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1.
J Biomed Mater Res B Appl Biomater ; 106(5): 1887-1896, 2018 07.
Article in English | MEDLINE | ID: mdl-28941021

ABSTRACT

Functionalizing medical devices with polypeptides to enhance their performance has become important for improved clinical success. The extracellular matrix (ECM) adhesion protein vitronectin (VN) is an effective coating, although the chemistry used to attach VN often reduces its bioactivity. In vivo, VN binds the ECM in a sequence-dependent manner with heparan sulfate (HS) glycosaminoglycans. We reasoned therefore that sequence-based affinity chromatography could be used to isolate a VN-binding HS fraction (HS9) for use as a coating material to capture VN onto implant surfaces. Binding avidity and specificity of HS9 were confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)-based assays. Plasma polymerization of allylamine (AA) to tissue culture-treated polystyrene (TCPS) was then used to capture and present HS9 as determined by radiolabeling and ELISA. HS9-coated TCPS avidly bound VN, and this layered surface supported the robust attachment, expansion, and maintenance of human pluripotent stem cells. Compositional analysis demonstrated that 6-O- and N-sulfation, as well as lengths greater than three disaccharide units (dp6) are critical for VN binding to HS-coated surfaces. Importantly, HS9 coating reduced the threshold concentration of VN required to create an optimally bioactive surface for pluripotent stem cells. We conclude that affinity-purified heparan sugars are able to coat materials to efficiently bind adhesive factors for biomedical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1887-1896, 2018.


Subject(s)
Coated Materials, Biocompatible/chemistry , Extracellular Matrix Proteins/chemistry , Heparitin Sulfate/chemistry , Pluripotent Stem Cells/metabolism , Vitronectin/chemistry , Cell Adhesion , Cell Line , Humans , Pluripotent Stem Cells/cytology
2.
Spine J ; 18(5): 818-830, 2018 05.
Article in English | MEDLINE | ID: mdl-29269312

ABSTRACT

BACKGROUND CONTEXT: Interbody spinal fusion relies on the use of external fixation and the placement of a fusion cage filled with graft materials (scaffolds) without regard for their mechanical performance. Stability at the fusion site is instead reliant on fixation hardware combined with a selected cage. Ideally, scaffolds placed into the cage should both support the formation of new bone and contribute to the mechanical stability at the fusion site. PURPOSE: We recently developed a scaffold consisting of silane-modified PCL-TCP (PCL-siTCP) with mechanical properties that can withstand the higher loads generated in the spine. To ensure the scaffold more closely mimicked the bone matrix, we incorporated collagen (Col) and a heparan sulfate glycosaminoglycan sugar (HS3) with increased affinity for heparin-binding proteins such as bone morphogenetic protein-2 (BMP-2). The osteostimulatory characteristic of this novel device delivering exogenous BMP2 was assessed in vitro and in vivo as a prelude to future spinal fusion studies with this device. STUDY DESIGN/SETTING: A combination of cell-free assays (BMP2 release), progenitor cell-based assays (BMP2 bioactivity, cell proliferation and differentiation), and rodent ectopic bone formation assays was used to assess the osteostimulatory characteristics of the PCL-siTCP-based scaffolds. MATERIALS AND METHODS: Freshly prepared rat mesenchymal stem cells were used to determine reparative cell proliferation and differentiation on the PCL-siTCP-based scaffolds over a 28-day period in vitro. The bioactivity of BMP2 released from the scaffolds was assessed on progenitor cells over a 28-day period using ALP activity assays and release kinetics as determined by enzyme-linked immunosorbent assay. For ectopic bone formation, intramuscular placement of scaffolds into Sprague Dawley rats (female, 4 weeks old, 120-150 g) was achieved in five animals, each receiving four treatments randomized for location along the limb. The four groups tested were (1) PCL-siTCP/Col (5-mm diameter×1-mm thickness), PCL-siTCP/Col/BMP2 (5 µg), (3) PCL-siTCP/Col/HS3 (25 µg), and (4) PCL-siTCP/Col/HS3/BMP2 (25 and 5 µg, respectively). Bone formation was evaluated at 8 weeks post implantation by microcomputed tomography (µCT) and histology. RESULTS: Progenitor cell-based assays (proliferation, mRNA transcripts, and ALP activity) confirmed that BMP2 released from PCL-siTCP/Col/HS3 scaffolds increased ALP expression and mRNA levels of the osteogenic biomarkers Runx2, Col1a2, ALP, and bone gla protein-osteocalcin compared with devices without HS3. When the PCL-siTCP/Col/HS3/BMP2 scaffolds were implanted into rat hamstring muscle, increased bone formation (as determined by two-dimensional and three-dimensional µCTs and histologic analyses) was observed compared with scaffolds lacking BMP2. More consistent increases in the amount of ectopic bone were observed for the PCL-siTCP/Col/HS3/BMP2 implants compared with PCL-siTCP/Col/BMP2. Also, increased mineralizing tissue within the pores of the scaffold was seen with modified-tetrachrome histology, a result confirmed by µCT, and a modest but detectable increase in both the number and the thickness of ectopic bone structures were observed with the PCL-siTCP/Col/HS3/BMP2 implants. CONCLUSIONS: The combination of PCL-siTCP/Col/HS3/BMP2 thus represents a promising avenue for further development as a bone graft alternative for spinal fusion surgery.


Subject(s)
Bone Regeneration , Guided Tissue Regeneration/methods , Mesenchymal Stem Cell Transplantation/methods , Spinal Fusion/methods , Tissue Scaffolds/chemistry , Animals , Bone Morphogenetic Protein 2/pharmacology , Calcium Phosphates/chemistry , Cell Proliferation , Cells, Cultured , Collagen/metabolism , Female , Heparitin Sulfate/chemistry , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Polyesters/chemistry , Rats , Rats, Sprague-Dawley
3.
Biointerphases ; 10(4): 04A308, 2015 Dec 16.
Article in English | MEDLINE | ID: mdl-26474791

ABSTRACT

Heparin has a high affinity for bone morphogenetic protein-2 (BMP-2), which is a key growth factor in bone regeneration. The aim of this study was to investigate how the rate of release of BMP-2 was affected when adsorbed to nanosized hydroxyapatite (HAP) particles functionalized with heparin by different methods. Heparin was attached to the surface of HAP, either via adsorption or covalent coupling, via a 3-aminopropyltriethoxysilane (APTES) layer. The chemical composition of the particles was evaluated using X-ray photoelectron spectroscopy and elemental microanalysis, revealing that the heparin grafting densities achieved were dependent on the curing temperature used in the fabrication of APTES-modified HAP. Comparable amounts of heparin were attached via both covalent coupling and adsorption to the APTES-modified particles, but characterization of the particle surfaces by zeta potential and Brunauer-Emmett-Teller measurements indicated that the conformation of the heparin on the surface was dependent on the method of attachment, which in turn affected the stability of heparin on the surface. The release of BMP-2 from the particles after 7 days in phosphate-buffered saline found that 31% of the loaded BMP-2 was released from the APTES-modified particles with heparin covalently attached, compared to 16% from the APTES-modified particles with the heparin adsorbed. Moreover, when heparin was adsorbed onto pure HAP, it was found that the BMP-2 released after 7 days was 5% (similar to that from unmodified HAP). This illustrates that by altering the mode of attachment of heparin to HAP the release profile and total release of BMP-2 can be manipulated. Importantly, the BMP-2 released from all the heparin particle types was found by the SMAD 1/5/8 phosphorylation assay to be biologically active.


Subject(s)
Adsorption , Bone Morphogenetic Protein 2/metabolism , Drug Liberation , Durapatite/chemistry , Heparin/metabolism , Nanoparticles/chemistry , Protein Binding , Animals , Bone Morphogenetic Protein 2/pharmacokinetics , Electron Probe Microanalysis , Humans , Photoelectron Spectroscopy , Swine , Time Factors
4.
Spine J ; 15(12): 2552-63, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26342750

ABSTRACT

BACKGROUND CONTEXT: The combination of potent osteoinductive growth factor, functional osteoblastic cells, and osteoconductive materials to induce bone formation is a well-established concept in bone tissue engineering. However, supraphysiological dose of growth factor, such as recombinant human bone morphogenetic protein 2 (rhBMP-2), which is necessary in contemporary clinical application, have been reported to result in severe side effects. PURPOSE: We hypothesize that the synergistic osteoinductive capacity of low-dose bone morphogenetic protein 2 (BMP-2) combined with undifferentiated bone marrow-derived stromal cells (BMSCs) is comparable to that of osteogenically differentiated BMSCs when used in a rodent model of posterolateral spinal fusion. STUDY DESIGN/SETTING: A prospective study using a rodent model of posterolateral spinal fusion was carried out. PATIENT SAMPLE: Thirty-six syngeneic Fischer rats comprised the patient sample. METHODS: Six groups of implants were evaluated as follows (n=6): (1) 10 µg BMP-2 with undifferentiated BMSCs; (2) 10 µg BMP-2 with osteogenic-differentiated BMSCs; (3) 2.5 µg BMP-2 with undifferentiated BMSCs; (4) 2.5 µg BMP-2 with osteogenic-differentiated BMSCs; (5) 0.5 µg BMP-2 with undifferentiated BMSCs; and (6) 0.5 µg BMP-2 with osteogenic-differentiated BMSCs. Optimal in vitro osteogenic differentiation of BMSCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR) gene analysis whereas in vivo bone formation capacity was evaluated by manual palpation, micro-computed tomography, and histology. RESULTS: Rat BMSCs cultured in fibrin matrix that was loaded into the pores of medical-grade poly epsilon caprolactone tricalcium phosphate scaffolds differentiated toward osteogenic lineage by expressing osterix, runt-related transcription factor 2, and osteocalcium mRNA when supplemented with dexamethasone, ascorbic acid, and ß-glycerophosphate. Whereas qRT-PCR revealed optimal increase in osteogenic genes expression after 7 days of in vitro culture, in vivo transplantation study showed that pre-differentiation of BMSCs before transplantation failed to promote posterolateral spinal fusion when co-delivered with low-dose BMP-2 (1/6 or 17% fusion rate). In contrast, combined delivery of undifferentiated BMSCs with low-dose BMP-2 (2.5 µg) demonstrated significantly higher fusion rate (4/6 or 67%) as well as significantly increased volume of new bone formation (p<.05). CONCLUSION: In summary, this study supports the combination of undifferentiated BMSCs and low-dose rhBMP-2 for bone tissue engineering construct.


Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Mesenchymal Stem Cells/cytology , Spinal Fusion/methods , Transforming Growth Factor beta/pharmacology , Animals , Cells, Cultured , Humans , Male , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/drug effects , Osteogenesis , Rats , Rats, Inbred F344 , Recombinant Proteins/pharmacology , Tissue Engineering/methods
5.
Spine (Phila Pa 1976) ; 40(9): 613-21, 2015 May 01.
Article in English | MEDLINE | ID: mdl-25705961

ABSTRACT

STUDY DESIGN: A rodent posterolateral spinal fusion model. OBJECTIVE: This study evaluated a protamine-based polyelectrolyte complex (PEC) developed to use heparin in enhancing the biological activity of low-dose recombinant human bone morphogenetic protein-2 (rhBMP-2) in spinal fusion. SUMMARY OF BACKGROUND DATA: rhBMP-2 is commonly regarded as the most potent bone-inducing molecule. However, poor pharmacokinetics and short in vivo half-life means that large amounts of the bioactive growth factor are required for consistent clinical outcomes. This has been associated with a number of adverse tissue reactions including seroma and heterotopic ossification. Glycosaminoglycans including heparin are known to stabilize rhBMP-2 bioactivity. Previous studies with poly-L-lysine (PLL) and heparin-based PEC carriers amplified the therapeutic efficacy of low-dose BMP-2. However, questions remained on the eventual clinical applicability of relatively cytotoxic PLL. In the present study, a protamine-based PEC carrier was designed to further enhance the safety and efficacy of BMP-2 by delivering lower dose within the therapeutic window. METHODS: A polyelectrolyte shell was deposited on the surface of alginate microbead templates using the polycation (protamine)/polyanion (heparin) layer-by-layer polyelectrolyte self-assembly protocol. rhBMP-2 was loaded onto the outermost layer via heparin affinity binding. Loading and release of rhBMP-2 were evaluated in vitro. The bone-inductive ability of 20-fold reduction of rhBMP-2 with the different carrier vehicle was evaluated using a posterolateral spinal fusion model in rats. RESULTS: In vitro uptake and release analysis, protamine-based PEC showed higher uptake and significantly enhanced control release than PLL-based PEC (P < 0.05). In vivo implantation with protamine-based and PLL-based PEC showed better fusion performances than absorbable collagen sponge-delivered same dose of rhBMP-2, and negative control group through manual palpation, micro-computed tomography, and histological analyses. CONCLUSION: Solid posterolateral spinal fusion was achieved with 20-fold reduction of rhBMP-2 when delivered using protamine-based PEC carrier in the rat posterolateral spinal fusion model. LEVEL OF EVIDENCE: N/A.


Subject(s)
Bone Morphogenetic Protein 2/pharmacokinetics , Drug Carriers/chemistry , Protamines/chemistry , Spinal Fusion/methods , Transforming Growth Factor beta/pharmacokinetics , Alginates/chemistry , Animals , Bone Morphogenetic Protein 2/administration & dosage , Bone Morphogenetic Protein 2/chemistry , Glucuronic Acid/chemistry , Heparin/chemistry , Hexuronic Acids/chemistry , Male , Microspheres , Rats, Sprague-Dawley , Recombinant Proteins/administration & dosage , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacokinetics , Tissue Engineering , Transforming Growth Factor beta/administration & dosage , Transforming Growth Factor beta/chemistry
6.
Results Immunol ; 4: 46-53, 2014.
Article in English | MEDLINE | ID: mdl-24936399

ABSTRACT

The efficacy of pEGFP (plasmid expressing enhanced green fluorescent protein)-encapsulated PEGylated (meaning polyethylene glycol coated) magnesium phosphate nanoparticles (referred to as MgPi-pEGFP nanoparticles) for the induction of immune responses was investigated in a mouse model. MgPi-pEGFP nanoparticles induced enhanced serum antibody and antigen-specific T-lymphocyte responses, as well as increased IFN-? and IL-12 levels compared to naked pEGFP when administered via intravenous, intraperitoneal or intramuscular routes. A significant macrophage response, both in size and activity, was also observed when mice were immunized with the nanoparticle formulation. The response was highly specific for the antigen, as the increase in interaction between macrophages and lymphocytes as well as lymphocyte proliferation took place only when they were re-stimulated with recombinant green fluorescence protein (rGFP). Thus the nanoparticle formulation elicited both humoral as well as cellular responses. Cytokine profiling revealed the induction of Th-1 type responses. The results suggest DNA-encapsulated magnesium phosphate (MgPi) nanoparticles may constitute a safer, more stable and cost-efficient DNA vaccine formulation.

7.
Acta Biomater ; 9(11): 9098-106, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23871940

ABSTRACT

Bone morphogenetic protein-2 (BMP-2) is known to enhance fracture healing when delivered via a bovine collagen sponge. However, collagen rapidly releases BMP-2 with a high burst phase that is followed by a low sustained phase. As a result, supra-physiological doses of BMP-2 are often required to successfully treat bone defects. High BMP-2 dosing can introduce serious side effects that include edema, bone overgrowth, cyst-like bone formation and significant inflammation. As the release behavior of BMP-2 carriers significantly affects the efficacy of fracture healing, we sought to compare the influence of two BMP-2 delivery matrices with contrasting release profiles on BMP-2 bioactivity and ectopic bone formation. We compared a thiol-modified hyaluronan (Glycosil™) hydrogel that exhibits a low burst followed by a sustained release of BMP-2 to a collagen sponge for the delivery of three different doses of BMP-2, the bioactivities of released BMP-2 and ectopic bone formation. Analysis of bone formation by micro-computed tomography revealed that low burst followed by sustained release of BMP-2 from a hyaluronan hydrogel induced up to 456% more bone compared to a BMP-2 dose-matched collagen sponge that has a high burst and sustained release. This study demonstrates that BMP-2 released with a low burst followed by a sustained release of BMP-2 is more desirable for bone formation. This highlights the therapeutic potential of hydrogels, particularly hyaluronan-based, for the delivery of BMP-2 for the treatment of bone defects and may help abrogate the adverse clinical effects associated with high dose growth factor use.


Subject(s)
Biocompatible Materials/pharmacology , Bone Morphogenetic Protein 2/administration & dosage , Bone Morphogenetic Protein 2/therapeutic use , Collagen/pharmacology , Drug Delivery Systems , Hyaluronic Acid/pharmacology , Ossification, Heterotopic/drug therapy , Transforming Growth Factor beta/administration & dosage , Transforming Growth Factor beta/therapeutic use , Animals , Bone Morphogenetic Protein 2/pharmacology , Bone and Bones/diagnostic imaging , Bone and Bones/drug effects , Cattle , Cell Line , Female , Hydrogels , Mice , Prosthesis Implantation , Rats , Rats, Sprague-Dawley , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Transforming Growth Factor beta/pharmacology , X-Ray Microtomography
8.
Biomaterials ; 33(26): 6113-22, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22687758

ABSTRACT

Bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive factor, yet its clinical use is limited by a short biological half-life, rapid local clearance and propensity for side effects. Heparin (HP), a highly sulfated glycosaminoglycan (GAG) that avidly binds BMP-2, has inherent biological properties that may circumvent these limitations. Here, we compared hyaluronan-based hydrogels formulated to include heparin (Heprasil™) with similar gels without heparin (Glycosil™) for their ability to deliver bioactive BMP-2 in vitro and in vivo. The osteogenic activity of BMP-2 released from the hydrogels was evaluated by monitoring alkaline phosphatase (ALP) activity and SMAD 1/5/8 phosphorylation in mesenchymal precursor cells. The osteoinductive ability of these hydrogels was determined in a rat ectopic bone model by 2D radiography, 3D µ-CT and histological analyses at 8 weeks post-implantation. Both hydrogels sustain the release of BMP-2. Importantly, the inclusion of a small amount of heparin (0.3% w/w) attenuated release of BMP-2 and sustained its osteogenic activity for up to 28 days. In contrast, hydrogels lacking heparin released more BMP-2 initially but were unable to maintain BMP-2 activity at later time points. Ectopic bone-forming assays using transplanted hydrogels emphasized the therapeutic importance of the initial burst of BMP-2 rather than its long-term osteogenic activity. Thus, tuning the burst release phase of BMP-2 from hydrogels may be advantageous for optimal bone formation.


Subject(s)
Bone Morphogenetic Protein 2/chemistry , Heparin/chemistry , Hyaluronic Acid/chemistry , Hydrogels/chemistry , Animals , Female , Osteogenesis/physiology , Rats , Rats, Sprague-Dawley , Tissue Engineering/methods
9.
Nanomedicine ; 7(4): 472-9, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21215332

ABSTRACT

This article reports the method of preparation of gadolinium oxide-doped silica nanoparticles (NPs) whose surface has been functionalized to anchor DNA onto it. The silica NP's surface was modified by 3-aminopropyltrimethoxysilane for DNA to bind electrostatically. Silica NPs with low polydispersity and encapsulating gadolinium oxide were prepared in the aqueous core of the reverse micelles. The average size of these spherical silica NPs doped with gadolinium oxide and dispersed in water is ∼ 50 nm as measured by dynamic light scattering and transmission electron microscopy. The plasmid DNA electrostatically held over NP's surface was firmly immobilized and protected from DNase attack. The gadolinium oxide-doped silica NPs are paramagnetic as observed from the nuclear magnetic resonance (NMR) line-broadening effect on proton spectrum of the surrounding water. In vitro transfection efficiencies of these gadolinium oxide-doped and DNA-conjugated silica NPs in COS-7 and 293T cells were found to be about 75% and 77% respectively of that of 'Polyfect®' as positive control. FROM THE CLINICAL EDITOR: This article reports the method of preparation of gadolinium oxide-doped silica nanoparticles (NPs) whose surface has been functionalized to anchor DNA. These NPs are paramagnetic with in vitro transfection efficiencies in COS-7 and 293T cells of about 75% and 77% compared to 'Polyfect®' as positive control.


Subject(s)
Contrast Media/chemistry , Gadolinium/chemistry , Genetic Vectors/chemistry , Nanoparticles/chemistry , Silicon Dioxide/chemistry , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Contrast Media/adverse effects , Gadolinium/adverse effects , Genetic Vectors/ultrastructure , Humans , Magnetic Resonance Spectroscopy , Microscopy, Electron, Transmission , Nanoparticles/adverse effects , Nanoparticles/ultrastructure , Plasmids , Transfection
10.
Tissue Eng Part C Methods ; 15(1): 105-14, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19196127

ABSTRACT

Development of an effective preservation strategy to fulfill off-the-shelf availability of tissue-engineered constructs (TECs) is demanded for realizing their clinical potential. In this study, the feasibility of vitrification, ice-free cryopreservation, for precultured ready-to-use TECs was evaluated. To prepare the TECs, bone marrow-derived porcine mesenchymal stromal cells (MSCs) were seeded in polycaprolactone-gelatin nanofibrous scaffolds and cultured for 3 weeks before vitrification treatment. The vitrification strategy developed, which involved exposure of the TECs to low concentrations of cryoprotectants followed by a vitrification solution and sterile packaging in a pouch with its subsequent immersion directly into liquid nitrogen, was accomplished within 11min. Stepwise removal of cryoprotectants, after warming in a 38 degrees C water bath, enabled rapid restoration of the TECs. Vitrification did not impair microstructure of the scaffold or cell viability. No significant differences were found between the vitrified and control TECs in cellular metabolic activity and proliferation on matched days and in the trends during 5 weeks of continuous culture postvitrification. Osteogenic differentiation ability in vitrified and control groups was similar. In conclusion, we have developed a time- and cost-efficient cryopreservation method that maintains integrity of the TECs while preserving MSCs viability and metabolic activity, and their ability to differentiate.


Subject(s)
Cryopreservation/methods , Mesenchymal Stem Cells/cytology , Nanostructures/chemistry , Stromal Cells/cytology , Tissue Engineering , Alkaline Phosphatase/metabolism , Animals , Anthraquinones , Calcium/metabolism , Cell Proliferation , Cell Shape , Cell Survival , Cells, Cultured , Collagen Type I/metabolism , Mesenchymal Stem Cells/enzymology , Nanostructures/ultrastructure , Osteogenesis , Stromal Cells/enzymology , Surface Properties , Sus scrofa , Tissue Scaffolds
11.
J Biomed Nanotechnol ; 5(1): 106-14, 2009 Feb.
Article in English | MEDLINE | ID: mdl-20055113

ABSTRACT

Magnesium phosphate (MgPi) nanoparticles (NPs) encapsulating pSVbetagal and pEGFP have been used as novel non-viral vector for targeted gene delivery. These plasmid DNA loaded magnesium phosphate nanoparticles of diameter 100-130 nm were prepared in water-in-oil microemulsion. In vitro cell viability study carried out on MCF-7, HEK, and COS-7 cells demonstrated that magnesium phosphate nanoparticles have no cytotoxic effect against cell proliferation. In vivo cytotoxicity conducted on Swiss Albino mice indicated no cytotoxic effect 3 months after intraperitoneal administration of 600 mg of void magnesium phosphate nanoparticles per Kg of body weight one-time only. In vitro transfection in COS-7 cells demonstrated that magnesium phosphate nanoparticles showed approximately 100% efficiency when compared to commercial transfecting reagent Polyfect as well as the transfection efficiency of calcium phosphate (CaPi) nanoparticles recently reported. Moreover, to explore the possibility of targeting these nanoparticles to specific tissue, we have surface modified these particles by adsorbing highlyt adhesive polymer, polyacrylic acid (PAA), followed by conjugating the carboxylic groups of the polymer with p-amino-thio-beta-galactopyranoside (PAG) using a cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and used these particles to target to liver in vivo successfully and more efficiently.


Subject(s)
DNA, Viral/genetics , DNA, Viral/pharmacokinetics , Drug Carriers/chemistry , Gene Targeting/methods , Genetic Vectors/genetics , Magnesium Compounds/chemistry , Nanoparticles/chemistry , Phosphates/chemistry , Transfection/methods , Animals , COS Cells , Chlorocebus aethiops , Crystallization/methods , DNA, Viral/administration & dosage , Macromolecular Substances/chemistry , Materials Testing , Molecular Conformation , Surface Properties
12.
Biomaterials ; 30(3): 336-43, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18930316

ABSTRACT

Application of cell--biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate-fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell-biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreservation of "ready-to-use" cell-biomaterial constructs.


Subject(s)
Alginates/metabolism , Bone Marrow Cells/cytology , Cryopreservation/methods , Fibrin/metabolism , Mesoderm/cytology , Microspheres , Stromal Cells/cytology , Alginates/ultrastructure , Animals , Bone Marrow Cells/metabolism , Calcium/metabolism , Cell Proliferation , Cell Shape , Cell Survival , Cells, Cultured , Fibrin/ultrastructure , Glucuronic Acid/metabolism , Hexuronic Acids/metabolism , Mesoderm/metabolism , Microscopy, Confocal , Minerals , Osteogenesis , Staining and Labeling , Stromal Cells/metabolism , Stromal Cells/ultrastructure , Sus scrofa
13.
Int J Pharm ; 288(1): 157-68, 2005 Jan 06.
Article in English | MEDLINE | ID: mdl-15607268

ABSTRACT

Nanoparticles of calcium phosphate encapsulating plasmid DNA (pDNA) of size 100-120 nm in diameter were prepared. XRD studies of these nanoparticles showed them to be crystalline in nature having hydroxyapatite structure. The maximum loading of pDNA and its release from nanoparticles were studied using gel electrophoresis. The time dependent size measurement of these particles demonstrated that these particles show strong aggregational behaviour in aqueous dispersion. Calcium phosphate nanoparticles were found to be dissolved even in low acidic buffer (pH 5.0) releasing the pDNA, which suggested that DNA release from these particles in the endosomal compartment was possible. In vitro transfection efficiency of these calcium phosphate nanoparticles was found to be higher than that of the commercial transfecting reagent Polyfect.


Subject(s)
Calcium Phosphates/administration & dosage , Genetic Vectors/administration & dosage , Nanostructures , Plasmids/administration & dosage , DNA/administration & dosage , DNA/genetics , Genetic Therapy/methods , Genetic Vectors/genetics , Plasmids/genetics
14.
Biomaterials ; 26(14): 2157-63, 2005 May.
Article in English | MEDLINE | ID: mdl-15576191

ABSTRACT

Nanoparticles of Mg and Mn (II) phosphates encapsulating pDNA were prepared. The sizes of these DNA loaded particles in aqueous dispersion were about 100-130 nm diameter, and they aggregated with the progression of time. Although magnesium phosphate nanoparticles were crystalline, the manganous phosphate nanoparticles were found to be amorphous in nature. Nanoparticle dissolution and pDNA release were studied using atomic absorption spectroscopy and gel electrophoresis experiments. These inorganic phosphate nanoparticles dissolved in mild acidic pH ( approximately 5) releasing pDNA indicating that DNA release in the endosomal compartment is possible. In vitro transfection in HeLa cells demonstrated that while magnesium phosphate nanoparticles showed 100% efficiency, manganous phosphate nanoparticles exhibited about 85% transfection efficiency compared to that of 'polyfect', as control.


Subject(s)
Drug Delivery Systems/methods , Magnesium Compounds/chemistry , Magnesium/chemistry , Nanotubes/chemistry , Nanotubes/ultrastructure , Phosphates/chemistry , Plasmids/administration & dosage , Plasmids/chemistry , Transfection/methods , Coated Materials, Biocompatible/administration & dosage , Coated Materials, Biocompatible/chemistry , Feasibility Studies , Genetic Vectors , HeLa Cells , Humans , Materials Testing , Particle Size , Viruses
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