 , Xiu-Ping Chen 1 , Yi-Tao Wang 1 , Wei Zheng 4 and Jia-Hong Lu 1 1 State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR, China 2 State Key Laboratory of Medical Genetics, Xiangya Medical School, Central South University, Changsha, Hunan, China 3 Mr. and Mrs. Ko Chi Ming Centre for Parkinson’s Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China 4 Department of Thyroid and Breast Surgery, The Third People’s Hospital of Shenzhen, Shenzhen, Guangdong, China * Co-first authors Correspondence to: Jia-Hong Lu, email: jiahonglu@umac.mo Keywords: dauricine, daurisoline, autophagy blocker, cancer cells, cell toxicity Received: March 21, 2017 Accepted: August 02, 2017 Published: September 08, 2017 ABSTRACT Autophagy is a cellular bulk degradation pathway implicated in various diseases. Inhibition of autophagy has been regarded as a new therapeutic strategy for cancer treatment, especially in combination with chemotherapy. In our study, we identified two natural compounds, dauricine (DAC) and daurisoline (DAS), as two potent autophagy blockers through a high-content screening. DAC and DAS are alkaloids isolated from traditional Chinese medicine Rhizoma Menispermi . We systematically examined the effects of DAC and DAS on autophagy function in HeLa cells and found that DAC and DAS induced massive formation of autophagic vacuoles and lipidation of LC3. The accumulation of autophagic vacuoles and LC3 lipidation are due to blockage of autophagosome maturation as evidenced by interrupted colocalization of autophagsosome and lysosome, increased GFP-LC3/RFP-LC3 ratio and accumulation of autophagic substrate p62. Moreover, DAC and DAS impaired lysosomal function, as indicated by reduced lysosomal protease activity and increased lysosomal pH values. Importantly, we showed that DAC and DAS strongly inhibited the lysosome V-type ATPase activity. For the therapeutic potential, we found that DAC and DAS blocked the campothecin (CPT)-induced protective autophagy in HeLa cells, and dramatically sensitized the multiple cancer cells to CPT-induced cell death. In conclusion, our result shows that DAC and DAS are autophagy inhibitors which inhibit the lysosomal degradation of auophagic vacuoles, and sensitize the CPT-induced cancer cell death. The study implies the therapeutic potential of DAC and DAS in the treatment of cancers in combination of chemotherapy by inhibiting autophagy.

Nitric oxide (NO) is an important mediator of inflammation response and the production of NO has been linked to a variety of diseases, including tumors, inflammation and central nervous system diseases. In macrophages, a high level of NO is generated by iNOS during inflammatory responses triggered by cytokines or pathogens. Autophagy, a cellular bulk degradation process via lysosome, has been implicated in many disease conditions including inflammation. In this study, we have reported the previously unknown role of autophagy in regulating iNOS levels in macrophages, both under basal and Lipopolysaccharides (LPS)-induced conditions. Our data showed that iNOS levels accumulated upon autophagy inhibition and decreased upon autophagy induction. iNOS interacted and co-localized with autophagy receptor p62/SQSTM1, especially under LPS-stimulated condition in macrophages. Moreover, the immunostaining data revealed that iNOS also co-localizes with the autophagosome marker LC3 and lysosome marker LAMP1, especially under lysosomal inhibition conditions, indicating iNOS is an autophagy substrate. Finally, we showed that autophagy negatively regulated the generation of NO in macrophages, which is consistent with the changes of iNOS levels. Collectively, our study revealed a previously unknown mechanism by which autophagy regulates iNOS levels to modulate NO production during inflammation.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by the accumulation of protein aggregates (namely Lewy bodies) in dopaminergic neurons in the substantia nigra region of the brain. Alpha-synuclein (α-syn) is the major component of Lewy bodies in PD patients, and impairment of the ubiquitin-proteasome system has been linked to its accumulation. In this work, we developed a tetracycline–inducible expression system, with simultaneous induced expression of α-syn-EGFP and a bright red fluorescent protein marker (mCherry) to screen for potential compounds for degrading α-syn. We identified canthin-6-one as an α-syn lowering compound which promoted both wild type and mutants α-syn degradation in an ubiquitin-proteasome-system (UPS) dependent manner. By CRISPR/Cas9 genome-wide screening technology, we identified RPN2/PSMD1, the 26S proteasome non-ATPase regulatory subunit 1, as the targeting gene for pharmacological activity of canthin-6-one. Finally, we showed that canthin-6-one up-regulates PSMD1 and enhances UPS function by activating PKA.

Background Parkinson's disease (PD) is an age-dependent progressive movement disorder characterized by a profound and selective loss of nigrostriatal dopaminergic neurons. Accumulation of -synuclein ( -syn) positive protein aggregates in the substantia nigra is a pathological hallmark of PD, indicating that protein turnover defect is implicated in PD pathogenesis. Purpose This study aims to identify neuroprotective compounds which can alleviate the accumulation of -syn in neuronal cells and dissect the underlying mechanisms. Methods High throughput screening was performed by dot blot assay. The degradation of different forms of -syn by candidate compounds were assessed by western blot. The autophagy lysosome pathway and ubiquitin-proteasome system were examined to dissect the degradation pathway. The UPS activity was assessed by cellular UPS substrates degradation assay and biochemical proteasome activity assay. Q-PCR was performed to test the mRNA level of different proteasome subunits. Furthermore, Neuroprotective effect of candidate compound was tested by LDH assay and PI staining. Results Through the high throughput screening, harmine was identified as a potent -syn lowering compound. The time-dependent and dose-dependent effects of harmine on the degradation of different forms of -syn were further confirmed. Harmine could dramatically promote the degradation of UPS substrates GFP-CL1, Ub-R-GFP and Ub-G76V-GFP, and activate cellular proteasome activity. Mechanistically, harmine dramatically enhanced PKA phosphorylation to enhance proteasome subunit PSMD1 expression. PKA inhibitor blocked the effects of harmine in activating UPS, up regulating PSMD1 and promoting -syn degradation, indicating that harmine enhances UPS function via PKA activation. Moreover, harmine efficiently rescued cell death induced by over-expression of -syn, via UPS-dependent manner. Conclusion Harmine, as a new proteasome enhancer, may have potential to be developed into therapeutic agent against neurodegenerative diseases associated with UPS dysfunction and aberrant proteins accumulation.

6-OHDA plus ascorbic acid (AA) has long been used to induce Parkinson’s disease in rodents, while only 6-OHDA is commonly used to induce cell damage in cellular PD models. AA was believed to act as an anti-oxidant to prevent the degradation of 6-OHDA; however, some studies suggested that AA dramatically enhanced the selectivity and toxicity of 6-OHDA. To understand the mechanisms by which 6-OHDA/AA induces cell death, we established a 6-OHDA/AA cell toxicity model in human dopaminergic neuroblastoma SH-SY5Y cells. We confirmed that the toxicity of 6-OHDA was dramatically increased in the presence of AA, and the toxicity can be prevented by a flavonoid, baicalein. Mechanistically, our research reveals that 6-OHDA/AA induces cell death mainly through the interruption of intracellular calcium homeostasis, which leads to calpain activation and mitochondrial damage. Baicalein prevents 6-OHDA/AA-induced intracellular calcium elevation as well as consequent mitochondria damage. Taken together, our study confirms that 6-OHDA/AA is a more sensitive model for inducing neuronal lesion in vitro and reveals the central role of intracellular calcium in 6-OHDA/AA-induced cell death. Our studies further show that baicalein prevents 6-OHDA/AA-induced cell death by inhibiting intracellular calcium elevation.

Mig1p, a zinc finger class of DNA-binding protein, plays a critical role in glucose repression for maltose utilization in Baker’s yeast. Maltose is the hydrolyzate of starch, which is the main component of dough. In this study, the recombinant yeast strains with MIG1 gene deletion were constructed, and the maltose metabolism of the parental and mutant strains in batch cultivations were investigated. Our results show that the degree of glucose repression of mutants △MIG1α and △MIG1a are reduced by 49.88% and 41.59% respectively compared to their parental strains, suggesting that MIG1 deletion can partially relieve glucose repression of maltose metabolism.

The bioactivity of a novel composite of carbonate-containing low-crystallinity nanoparticle hydroxyapatite (HA) and a chitosan—phosphorylated chitosan polyelectrolyte complex (PEC) was evaluated in vitro and in vivo. The HA—PEC nanocomposite with complicated porous structure was prepared by a biomimetic method. An acidic chitosan (polycation) solution containing calcium and phosphate ions (6 mM Ca2+, Ca/P: 1.67) was added into phosphorylated chitosan (polyanion) solution; the formation of PEC and the controlled HA crystal growth were co-organized in alkaline solution. The material was co-cultured with rat osteoblasts in vitro, and implanted into rabbit femur marrow cavities. The results indicate that the PEC—HA composite promoted osteoblast adhesion, morphology, proliferation, and differentiation in vitro; the bone tissue response to the material histologically showed that it was bioactive, as well as biodegradable. The HA—PEC composite shows promise as a bone-repair material.

A polyelectrolyte complex (PEC) composed of chitosan (CS) and phosphorylated chitosan (PCS) was used to encapsulate a calcium phosphate by a biomimetic method. An acidic CS (polycation) solution containing calcium and phosphate ions (Ca2+: 6mM, Ca/P = 1.67) was added into PCS (polyanion) solution leading to the formation of a polyelectrolyte complex (PEC) with nanoscopic carbonate-containing, low-crystallinity hydroxyapatite (HA) distributed evenly in the fibrils of the PEC by controlled crystal growth. The resulting composite material, PEC-HA, has a complicated, hierarchical porous structure that is expected to have high bio-compatibity and that may be of use as a carrier for controlled-release therapetic agents.

A polyelectrolyte complex (PEC) composed of chitosan (CS) and phosphorylated chitosan (PCS) was used to encapsulate a calcium phosphate by a biomimetic method. An acidic CS (polycation) solution containing calcium and phosphate ions (Ca 2+ : 6 mM, Ca/P = 1.67) was added into a PCS (polyanion) solution leading to the formation of a polyelectrolyte complex (PEC) with nanoscopic carbonate-containing, low-crystallinity hydroxyapatite (HA) distributed evenly in the fibrils of the PEC by controlled crystal growth. The resulting composite material, PEC–HA, has a complicated porous structure that is expected to have high biocompatibility and that may be of use as materials for bone replacement and a carrier for controlled-release therapeutic agents.

In this study, orthophosphoric acid (H3PO4) in the treatment of porous titanium (Ti) is investigated and the ability of rat bone marrow stromal cells (BMSCs) is assessed to proliferate and differentiate on these modified surfaces in vitro. To improve the cytocompatibility of Ti surfaces, pure Ti was activated commercially by simple chemical pretreatment in H3PO4 with different densities. Next, the phosphorylated specimens were soaked in simulated body fluid (SBF) to study the effect of biomineralization. The3-(4,5-dimethylthiazol-2-y1)-2, 5-diphenytetrazolium bromide (MTT) assay and the measurement of alkaline phosphatase (ALP) activity utilized to assess proliferation and differentiation of BMSCs on exposure to modified Ti surfaces. Scanning electron microscopic (SEM) images showed that the surfaces of the pre-treated samples were characterized by a complex structure which consisted of a mesh-like morphological matrix and an uniform surface with different morphic crystals of titanium dihydrogen orthophosphate (Ti(H2PO4)3). These crystals contained hydroxyl with phosphate residues that resulted in biomineralization of cells. Therefore, BMSCs reveales a well-dispersed morphology on these modified and functionalized Ti surfaces. The viability and ALP activity of BMSCs on these altered biomimetic surfaces are found to be greater than those of the controls. It is concluded that the treatment of Ti by acid etching in orthophosphoric acid is a suitable method to enhance the in vitro proliferation and differentiation of BMSCs.