Cancer biomarkers in atherosclerotic plaque: Evidenced from structural and proteomic analyses.

Atherosclerosis and cancer are the leading causes of mortality around the world that share common pathogenic pathways. The aim of this study is the investigation of the protein profile of atherosclerotic plaque in order to find similar biomarker between cancer and atherosclerosis. The small pieces of human coronary artery containing advanced atherosclerotic plaque is obtained from patients during bypass surgery. Structural characterization of type V plaque, including fibrous connective tissue, necrotic lipid core, cholesterol clefts and calcium deposits are performed using high resolution transmission electron microscopy (HR-TEM). The protein profile of atherosclerosis plaque is also analyzed using 2-dimensional electrophoresis and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF). TEM analysis shows that vascular smooth muscle cells (VSMCs) exhibit different and uncommon morphologies in atherosclerotic plaque which is correlated to the proliferative state of the cells. The proteomics analysis reveals proteins related to atherosclerosis formation including Mimecan, Ras Suppressor Protein-1 (RSUP-1) and Cathepsin D which identified as biomarker of cancerous tumors. The expression of Mimecan and RSUP-1 is down-regulated in atherosclerotic plaque while the expression of Cathepsin D is up-regulated. These data support that atherosclerotic plaque presents some degree of tumorgenesis with the significant activity of VSMCs as the key player in atherogenesis.

Graphene oxide incorporated polycaprolactone/chitosan/collagen electrospun scaffold: Enhanced osteogenic properties for bone tissue engineering.

This in vitro study aimed to evaluate the physicochemical and biological activity of the polycaprolactone/chitosan/collagen scaffolds incorporated with 0, 0.5, 3, and 6 wt% of graphene oxide (GO). Using standard tests and MG-63 cells, the characteristics of scaffolds were evaluated, and the behavior of osteoblasts were simulated, respectively. A non-significant decrease in nanofibers diameter was noted in scaffolds with a higher ratio of GO. The hydrophilicity and bioactivity of the scaffold surface, as well as cell attachment and proliferation, increased in correspondence to an increase in GO. The higher ratio of GO also improved the osteogenesis activity. GO increased the degradation rate, but it was negligible and seemed not enough to endanger stability. Modifying the scaffolds with GO did not make a significant change to the antibacterial effect.

The Effect of Bio-Conditioning of Titanium Implants for Enhancing Osteogenic Activity.

Early and effective integration of titanium-based materials into bone tissue is of vital importance for long-term stability of implants. Surface modification is commonly used to enhance cell-substrate interactions for improving cell adhesion, proliferation, and activity. Here, the surface of titanium substrates and commercial implants were coated with blood (TiB), fetal bovine serum (TiF), and phosphate-buffered saline (TiP) solution using a spin coating process. Surface roughness and wettability of samples were measured using contact angle measurements and atomic force microscopy. The samples were then exposed to human osteoblast-like MG63 cells in order to evaluate adhesion, growth, differentiation, and morphology on the surface of modified samples. Untreated titanium disks were used as controls. The lowest roughness and wettability values were found in unmodified titanium samples followed by TiP, TiF, and TiB. The percentage of cellular attachment and proliferation for each sample was measured using an MTT (3-[4,5-dimethylthiazol-2yl] 2,5diphenyl-2H-tetrazoliumbromide) assay. Cell adhesion and proliferation were most improved on TiB followed closely by TiF. The results of this study revealed an increased expression of the osteogenic marker protein alkaline phosphatase on TiB and the coated commercial titanium implants. These results suggested that precoating titanium samples with blood may improve cellular response by successfully mimicking a physiological environment that could be beneficial for clinical implant procedures.

Influence of hydrodynamic pressure on chondrogenic differentiation of human bone marrow mesenchymal stem cells cultured in perfusion system.

The natural conditions of chondrocytes in native cartilage including mechanical forces and surface topology could be simulated to enhance chondrogenesis. A perfusion system recapitulating the hydrodynamic pressure of cartilage tissue is designed. Mesenchymal stem cells (MSCs) are isolated and seeded on aligned nanofibrous PCL/PLGA scaffolds that mimic the structure of superficial zone of articular cartilage. The cell-seeded scaffolds are placed into the perfusion bioreactor and exposed to chondrogenic differentiating medium. The chondrogenesis is then investigated by histological analysis and real time PCR for cartilage-specific genes. The highest expression levels of aggrecan and type II collagen are observed in the cells cultured in the presence of differentiating medium and mechanical stimulation. The expression level of type II collagen is higher than aggrecan in presence of differentiating medium and absence of mechanical stimulation. On the contrary, the expression ratio of aggrecan is higher than type II collagen in presence of mechanical stimulation and absence of differentiating medium. These results show the dominant role of mechanical stimulation and differentiating medium on upregulated expression of aggrecan and type II collagen, respectively. The application of mechanical stimulation upon cells-seeded scaffolds could mimic superficial zone of articular cartilage tissue and increase derivation of chondrocytes from MSCs.

Delivery of disulfiram into breast cancer cells using folate-receptor-targeted PLGA-PEG nanoparticles: in vitro and in vivo investigations.

BACKGROUND: A folate-receptor-targeted poly (lactide-co-Glycolide) (PLGA)-Polyethylene glycol (PEG) nanoparticle is developed for encapsulation and delivery of disulfiram into breast cancer cells. After a comprehensive characterization of nanoparticles, cell cytotoxicity, apoptosis induction, cellular uptake and intracellular level of reactive oxygen species are analyzed. In vivo acute and chronic toxicity of nanoparticles and their efficacy on inhibition of breast cancer tumor growth is studied. RESULTS: The folate-receptor-targeted nanoparticles are internalized into the cells, induce reactive oxygen species formation, induce apoptosis and inhibit cell proliferation more efficiently compared to the untargeted nanoparticles. The acute and toxicity test show the maximum dose of disulfiram equivalent of nanoparticles for intra-venous injection is 6 mg/kg while show significant decrease in the breast cancer tumor growth rate. CONCLUSION: It is believed that the developed formulation could be used as a potential vehicle for successful delivery of disulfiram, an old and inexpensive drug, into breast cancer cells and other solid tumors.

Synthesis and characterization of an octaarginine functionalized graphene oxide nano-carrier for gene delivery applications.

The success of gene therapy is largely dependent on the development of a gene carrier. Recently cell-penetrating peptides (CPPs) have been employed for enhancing the gene and drug delivery efficacy of nano-particles. The feasibility of octaarginine (R8) functionalized graphene oxide (GO) as a novel nano-carrier for gene delivery is investigated. A DNA plasmid expressing enhanced green fluorescent protein (pEGFP) is used as a model gene to study the R8-GO transfection ability into mammalian cells. R8 peptide is conjugated in different ratios (0.1-1.5 µmol per mg of GO) to carboxylated graphene oxide by a two step amidation process. The process of peptide conjugation is analyzed by Fourier transform infrared (FTIR), atomic force microscopy (AFM), UV-vis spectroscopy and X-ray diffraction (XRD). In order to obtain the highest transfection of pEGFP into the cells, the amount of peptide bound to GO is optimized which is evidenced by dynamic light scattering (DLS), zeta potential, TNBS and gel retardation assays. The cytotoxicity of R8-functionalized GO is also tested by MTT assay. The results confirm the successful attachment of R8 peptide to GO. The AFM and XRD results show a significant increase in the thickness of nano graphene oxide sheets (NGOS) from 0.8 to 2-7 nm as well as an increase in the GO interlying space after the R8-functionalization process. A reduction in nano-carrier stability in both aqueous solution and cell culture media is observed when the amount of peptide is increased to more than 1 µmol mg(-1). Gel electrophoresis analysis shows the highest DNA loading on the peptide functionalized GO at the ratios of 0.5 and 1 µmol mg(-1). As a result, the conjugated peptide sample with a peptide molar ratio of 1 µmol per mg of GO shows the highest conjugational efficiency and EGFP gene expression along with improved dispersibility and biocompatibility. Overall, the findings reveal the importance of peptide density on the surface of NGOS in order to obtain the most efficient cell transfection. It is concluded that the R8-conjugated GO could be a promising nano-carrier for gene delivery with relevance in biotechnology therapeutics and clinical applications.

C-terminal amidation of an osteocalcin-derived peptide promotes hydroxyapatite crystallization.

Genesis of natural biocomposite-based materials, such as bone, cartilage, and teeth, involves interactions between organic and inorganic systems. Natural biopolymers, such as peptide motif sequences, can be used as a template to direct the nucleation and crystallization of hydroxyapatite (HA). In this study, a natural motif sequence consisting of 13 amino acids present in the first helix of osteocalcin was selected based on its calcium binding ability and used as substrate for nucleation of HA crystals. The acidic (acidic osteocalcin-derived peptide (OSC)) and amidic (amidic osteocalcin-derived peptide (OSN)) forms of this sequence were synthesized to investigate the effects of different C termini on the process of biomineralization. Electron microscopy analyses show the formation of plate-like HA crystals with random size and shape in the presence of OSN. In contrast, spherical amorphous calcium phosphate is formed in the presence of OSC. Circular dichroism experiments indicate conformational changes of amidic peptide to an open and regular structure as a consequence of interaction with calcium and phosphate. There is no conformational change detectable in OSC. It is concluded that HA crystal formation, which only occurred in OSN, is attributable to C-terminal amidation of a natural peptide derived from osteocalcin. It is also proposed that natural peptides with the ability to promote biomineralization have the potential to be utilized in hard tissue regeneration.

A morphology-based method for the diagnosis of red blood cells parasitized by Plasmodium malariae and Plasmodium ovale.

BACKGROUND: The morphology of red blood cells (RBCs) is altered significantly during the maturation stages of malaria parasites, which include ring, trophozoite, and schizont. There is dissimilarity in terms of the morphological characteristics of parasitized RBCs infected by the 4 species of Plasmodium, including falciparum, vivax, malariae, and ovale. This makes the process of diagnosis very difficult, which may lead to a wrong treatment method and substantial damage to the health of the patient. An innovative technique in introduced that accurately defines the shape of parasitized RBCs at each stage of infection as a potential method of diagnosis. METHODS: Giemsa-stained thin blood films were prepared using blood samples collected from healthy donors as well as patients infected with P. malariae and P. ovale. The diameter and thickness of healthy and infected RBCs at each stage of infection were measured from their optical images using Olysia and Scanning Probe Image Processor (SPIP) software, respectively. A shape equation was fitted based on the morphological characteristics of RBCs, and their relative 2-dimensional shapes were plotted using Wolfram Mathematica. RESULTS: At the ring stage, the thicknesses of RBCs parasitized by P. malariae (Pm-RBCs) and P. ovale (Po-RBCs) increased by 42% and 51%, respectively. Both Pm-RBCs and Po-RBCs remained nearly biconcave throughout parasite development even though their volumes increased. CONCLUSIONS: It is proposed that the morphology-based characterization technique introduced here could be used to intensify the accuracy of the Giemsa staining diagnosis method for the detection of the Plasmodium genus and infection stage. Based on the significant morphological alterations induced by different Plasmodium species, the results may also find practical use for faster prediction and treatment of human malaria.

An innovative shape equation to quantify the morphological characteristics of parasitized red blood cells by Plasmodium falciparum and Plasmodium vivax.

The morphology of red blood cells is affected significantly during maturation of malaria parasites, Plasmodium falciparum and Plasmodium vivax. A novel shape equation is presented that defines shape of parasitized red blood cells by P. falciparum (Pf-red blood cells) and P. vivax (Pv-red blood cells) at four stages of infection. The Giemsa-stained thin blood films are prepared using blood samples collected from healthy donors, patients having P. falciparum and P. vivax malaria. The diameter and thickness of healthy red blood cells plus Pf-red blood cells and Pv-red blood cells at each stage of infection are measured from their optical images using Olysia and Scanning Probe Image Processor softwares, respectively. Using diameters and thicknesses of parasitized red blood cells, a shape equation is fitted and relative two-dimensional shapes are plotted using MATHEMATICA. The shape of Pf-red blood cell drastically changes at ring stage as its thickness increases by 82%, while Pv-red blood cell remains biconcave (30% increase in thickness). By trophozoite and subsequent schizont stage, the Pf-red blood cell entirely loses its biconcave shape and becomes near spherical (diameter and thickness of ~8 µm). The Pv-red blood cell remains biconcave throughout the parasite development even though its volume increases. These results could have practical use for faster diagnosis, prediction, and treatment of human malaria and sickle-cell diseases.

A finite element investigation on plaque vulnerability in realistic healthy and atherosclerotic human coronary arteries.

Atherosclerosis is the most common arterial disease. It has been shown that stresses that are induced during blood circulation can cause plaque rupture and, in turn, lead to thrombosis and stroke. In this study, finite element method is used to predict plaque vulnerability based on peak plaque stress using human samples. A total of 23 healthy and atherosclerotic human coronary arteries of 14 healthy and 9 atherosclerotic patients are excised within 5 h postmortem. The samples are mounted on an uniaxial tensile test machine, and the obtained mechanical properties are used in two-dimensional and three-dimensional finite element models. The results including the Neo-Hookean hyperelastic coefficients of the samples as well as peak plaque stresses are analyzed. The results indicate that the atherosclerotic human coronary arteries have significantly (p < 0.05) higher stiffness compared with the healthy ones. The hypocellular plaque also has the highest stress values and, as a result, is most likely (vulnerable) to rupture, while the calcified type has the lowest stress values and, consequently, is expected to remain stable. The results could be used in the plaque vulnerability anticipation and have clinical implications in interventions and surgeries, including balloon angioplasty, bypass, and stenting.

An inspired microenvironment of cell replicas to induce stem cells into keratocyte-like dendritic cells for corneal regeneration.

Corneal stromal disorders due to the loss of keratocytes can affect visual impairment and blindness. Corneal cell therapy is a promising therapeutic strategy for healing corneal tissue or even enhancing corneal function upon advanced disorders, however, the sources of corneal keratocytes are limited for clinical applications. Here, the capacity of cell-imprinted substrates fabricated by molding human keratocyte templates to induce differentiation of human adipose-derived stem cells (hADSCs) into keratocytes, is presented. Keratocytes are isolated from human corneal stroma and grown to transmit their ECM architecture and cell-like topographies to a PDMS substrate. The hADSCs are then seeded on cell-imprinted substrates and their differentiation to keratocytes in DMEM/F12 (with and without chemical factors) are evaluated by real-time PCR and immunocytochemistry. The mesenchymal stem cells grown on patterned substrates present gene and protein expression profiles similar to corneal keratocytes. In contrast, a negligible expression of myofibroblast marker in the hADSCs cultivated on the imprinted substrates, is observed. Microscopic analysis reveals dendritic morphology and ellipsoid nuclei similar to primary keratocytes. Overall, it is demonstrated that biomimetic imprinted substrates would be a sufficient driver to solely direct the stem cell fate toward target cells which is a significant achievement toward corneal regeneration.

Effect of electrochemical oxidation and drug loading on the antibacterial properties and cell biocompatibility of titanium substrates.

A combination of [Formula: see text] nanotube array (TON) and controlled drug release system is employed to provide enhanced surface properties of titanium implants. Electrochemical anodization process is used to generate TON for introducing, vancomycin, an effective antibacterial drug against Staphylococcus aureus. TON loaded vancomycin is then coated with a number of layers of 10% gelatin using spin coating technique. The gelatin film is reinforced with graphene oxide (GO) nanoparticles to improve the surface bioactivity. The surface of the samples is characterized by field emission electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDS), and contact angle measurement. The results illustrate that the TON was constructed and vancomycin molecules are successfully loaded. The drug release study shows that the amount of released vancomycin is controlled by the thickness of gelatin layers. With an increase in gelatin film layers from 3 to 7, the release of vancomycin in the burst release phase decreased from 58 to 31%, and sustained release extended from 10 to 17 days. The addition of GO nanoparticles seems to reduce drug release in from 31 to 22% (burst release phase) and prolonged drug release (from 17 to 19 days). MTT assay indicates that samples show no cytotoxicity, and combination of GO nanoparticles with gelatin coating could highly promote MG63 cell proliferation. Soaking the samples in SBF solution after 3 and 7 days demonstrates that hydroxy apatite crystals were deposited on the TON surface with GO-gelatin coating more than surface of TON with gelatin. Moreover, based on the results of disc diffusion assay, both samples (loaded with Vancomycin and coated with gelatin and gelatin-GO) with the inhibition zones equaled to 20 mm show effective antibacterial properties against S. aureus. The evidence demonstrates that titania nanotube loaded with vancomycin and coated with gelatin-GO has a great potential for general applicability to the orthopedic implant field.

Microencapsulated Multifunctionalized Graphene Oxide Equipped with Chloroquine for Efficient and Sustained siRNA Delivery.

A multifunctionalized graphene oxide (GO)-based carrier with conjugation of aminated-polyethylene glycol (PEG-diamine), octaarginine (R8), and folic acid (FA), which also contains chloroquine (CQ), a lysosomotropic agent, is introduced. The cellular uptake mechanisms and intracellular targeting of FA-functionalized nanocarriers are examined. The localized releases of CQ and siRNA intracellular delivery are evaluated. Microencapsulation of the nanocarrier complexed with genes in layer-by-layer coating of alginate microbeads is also investigated. The covalently coconjugated FA with PEG and R8 provides a stable formulation with increased cellular uptake compared to FA-free carrier. The CQ-equipped nanocarrier shows a 95% release of CQ at lysosomal pH. The localized release of the drug inside the lysosomes is verified which accelerates the cargo discharge into cytoplasm.

Emerging tissue engineering strategies for the corneal regeneration.

Cornea as the outermost layer of the eye is at risk of various genetic and environmental diseases that can damage the cornea and impair vision. Corneal transplantation is among the most applicable surgical procedures for repairing the defected tissue. However, the scarcity of healthy tissue donations as well as transplantation failure has remained as the biggest challenges in confront of corneal grafting. Therefore, alternative approaches based on stem-cell transplantation and classic regenerative medicine have been developed for corneal regeneration. In this review, the application and limitation of the recently-used advanced approaches for regeneration of cornea are discussed. Additionally, other emerging powerful techniques such as 5D printing as a new branch of scaffold-based technologies for construction of tissues other than the cornea are highlighted and suggested as alternatives for corneal reconstruction. The introduced novel techniques may have great potential for clinical applications in corneal repair including disease modeling, 3D pattern scheming, and personalized medicine.

Alendronate Sodium Intercalation in Layered Double Hydroxide/Poly (ε-caprolactone): Application in Osteoporosis Treatment.

BACKGROUND: Osteoporosis is a bone disease alters the amount and variety of proteins in bone tissue and increases the potential of bone fracture. Antiresorptive therapy is one of the most popular treatment methods for osteoporosis. To reduce side effects and enhance the bioavailability of drug agents, the controlled delivery of drug is commonly utilized. OBJECTIVES: We investigated the controlled release of Alendronate in different composites of layered double hydroxide (LDH) using poly (ε-caprolactone) (PCL) as a matrix. MATERIALS AND METHODS: We prepared different microsphere composites of ALD intercalated in various amounts of LDH, using PCL as a matrix. The controlled release of ALD from these composites is subsequently investigated. Samples are characterized and in vitro cell cytotoxicity, attachment, osteogenic activity including alkaline phosphatase activity and mineralization are examined using MG-63 human osteosarcoma cells. RESULTS: The results showed that the release of ALD is more desirable and controlled in the samples having a higher amount of LDH incorporated into the PCL matrix. MG63 cells show a significant increase in viability, attachment, and mineralization while alkaline phosphatase activity remains almost at a constant level after 3 weeks. CONCLUSIONS: Overall, the findings showed that by incorporation of 15 wt% of LDH, the composite microsphere is capable of holding the antiresorptive drug longer and release it in a more controlled manner. This is an advantageous and promising characteristic for a carrier that could be used as a potential candidate for osteoporosis treatment.

Chitosan Coating of TiO2 Nanotube Arrays for Improved Metformin Release and Osteoblast Differentiation.

BACKGROUND: Ineffective integration has been recognized as one of the major causes of early orthopedic failure of titanium-based implants. One strategy to address this problem is to develop modified titanium surfaces that promote osteoblast differentiation. This study explored titanium surfaces modified with TiO2 nanotubes (TiO2 NTs) capable of localized drug delivery into bone and enhanced osteoblast cell differentiation. MATERIALS AND METHODS: Briefly, TiO2 NTs were subjected to anodic oxidation and loaded with Metformin, a widely used diabetes drug. To create surfaces with sustainable drug-eluting characteristics, TiO2 NTs were spin coated with a thin layer of chitosan. The surfaces were characterized via scanning electron microscopy, atomic force microscopy, and contact angle measurements. The surfaces were then exposed to mesenchymal bone marrow stem cells (MSCs) to evaluate cell adhesion, growth, differentiation, and morphology on the modified surfaces. RESULTS: A noticeable increase in drug release time (3 days vs 20 days) and a decrease in burst release characteristics (85% to 7%) was observed in coated samples as compared to uncoated samples, respectively. Chitosan-coated TiO2 NTs exhibited a considerable enhancement in cell adhesion, proliferation, and genetic expression of type I collagen, and alkaline phosphatase activity as compared to uncoated TiO2 NTs. CONCLUSION: TiO2 NT surfaces with a chitosan coating are capable of delivering Metformin to a bone site over a sustained period of time with the potential to enhance MSCs cell attachment, proliferation, and differentiation.

Engineered substrates with imprinted cell-like topographies induce direct differentiation of adipose-derived mesenchymal stem cells into Schwann cells.

Differentiation of stem cells to Schwann is considered efficient way for nerve regeneration since the sources of human Schwann cells are limited for clinical application. It is demonstrated that mimicking micromechanical forces or micro/nanotopographical environments that stem cells are experienced in vivo could control their fate. Here, the potency of substrates with imprinted cell-like topographies for direct differentiation of adipose-derived mesenchymal stem cells (ADSCs) into Schwann cells (SCs) is reported. For the preparation of substrates with imprinted SC-Like topographies, SCs are isolated from the sciatic nerve, grown, fixed, and then SC morphologies are transferred to polydimethylsiloxane (PDMS) substrates by mold casting. Subsequently, mesenchymal stem cells (MSCs) are seeded on the SC-imprinted substrates and their differentiation to SCs is evaluated by immunocytochemistry, real-time PCR, and western blotting. Analysis of morphology and expression of SC-specific markers show that MSCs cultured on the imprinted substrates have the typical SC-like morphology and express SC-specific markers including S100b, p75NTR, and Sox10. It is believed that specific cell-like topographies and related micromechanical cues can be sufficient for direct differentiation of ADSCs into Schwann cells by cell-imprinting method as a physical technique.

The Effect of Physical Cues on the Stem Cell Differentiation.

Development of multicellular organisms is a very complex and organized process during which cells respond to various factors and features in extracellular environments. It has been demonstrated that during embryonic evolvement, under certain physiological or experimental conditions, unspecialized cells or stem cells can be induced to become tissue or organ-specific cells with special functions. Considering the importance of physical cues in stem cell fate, the present study reviews the role of physical factors in stem cells differentiation and discusses the molecular mechanisms associated with these factors.

Contribution of osteocalcin-mimetic peptide enhances osteogenic activity and extracellular matrix mineralization of human osteoblast-like cells.

A natural peptide motif in the first helix of osteocalcin (OCN) is used to promote nucleation and crystallization of hydroxyapatite (HA) in hard tissue. The capability of osteocalcin mimetic peptides to induce osteogenic activity of osteoblast cells leading to in-vitro mineralization is demonstrated. An osteocalcin-derived peptide consisting of thirteen amino acids is synthesized in both acidic (OSC) and amidic (OSN) forms and added into the human osteoblast-like cells (MG63) culture. The viability, proliferation, alkaline phosphatase activity, HA deposition and osteogenic gene expression by osteoblast cells are evaluated. It is revealed that the addition of 100 µg/ml of peptides enhances the proliferation rate and total protein content of osteoblast cells. Alkaline phosphatase activity is significantly higher in the presence of peptides which in turn stimulated RNA expression of collagen type I and osteopontin in a phosphate-dependent manner. Alizarin red staining and calcium content measurement show that mineral deposition is considerably increased. Ultrastructural characterization of MG63 cultures confirms the crystalline nature and chemical composition of HA mineral formation in the presence of peptides. It is confirmed that the osteocalcin-derived peptide, particularly in amidic form (OSN), is able to act as a bioactive inducer of mineralization process and hence accelerating bone tissue regeneration.

Development of a PCL/gelatin/chitosan/ß-TCP electrospun composite for guided bone regeneration.

Many approaches have been developed to regenerate biological substitutes for repairing damaged tissues. Guided bone/tissue regeneration (GBR/GTR) that employs a barrier membrane has received much attention in recent years. Regardless of substantial efforts for treatment of damaged tissue in recent years, an effective therapeutic strategy is still a challenge for tissue engineering researchers. The aim of the current study is to fabricate a GBR membrane consisting of polycaprolactone (PCL)/gelatin/chitosan which is modified with different percentages of ß-tricalcium phosphate (ß-TCP) for improved biocompatibility, mechanical properties, and antibacterial activity. The membranes are examined for their mechanical properties, surface roughness, hydrophilicity, biodegradability and biological response. The mechanical properties, wettability and roughness of the membranes are improved with increases in ß-TCP content. An increase in the elastic modulus of the substrates is obtained as the amount of ß-TCP increases to 5% (145-200 MPa). After 5 h, the number of attached cells is enhanced by 30%, 40% and 50% on membranes having 1%, 3% and 5% ß-TCP, respectively. The cell growth on a membrane with 3% of ß-TCP is also 50% and 20% higher than those without ß-TCP and 5% ß-TCP, respectively. Expression of type I collagen is increased with addition of ß-TCP by 3%, while there is no difference in ALP activity. The results indicated that a composite having (3%) ß-TCP has a potential application for guided bone tissue regeneration.