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Hasan Uludag

Publications and source records attributed to Hasan Uludag.

17 recordsLinked to original sources
PubMed2006

RGD conjugation to polyethyleneimine does not improve DNA delivery to bone marrow stromal cells.

Bone marrow stromal cells (BMSC) modified with therapeutic genes are being actively pursued for gene therapy protocols. To develop safe and effective nonviral methods for BMSC modification, the cationic polymer polyethyleneimine (PEI) has been utilized to condense plasmid DNA for intracellular delivery. This study was conducted to explore the feasibility of increasing the PEI's effectiveness by coupling integrin-binding arginine-glycine-aspartic acid (RGD) peptides to the polymer. BMSC from rats were isolated and expanded in culture for gene transfer studies. In contrast to our expectations, RGD-conjugated PEI did not exhibit an enhanced binding to BMSC. This was the case where the peptides were conjugated to PEI by short, disulfide linkages or long poly(ethylene glycol) linkages. Using a reporter gene for the enhanced green fluorescent protein, the transfection efficiency of RGD-conjugated PEI was also lower than the delivery by the native PEI, which exhibited equivalent transfection efficiency to that of an adenovirus. We conclude that native PEI was sufficient for the transformation of BMSC and that coupling of the integrin-binding RGD-peptides did not improve the effectiveness of this polymer for BMSC transfection.

Amino Acid Sequence↗
PubMed2005

Imparting mineral affinity to proteins with thiol-labile disulfide linkages.

Chemical conjugation of bisphosphonates (BPs) to proteins is an effective means to enhance binding of proteins to mineral-containing biomaterials. BPs linked to proteins with reversible (i.e., cleavable) linkages were considered desirable over the conjugates linked with stable linkages because cleavable linkages allow protein release in free form from the mineral-containing biomaterials. To explore the feasibility of creating cleavable BP-protein conjugates, an amine- and a thiol-containing BP were conjugated to the model protein Bovine Serum Albumin (BSA) with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), which resulted in disulfide-linked BP-BSA conjugates. Although disulfide-linked conjugates were stable under aqueous conditions, the conjugates in solution were readily cleaved in the presence of physiological concentrations (approximately 0.3 mM) of the thiol compound, cysteine. The imparted mineral affinity as a result of BP conjugation, as assessed by hydroxyapatite (HA) binding in vitro, was lost upon cleavage of the disulfide-linked BP. The conjugates bound to HA were also cleavable with cysteine, but their cleavage rate was significantly reduced as compared to the conjugates in solution. In conclusion, disulfide-linked BP conjugates were shown to be readily cleavable by the amino acid cysteine and this resulted in the loss of imparted mineral affinity of the proteins. The proposed approach will be useful for modulating in vivo delivery of proteins implanted with mineral-containing biomaterials.

Animals↗
PubMed2005

RGD-grafted thermoreversible polymers to facilitate attachment of BMP-2 responsive C2C12 cells.

The purpose of this study was to design thermoreversible biomaterials for enhanced adhesion of bone morphogenetic protein-2 (BMP-2)-responsive cells. Peptides containing the arginine-glycine-aspartic acid (RGD) sequence were conjugated to N-isopropylacrylamide (NiPAM) polymers via amine-reactive N-acryloxysuccinimide (NASI) groups. In monolayer cultures, the adhesion of BMP-2-responsive C2C12 cells to RGD-grafted NiPAM/NASI surfaces was significantly higher than adhesion on ungrafted NiPAM/NASI surfaces. Although the morphology of cells adhered to RGD-grafted NiPAM/NASI surfaces was comparable to cells adhered on tissue culture polystyrene (TCPS), long-term cell growth was limited on the NiPAM/NASI surfaces, even for RGD-grafted surfaces. Treatment of C2C12 cells with recombinant BMP-2 induced dose-dependent osteoblastic differentiation as assessed by alkaline phosphatase (ALP) activity. In the absence of BMP-2, cells cultured on NiPAM/NASI polymers (either grafted with RGD peptide or not) expressed significantly higher levels of ALP activity than the cells cultured on TCPS, indicating that the polymer surfaces induced some osteoblastic activity in C2C12 cells without the need for BMP-2. We conclude that NiPAM-based thermoreversible biomaterials, despite their limited ability to support cell growth, allowed an enhanced expression of the chosen osteogenic marker (ALP) by C2C12 cells in vitro.

Acrylamides↗
PubMed2004

Imparting bone mineral affinity to osteogenic proteins through heparin-bisphosphonate conjugates.

Although numerous growth factors can promote the regeneration of bone upon parenteral administration, all exhibit undesirable side-effects that prevent their clinical utility. These side-effects arise due to the growth factors' inherent lack of bone affinity. The goal of this study was to develop a means to enhance the bone mineral affinity of osteogenic growth factors so as to minimize their extra-skeletal distribution. Heparin, a glycosaminoglycan that exhibits a high affinity to numerous growth factors, was modified with bisphosphonates to enhance its affinity to bone mineral (i.e. hydroxyapatite, HA). To this end, conjugation of 1-amino-1,1-diphosphonate methane (aminoBP) onto periodate-oxidized heparin using 4-(maleimidomethyl)cyclohexane-1-carboxyl-hydrazide was attempted. Using this chemistry, the number of aminoBPs conjugated onto heparin was modulated by varying the reacting reagent concentrations (to a maximum of 7 aminoBPs per heparin). Increasing the number of aminoBPs resulted in a more than twofold increase in heparin's affinity for HA in vitro. Subsequently, the ability of aminoBP-heparin conjugates to enhance the mineral affinity of basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) was explored. The results revealed that the complexation between the conjugates and growth factors had occurred and that the conjugates increased the bone mineral affinity of bFGF and BMP-2 in an aminoBP-dependent manner. In conclusion, the conjugation of aminoBP onto heparin is a feasible approach to enhance the affinity of heparin-binding, osteogenic growth factors to HA.

Bone Morphogenetic Protein 2↗
PubMed2004

Systemic bone formation with weekly PTH administration in ovariectomized rats.

PURPOSE: Weekly subcutaneous administration of 0 (vehicle), 10 and 80 microg/kg doses of human parathyroid hormone (1-34) [PTH (1-34)] were compared based on their capacity to induce systemic formation of bone in 9 month-old ovariectomized (OVX) Sprague-Dawley rats. METHODS: Changes elicited at bone tissue after 4 weeks of treatment were assessed using dual x-ray absorptiometry, micro-computed tomography (microCT), and ashing. RESULTS: The 10 microg/kg dose led to a significant increase (p<0.025) in femoral bone mineral density (BMD) over vehicle- and 80 microg/kg-treated groups. Similarly, structural analysis of the femoral neck trabecular bone by microCT revealed increases in bone volume fraction and trabecular thickness over the pre-treatment baseline, and vehicle- and 80 microg/kg-treated groups. CONCLUSIONS: The data suggest that the weekly administration of 10 microg/kg of PTH (1-34) was sufficient to significantly promote the bone mineral density systemically. The weekly administration of 10 microg/kg over a 4-week treatment period is, to our knowledge, one of the lowest reported total dose of PTH (1-34) shown to induce a net anabolic effect on skeletal tissue in OVX rats.

Animals↗
PubMed2004

Temperature-sensitive polymer-conjugated IFN-gamma induces the expression of IDO mRNA and activity by fibroblasts populated in collagen gel (FPCG).

Indoleamine 2,3-dioxygenase (IDO) is an intracellular tryptophan-catabolizing enzyme possessing various immunosuppressive properties. Here, we report the use of this enzyme to suppress the proliferation of peripheral blood mononuclear cells (PBMC) co-cultured with IDO-expressing fibroblasts of an allogeneic skin substitute in vitro. Fetal foreskin fibroblasts populated within collagen gel (FPCG) were treated with interferon-gamma (IFN-gamma) conjugated with a temperature-sensitive polymer to induce the expression of IDO mRNA and protein. SDS-PAGE showed successful conjugation of IFN-gamma with the temperature-sensitive polymer. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by the measurement of kynurenine levels. The results of Northern blot analysis showed an induction of IDO mRNA expression when treated with polymer-conjugated IFN-gamma. Kynurenine levels, as a measure of IDO bioactivity, were significantly higher in IFN-gamma-treated fibroblasts than in controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA in FPCG treated with polymer-conjugated IFN-gamma was significantly longer than in those treated with free (non-conjugated) IFN-gamma (P < 0.001). IFN-gamma radiolabeling showed a prolonged retention of IFN-gamma within collagen gel in its polymer-conjugated form, compared to its free form. Presence of IDO protein in FPCG was demonstrated by Western analysis even 16 days after removal of the conditioned medium (containing released IFN-gamma). To demonstrate the immunosuppressive effects of IDO on the proliferation of PBMC, IDO-expressing FPCG treated with polymer-conjugated IFN-gamma were co-cultured with PBMC for a period of 5 days. The results showed a significant reduction in proliferation of PBMC co-cultured with IFN-gamma-treated IDO-expressing fibroblasts, compared to those co-cultured with non-IDO-expressing fibroblasts (P < 0.001). The addition of an IDO inhibitor (1-methyl-D-tryptophan) reversed the suppressive effects of IDO on PBMC proliferation. In conclusion, IDO expression in FPCG suppresses the proliferation of immune cells in vitro. The use of a temperature-sensitive polymer further prolongs the effect of IFN-gamma on the expression of IDO. Therefore, modulating IDO levels in situ might be an alternative for prolonging the survival of skin allografts.

Antineoplastic Agents↗
PubMed2004

Growth factor delivery for bone tissue repair: an update.

Growth factors (GFs) are endogenous proteins capable of acting on cell-surface receptors and directing cellular activities involved in the regeneration of new bone tissue. The specific actions and long-term effects of GFs on bone-forming cells have resulted in exploration of their potential for clinical bone repair. The concerted efforts have led to the recent approval of two GFs, bone morphogenetic protein-2 and osteogenic protein-1, for clinical bone repair, and human parathryroid hormone (1-34) for augmentation of systemic bone mass. This review provides a selective summary of recent (2001-2004) attempts for GF delivery in bone tissue regeneration. First, a summary of non-human primate studies involving local regeneration and repair is provided, with special emphasis on the range of biomaterials used for GF delivery. Next, efforts to administer GFs for systemic augmentation of bone tissue are summarised. Finally, an alternative means of GF delivery, namely the delivery of genes coding for osteogenic proteins, rather than the delivery of the proteins, is summarised from rodent models. To conclude, future avenues of research considered promising to enhance the clinical application of GFs are discussed.

Animals↗
PubMed2003

Immune cell proliferation is suppressed by the interferon-gamma-induced indoleamine 2,3-dioxygenase expression of fibroblasts populated in collagen gel (FPCG).

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme, is an intracellular enzyme possessing various immunosuppressive properties. Here, we report the possible use of this enzyme to suppress proliferation of immune cells cocultured with IDO-expressing fibroblasts of an allogenic skin substitute. Fetal skin fibroblasts embedded within bovine collagen were treated with cytokine interferon-gamma (IFN-gamma) to induce expression of IDO mRNA and protein. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by measurement of kynurenine and tryptophan levels in the IFN-gamma untreated and treated fibroblasts. The results of Northern analysis showed a dose-dependent increase in expression of IDO mRNA in response to various concentrations of IFN-gamma used. The levels of kynurenine and tryptophan measured, as the bioactivity of IDO, were significantly different in the IFN-gamma treated fibroblasts, compared to those of controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA was gradually reduced to an undetectable level within 32 h of IFN-gamma removal. The results of Western blot analysis, however, revealed a significantly longer (192 h) lasting effect of IFN-gamma on IDO protein level, relative to that of mRNA expression. To demonstrate immunosuppressive effects of IDO on proliferation of immune cells, IDO-expressing fibroblasts were cocultured with peripheral blood mononuclear cells (PBMC) for a period of 5 days. The results of (3)H-thymidine incorporation showed a significant reduction in proliferation of PBMC when cocultured with IDO-expressing fibroblasts, compared to those cocultured with non-IDO-expressing fibroblasts (P < 0.001). Furthermore, addition of IDO-inhibitor (1-methyl-d-tryptophan) reversed the suppressive effects of IDO on PBMC proliferation in a dose-dependant fashion. To test the viability of immune cells cocultured with IDO-expressing fibroblasts, FACS analysis of the PI stained PBMC was conducted and no significant difference was found between these cells and the controls. In another set of experiments, we showed that migration rate and subsequent proliferation of IDO-expressing fibroblasts are also the same as those of control cells. In conclusion, IDO-expressing allogenic fibroblasts embedded within collagen gel suppress the proliferation of allogenic immune cells, while they still remain viable in this IDO-induced tryptophan-deficient culture environment.

Animals↗
PubMed2002

Synthetic thermoreversible polymers are compatible with osteoinductive activity of recombinant human bone morphogenetic protein 2.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is currently in clinical studies as part of an implantable device that contains a biomaterial carrier. Implant retention of rhBMP-2 by the biomaterial carrier is important for the osteoinductive activity. To control in situ retention of rhBMP-2, thermoreversible polymers were synthesized and characterized, and their compatibility with rhBMP-2-induced osteoinduction was investigated. The results indicated that polymers with a controlled "solubility <--> insolubility" transition temperature could be prepared from N-isopropylacrylamide, ethylmethacrylate, and N-acryloxysuccinimide (NASI). NASI-containing polymers were able to conjugate to rhBMP-2 without additional cross-linkers. Implantation in the rat ectopic model, where alkaline phosphatase and calcium deposition were utilized as markers of osteoinductive activity, indicated that rhBMP-2 mixed with the polymers were effective for osteoinduction. Moreover, rhBMP-2 conjugated to the chosen polymers was as effective as native rhBMP-2 in inducing ALP activity and calcium deposition. We conclude that thermoreversible polymers are compatible with rhBMP-2-induced osteogenesis and can serve as novel biomaterials for rhBMP-2 delivery.

Alkaline Phosphatase↗
PubMed2002

Bisphosphonates as a foundation of drug delivery to bone.

Bisphosphonates are organic analogues of pyrophosphate that are resistant to hydrolysis. Bisphosphonates exhibit an exceptional affinity to bone, which led to exploration of their utility for targeting pharmacological agents to bone. Among the pharmacological agents explored for bone delivery are radioisotopes, anti-neoplastic drugs, agents intended for augmentation of systemic bone mass, anti-inflammatory drugs and proteins. This review is intended to provide a summary of the literature on bisphosphonate-based drug delivery. A survey of therapeutic agents designed for bone targeting was provided in the first part of this review. Special emphasis was placed on the preclinical performance of the developed agents. In the second part, several aspects that were considered critical for the success of bisphosphonate-based drug delivery were explored. These aspects were oral bioavailability, protein binding of BPs, the nature of a BP-drug linkage and the choice of bone-affinity molecule. This article concludes with the author's perspective on the future of bisphosphonate-based drug delivery.

Anti-Inflammatory Agents↗
PubMedDate not supplied

Cleavage of disulfide-linked fetuin-bisphosphonate conjugates with three physiological thiols.

An effective therapeutic agent for treatment of bone diseases is expected to exhibit a high affinity to bone. Conjugating proteins to bisphosphonates (BPs), a class of molecules with an exceptional affinity to bone mineral hydroxyapatite (HA), is a feasible means to impart such a bone affinity. Protein-BP conjugates with cleavable linkages, which allow protein release from the mineral, are preferable over conjugates with stable linkages. To this end, 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (thiolBP) was conjugated onto fetuin, a model protein, using N-succinimidyl-3-(2-pyridyldithio)propionate to create disulfide-linked conjugates. Although the fetuin-thiolBP conjugates were stable under aqueous conditions, the disulfide linkage was readily cleaved in the presence of the physiological thiols l-cysteine, dl-homocysteine, and l-glutathione. dl-Homocysteine exhibited the highest cleavage of the disulfide linkage among these thiols. The imparted bone affinity as a result of thiolBP conjugation, as assessed by HA binding in vitro, was eliminated upon cleavage of the disulfide linkage. The cleavage of the conjugates bound to HA was as effective as the conjugate cleavage in solution, and even more so at high concentrations of l-glutathione. In conclusion, disulfide-linked fetuin-thiolBP conjugates exhibited a high affinity to HA, which was readily lost upon cleavage with thiols found in physiological milieu.

Biocompatible Materials↗
PubMedDate not supplied

Targeting systemically administered proteins to bone by bisphosphonate conjugation.

To develop a methodology for bone-specific delivery of proteins, a bone-seeking aminobisphosphonate (aminoBP) was previously conjugated to a model protein, bovine serum albumin (BSA). The conjugates were shown to exhibit a high affinity to bone in vitro and in vivo. This study was conducted to determine whether the systemic delivery of proteins to bone can be increased by aminoBP conjugation. Two model proteins used for this study were BSA and lysozyme (LYZ). For each protein, an unmodified (i.e., control) and aminoBP-conjugated protein were (125)I-labeled and injected into rats, and the organ delivery of the proteins were determined. Intravenous (IV) injection of aminoBP-BSA resulted in a 2.0- to 3.7-fold increased delivery to bones as compared to the control protein in young rats. In osteopenic, ovariectomized rats, aminoBP conjugation enhanced the bone delivery of BSA by 2.2- to 7.5-fold. A 3.7- to 5.6-fold increased delivery was also observed for LYZ after IV injection in normal rats. In addition to IV route of administration, subcutaneous injection was also effective in delivering a higher amount of aminoBP-conjugated proteins to bone. We conclude that conjugating bone-seeking aminoBPs to proteins improved their delivery to mineralized tissues. The proposed targeting approach has the potential to improve the efficacy of recombinant proteins capable of stimulating bone formation by enhancing their localization to bones.

Animals↗
PubMedDate not supplied

Imparting mineral affinity to fetuin by bisphosphonate conjugation: a comparison of three bisphosphonate conjugation schemes.

Protein conjugation to bisphosphonic acids (BPs), such as 1-amino-1,1-diphosphonate methane (aminoBP) and 3,5-di(ethylamino-2,2-bisphosphono)benzoic acid (diBP), was proposed as a foundation for bone-specific delivery of protein therapeutics. This study was performed to directly compare the mineral affinity of protein-BP conjugates prepared by three different approaches. Fetuin, serving as a model protein, was derivatized with BPs by the following approaches: (i) by attaching the aminoBPs onto protein lysines using succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC); (ii) by attaching the aminoBPs onto protein carbohydrates using 4-(maleimidomethyl)-cyclohexane-1-carboxyl-hydrazide (MMCCH), and (iii) by conjugating diBP to protein lysines using the carbodiimide chemistry. The results indicated that conjugation of aminoBP and diBP to fetuin by all three means unequivocally enhanced the protein's affinity for hydroxyapatite in vitro. Similarly, conjugation of aminoBP and diBP onto fetuin increased the protein's retention in a mineral-containing matrix (Pro-Osteon) when the proteins were implanted in a rat subcutaneous model. Upon parenteral administration, however, no discernible differences were found between the SMCC- or MMCCH-linked conjugates and unmodified fetuin to target to bony tissues. DiBP-fetuin conjugates, however, led to successful bone targeting after intravenous injection in rats. We conclude that all three conjugation schemes were equally effective in imparting an affinity to the proteins toward mineral-containing matrices. Bone targeting, however, was achieved only with diBP conjugation to fetuin, supportive of the superior ability of this BP with a higher density of bisphosphonic acid groups.

Animals↗
PubMedDate not supplied

Proliferation of peripheral blood mononuclear cells is suppressed by the indoleamine 2,3-dioxygenase expression of interferon-gamma-treated skin cells in a co-culture system.

Indoleamine 2,3-dioxygenase (IDO) is an intracellular tryptophan-oxidizing enzyme possessing various immunosuppressive characteristics. In this study, we report the possible use of this enzyme in an allogenic skin substitute to suppress the proliferation of immune cells. Human fetal skin fibroblasts and keratinocytes were treated with the cytokine interferon-gamma to induce expression of IDO mRNA and protein. IDO enzyme activity was evaluated by measurement of kynurenine levels in the interferon-gamma-treated and -untreated cells. Results of Northern analysis showed a dose-dependent response in expression of IDO mRNA to the various concentrations of interferon-gamma used. Northern blot analysis also showed a time-dependent expression of IDO in response to different durations of interferon-gamma treatment. The level of kynurenine measured, as the bioactivity of IDO enzyme, was significantly higher in the interferon-gamma-treated fibroblasts and keratinocytes compared to those of controls (p < 0.001). To illustrate the immunosuppressive effects of IDO on immune cell proliferation, IDO-expressing fibroblasts were cocultured with human peripheral blood mononuclear cells for a period of 5 days. Results of 3H-thymidine incorporation assays showed a significant reduction in proliferation of the mononuclear cells cocultured with IDO-expressing skin cells compared to monocytes cocultured with control (non-IDO-expressing) skin cells (p < 0.001). Furthermore, addition of the IDO-inhibitor (1-methyl-D-tryptophan) significantly reversed the immunosuppressive effects of IDO on monocyte proliferation (p < 0.001). In conclusion, suppression of peripheral blood mononuclear cell proliferation due to interferon-gamma-induced IDO-expression in allogenic human skin cells might shed new light on developing a nonrejectable allogenic skin substitute.

Cell Division↗
PubMedDate not supplied

Bone marrow cells from normal and ovariectomized rats respond differently to basic fibroblast growth factor and bone morphogenetic protein 2 treatment in vitro.

The protein growth factors basic fibroblast growth factor (bFGF) and bone morphogenetic protein 2 (BMP-2) are being actively pursued for bone tissue engineering. Although both proteins are capable of stimulating osteogenic activity of bone marrow cells (BMCs), no studies have addressed the effect of estrogen deficiency on the growth factor responsiveness of BMCs. This study investigated the osteogenic response of BMCs from normal and ovariectomized (OVX) rats to bFGF and BMP- 2. In the absence of growth factors, a higher number of total colony-forming units (t-CFU) and alkaline phosphatase-expressing CFU (ALP-CFU) were obtained with BMCs derived from OVX rats. The percentage of ALP-CFU, however, was not significantly different between BMCs from the two groups of rats. Whereas BMP-2 did not influence the t-CFU and percentage of ALP-CFU, bFGF decreased t-CFU in BMCs derived from OVX rats and reduced the percentage of ALP-CFU in BMCs from both types of rats. Consistent with the higher t-CFU, the number of mineralized colonies (min-CFU) was also higher for BMCs derived from OVX rats. The number of min-CFU was not influenced by BMP-2 treatment, but was reduced with bFGF treatment. Comparison of the growth factor effects on a per-cell (DNA) basis confirmed the expected stimulatory effect of BMP-2 on ALP activity and mineralization in BMCs from normal rats, but these two parameters were not unequivocally stimulated in BMCs from OVX rats. We conclude that BMCs derived from normal and OVX rats exhibited significant differences in their osteogenic response to bFGF and BMP-2 treatment.

Animals↗