Synthesis and characterization of poly(glycerol-co-sebacate-co-ε-caprolactone) elastomers.

In this study, poly(glycerol-co-sebacate-co-ε-caprolactone) (PGSCL) elastomers were synthesized for the first time from the respective monomers. The structural analysis of PGSCL elastomers by nuclear magnetic resonance ((1)H-NMR) and Fourier transform infrared spectroscopy (FTIR) revealed that the elastomers have a high number of hydrogen bonds and crosslinks. X-ray diffraction (XRD) and thermal analysis indicated an amorphous state. Differential scanning calorimetry (DSC) analysis showed that the elastomers has a glass transition temperature (T(g)) of -36.96°C. The Young's modulus and compression strength values were calculated as 46.08 MPa and 3.192 MPa, respectively. Calculations based on acid number and end groups analysis revealed a number average molecular weight of 148.15 kDa. Even though the foaming studies conducted by using supercritical CO2 resulted in a porous structure; the obtained morphology tended to disappear after 48 h, leaving small cracks on the surface. This phenomenon was interpreted as an indication of self-healing due to the high number of hydrogen bonds. The PGSCL elastomers synthesized in this study are flexible, robust to compression forces and have self-healing capacity. Thanks to good biocompatibility and poor cell-adhesion properties, the elastomers may find diverse applications where a postoperative adhesion barrier is required.

Influence of scaffold composition over in vitro osteogenic differentiation of hBMSCs and in vivo inflammatory response.

To understand the role of chitosan in chitosan-poly(butylene succinate) scaffolds (50% wt), 50%, 25%, and 0% of chitosan were used to produce different scaffolds. These scaffolds were in vitro seeded and cultured with human bone marrow stromal cells in osteogenic conditions, revealing that higher percentage of chitosan showed enhanced cell viability over time, adhesion, proliferation, and osteogenic differentiation. Scaffolds were also implanted in cranial defects and iliac submuscular region in Wistar rats, and the results evidenced that chitosan-containing scaffolds displayed mild inflammatory response and good integration with surrounding tissues, showed by connective tissue colonization and the presence of new blood vessels. Scaffolds without chitosan-evidenced necrotic tissue in scaffolds' interior, proving that chitosan exerts a positive effect over cell behavior and displays a milder host inflammatory response in vivo.

Auricular cartilage repair using cryogel scaffolds loaded with BMP-7-expressing primary chondrocytes.

The loss of cartilage tissue due to trauma, tumour surgery or congenital defects, such as microtia and anotia, is one of the major concerns in head and neck surgery. Recently tissue-engineering approaches, including gene delivery, have been proposed for the regeneration of cartilage tissue. In this study, primary chondrocytes were genetically modified with plasmid-encoding bone morphogenetic protein-7 (BMP-7) via the commercially available non-viral Turbofect vector, with the aim of bringing ex vivo transfected chondrocytes to resynthesize BMP-7 in vitro as they would in vivo. Genetically modified cells were implanted into gelatin-oxidized dextran scaffolds and cartilage tissue formation was investigated in 15 × 15 mm auricular cartilage defects in vivo in 48 New Zealand (NZ) white rabbits for 4 months. The results were evaluated via histology and early gene expression. Early gene expression results indicated a strong effect of exogenous BMP-7 on matrix synthesis and chondrocyte growth. In addition, histological analysis results exhibited significantly better cartilage healing with BMP-7-modified (transfected) cells than in the non-modified (non-transfected) group and as well as the control.

Microwave-assisted rapid synthesis of poly(glycerol-sebacate) elastomers.

Poly(glycerol-sebacate) (PGS) was introduced a decade ago as a potential material for soft tissue repair. All of the proposed copolymerization reactions in the literature include a two-stage (prepolymerization and curing) synthesis where the reaction times can take as long as several days. This study, on the other hand, proposes a new route that eliminates these disadvantages and enables a rapid synthesis of PGS elastomers via microwave-assisted prepolymerization in minutes instead of days. No purge gas, catalyst or vacuum is needed in the first prepolymerization step. The curing stage was carried out at 150 °C for 4, 8, 16, and 24 hours. The glass transition temperature (Tg) and melting temperatures for the glycerol and sebacic acid fragments (Tm1 and Tm2 ) of these PGS elastomers were found as -35.61 °C, -15.82 °C, and 61.70 °C, respectively. The Young's modulus and tensile strength values were found as 0.50 ± 0.02 MPa and 0.27 ± 0.06 MPa, respectively.

3D ingrowth of bovine articular chondrocytes in biodegradable cryogel scaffolds for cartilage tissue engineering.

A feasibility study was undertaken to examine the potential of biodegradable HEMA-lactate-dextran (HEMA-LLA-D)-based cryogels as scaffolds for cartilage tissue engineering. This was a preliminary in vitro study giving essential information on the biocompatibility of cryogels with cartilage cells. HEMA-lactate (HEMA-LLA) and HEMA-LLA-D were synthesized and characterized by different techniques. Cryogel scaffolds with supermacroporous structures were produced by cryogenic treatment of these macromers. Chondrocytes obtained from bovine articular cartilage were seeded onto cylindrical cryogels and cultured. The samples were examined by several microcopical techniques for cell viability and morphological analyses were performed at two culture points. Histological study of the constructs revealed the cells' growth on the surface and within the scaffolds. Confocal microscopical images demonstrated that the majority of live vs. dead cells had been attached to and integrated with the pores of the scaffold. SEM analysis showed round to oval-shaped chondrocytic cells interconnected with each other by communicating junctions. The chondrocytes rapidly proliferated in the cryogels, manifesting that they fully covered the scaffold surface after 9 days and almost filled the spaces in the pores of the scaffold after 15 days of culture. Chondrocytes secreted significant amount of extracellular matrix in the scaffolds and exhibited highly interconnective morphology. Light and transmission electron microscopy revealed groups of active cartilage cells closely apposed to the cryogel. We concluded that cryogel scaffolds could be excellent candidates for cartilage tissue regeneration with their extraordinary properties, including soft, elastic nature, highly open interconnected pore structure and very rapid, controllable swellability.

Growth inhibition of SK-MEL-30 human melanoma cells by antisense c-myc oligonucleotides delivered by poly(N-isopropylacrylamide)/ poly(ethyleneimine) copolymer.

The c-myc oncogene has been shown to be overexpressed in a number of malignancies and plays a key role in the abnormal growth regulation of melanoma cells. This study aimed to provide an efficient system for the in vitro manipulation of c-myc expression by antisense oligonucleotides. Therefore, we used poly(NIPA)/PEI2B copolymer as vector in order to improve the intracellular availability and stability of AS ODNs. We targeted oligonucleotide sequences within the human c-myc mRNA as free AS ODNs or conjugated with a thermosensitive copolymer, in an effort to inhibit the growth of human melanoma cells. The conjugates adopted more positive charge and smaller size at 37 degrees C and they had no toxic effects on human fibroblast cells. The conjugated AS ODNs showed increased antiproliferative effect on melanoma cells as compared to free AS ODNs. At a concentration of 100 ng, AS ODNs inhibited SK-MEL 30 human melanoma cell line proliferation maximally by 18.6%, whereas the same amount of conjugated AS ODN provided 52% inhibition. The greatest inhibition was obtained by conjugates having a polymer:AS ODN ratio of 9. Greatest inhibition was detected at 48 h and decreased after 96 h, which may be due to the depletion of AS ODNs. The results confirm the enhanced antiproliferative effects of poly(NIPA)/PEI2B-conjugated AS ODNs, which may provide improved intracellular availability for c-myc-directed antisense strategies.

Improving pore interconnectivity in polymeric scaffolds for tissue engineering.

A new scaffold fabrication technique aiming to enhance pore interconnectivity for tissue engineering has been developed. Medical grade poly(lactic acid) was utilized to generate scaffolds by a solvent-evaporating/particulate-leaching technique, using a new dual-porogen system. Water-soluble sodium chloride particles were used to control macro-pore size in the range 106-255 microm, while organic naphthalene was utilized as a porogen to increase pore interconnections. The three-dimensional (3D) morphology of the scaffolds manufactured with and without naphthalene was examined by optical coherence tomography and scanning electron microscopy. The mechanical properties of the scaffolds were characterized by compression tests. MG63 osteoblast cells were seeded in the scaffolds to study the cell attachment and viability evaluated by confocal microscopy. It was revealed that introducing naphthalene as the second porogen in the solvent-evaporating/particulate-leaching process resulted in improvement of the pore interconnectivity. Cells grew in both scaffolds fabricated with and without naphthalene. They exhibited strong green fluorescence when using a live/dead fluorescent dye kit, indicating that the naphthalene in the scaffold process did not affect cell viability.

Cathepsin K/TRAP: Can they be used to induce osteogenesis?

Most of the strategies developed in bone tissue engineering in the past three decades have been aimed to repair/regenerate the tissue with forming elements such as osteoblasts and bone morphogenetic proteins. All these materials are selected as they are known to induce bone formation. Since it is known that bone turnover in basic multicellular units (BMUs) is at equilibrium, inducing an imbalance in this process via molecules known to resorb and transmit resorption signals and therefore initiate activation in bone forming cells from adjacent tissue may offer a radical approach to bone regeneration. Possible targets for such an approach may include resorbing molecules such as tartrate resistant acid phosphatase (TRAP) and cathepsin K. Delivering these enzymes (TRAP and cathepsin K) and/or other molecules involved in bone resorption into bone defects and thus obtaining a concentration difference in the levels of these materials may induce bone forming cells to balance bone turnover, therefore inducing bone regeneration.

Controllable formation of nanoscale patterns on TiO2 by conductive-AFM nanolithography.

We report on the nanopatterning of double-bond-terminated silane (5-hexenyltrichlorosilane, HTCS) molecules on titania (TiO2) using conductive atomic force microscopy (AFM). The influences of tip electrostatic potential and scanning velocity, relative humidity and of the repeated application of voltage on the topographic height, width, and hydrophilic and hydrophobic contrast of the resultant patterns were investigated. Tip voltage and tip velocity ( v) were applied between -10 V

Separation of mesenchymal stem cells with magnetic nanosorbents carrying CD105 and CD73 antibodies in flow-through and batch systems.

The aim of this study is to develop magnetically loaded nanosorbents carrying specific monoclonal antibodies (namely CD105 and CD73) for separation of mesenchymal stem cells from cell suspensions. Super-paramagnetic magnetite (Fe3O4) nanoparticles were produced and then coated with a polymer layer containing carboxylic acid functional groups (average diameter: 153 nm and polydispersity index: 0.229). In order to obtain the nanosorbents, the monoclonal antibodies were immobilized via these functional groups with quite high coupling efficiencies up to 80%. These nanosorbents and also a commercially available one (i.e., microbeads carrying CD105 antibodies from Miltenyi Biotec., Germany) were used for separation of CD105+ and CD73+ mesenchymal stem cells from model cell suspension composed of peripheral blood (97.6%), human bone marrow cells (1.2%) and fibroblastic cells (1.2%). The initial concentrations of the CD105+ and CD73+ cells in this suspension were measured as 5.86% and 6.56%, respectively. A flow-through separation system and a very simple homemade batch separator unit were used. We were able to increase the concentration of CD105+ cells up to about 86% in the flow-through separation system with the nanosorbents produced in this study, which was even significantly better than the commercial one. The separation efficiencies were also very high, especially for the CD73+ cells (reached to about 64%) with the very simple and inexpensive homemade batch unit.

Smart and cationic poly(NIPA)/PEI block copolymers as non-viral vectors: in vitro and in vivo transfection studies.

In this study, in vitro and in vivo transfection of temperature-sensitive, polycationic poly(N-isopropylacrylamide) and polyethyleneimine copolymers (poly(NIPA)/PEI25L) were performed. Copolymer and copolymer-plasmid DNA (pDNA) complexes were positively charged as + 7.6 and + 12.8, respectively. Gel retardation assay confirmed good complex formation and release of plasmid DNA in response to temperature and pH. Cytotoxicity tests showed at least 80% smooth muscle cell (SMC) viability. The uptake of the complexes by SMCs was quite high; however, the best gene expression efficiency achieved with the copolymeric vectors was about 30% with the complex prepared with a polymer:plasmid ratio of 6. Gene expression efficiency was enhanced up to 50% by changing the temperature from 37 degrees C to 28 degrees C. Preliminary in vivo studies were performed above and below lower critical solution temperature (LCST) in lung, heart, liver, kidney, muscle and also subcutaneously in 5 week-old mice. The gene expression ratio was higher in lung, tibial muscle and subcutaneously than in other tissues (heart, liver and kidney) above LCST. Then, temperature decrease caused an increase in the amount of gene expression in tibial muscle and subcutaneously, revealing the contribution of temperature-sensitivity on DNA release and gene expression.

In vivo performance of antibiotic embedded electrospun PCL membranes for prevention of abdominal adhesions.

The aim of this study was to prepare nonwoven materials from poly(epsilon-caprolactone) (PCL) and their antibiotic containing forms by electrospinning, so as to prevent postsurgery induced abdominal adhesions in rats. epsilon-Caprolactone was first polymerized by ring-opening polymerization, and then it was processed into matrices composed of nanofibers by electrospinning. A model antibiotic (Biteral) was embedded within a group of PCL membranes. In the rat model, defects on the abdominal walls in the peritoneum were made to induce adhesion. The plain or antibiotic embedded PCL membranes were implanted on the right side of the abdominal wall. No membrane implantation was made on the left side of the abdominal wall that served as control. Macroscopical and histological evaluations showed that using these barriers reduces the extent, type, and tenacity of adhesion. The antibiotic embedded membranes significantly eliminated postsurgery abdominal adhesions, and also improved healing.

Attachment and growth of fibroblasts on poly(L-lactide/epsilon-caprolactone) scaffolds prepared in supercritical CO2 and modified by polyethylene imine grafting with ethylene diamine-plasma in a glow-discharge apparatus.

In this study, a copolymer of L-lactide and epsilon-caprolactone (Mn: 73,523, Mw: 127,990 and PI: 1.74) was synthesized by ring-opening polymerization by using stannous octoate as the catalyst. FTIR, 1H-NMR and DSC confirmed the copolymer formation. The copolymer films were prepared and a novel method was developed to produce highly porous sponges for potential use in tissue engineering. Films were subjected to supercritical CO2 at 3300 psi and 70 degrees C to create porous structures for production of possible tissue engineering scaffolds. The pore sizes were in the range of 40-80 microm. The copolymer films were pre-wetted with polyethylene imine (PEI) and then treated with ethylene diamine (EDA)-plasma in glow-discharge apparatus. Gas plasma surface modification of three-dimensional scaffolds fabricated by supercritical carbon dioxide technique was demonstrated to enhance cell adhesion, proliferation, and differentiation over 6 days in culture using L929 fibroblast cell line. Alkaline phosphatase (ALP) activity and glucose uptake in cell culture medium were followed in the cell culture experiments. Fibroblastic cell attachment and growth on the EDA-plasma treated scaffolds were rather low. However, both cell attachment and growth were significantly increased by PEI pre-treatment before EDA-plasma. The changes in ALP activity and glucose uptake also supported the cell growth behavior on these PEI and EDA-plasma treated scaffolds.

In vitro and in vivo degradation of non-woven materials made of poly(epsilon-caprolactone) nanofibers prepared by electrospinning under different conditions.

The aim of this study was to prepare non-woven materials from a biodegradable polymer, poly(epsilon-caprolactone) (PCL) by electrospinning. PCL was synthesized by ring-opening polymerization of epsilon-caprolactone in bulk using stannous octoate as the catalyst under nitrogen atmosphere. PCL was then processed into non-woven matrices composed of nanofibers by electrospinning of the polymer from its solution using a high voltage power supply. The effects of PCL concentration, composition of the solvent (a mixture of chloroform and DMF with different DMF content), applied voltage and tip-collector distance on fiber diameter and morphology were investigated. The diameter of fibers increased with the increase in the polymer concentration and decrease in the DMF content significantly. Applied voltage and tip-collector distance were found critical to control 'bead' formation. Elongation-at-break, ultimate strength and Young's modulus were obtained from the mechanical tests, which were all increased by increasing fiber diameter. The fiber diameter significantly influenced both in vitro degradation (performed in Ringer solution) and in vivo biodegradation (conducted in rats) rates. In vivo degradation was found to be faster than in vitro. Electrospun membranes were more hydrophobic than PCL solvent-casted ones; therefore, their degradation was a much slower process.

Nano- and micro-fiber combined scaffolds: a new architecture for bone tissue engineering.

One possible interesting way of designing a scaffold for bone tissue engineering is to base it on trying to mimic the biophysical structure of natural extracellular matrix (ECM). This work was developed in order to produce scaffolds for supporting bone cells. Nano and micro fiber combined scaffolds were originally produced from starch based biomaterials by means of a fiber bonding and a electrospinning, two step methodology. The cell culture studies with SaOs-2 human osteoblast-like cell line and rat bone marrow stromal cells demonstrated that presence of nanofibers influenced cell shape and cytoskeletal organization of the cells on the nano/micro combined scaffolds. Moreover, cell viability and Alkaline Phosphatase (ALP) activity for both cell types was found to be higher in nano/micro combined scaffolds than in control scaffolds based on fiber meshes without nanofibers. Consequently, the developed structures are believed have a great potential on the 3D organization and guidance of cells that is provided for engineering of 3-dimensional bone tissues.

Intelligent polymers as nonviral vectors.

The successful gene therapy largely depends on the vector type that allows a selective and efficient gene delivery to target cells with minimal toxicity. Nonviral vectors are much safer and cheaper, can be produced easily in large quantities, and have higher genetic material carrying capacity. However, they are generally less efficient in delivering DNA and initiating gene expression as compared to viral vectors, particularly when used in vivo. As nonviral vectors, polycations may work well for efficient cell uptake and endosomal escape, because they do form compact and smaller complexes with plasmid DNA and carry amine groups, which give positive charge and buffering ability that allows safe escape from endosome/lysosome. However, this is a disadvantage in the following step, which is releasing the plasmid DNA within the cytosol. In order to initiate transcription and enhance gene expression, the polymer/plasmid complex should dissociate after releasing from endosome safely and effectively. There are also other limitations with some of the polycationic carriers, for example, aggregation, toxicity, etc. Intelligent polymers, also called as 'stimuli responsive polymers', have a great potential as nonviral vectors to obtain site-, timing-, and duration period-specific gene expression, which is already exhibited in recent studies that are briefly summarized here.

Treatment of segmental bone defects in rats by the stimulation of bone marrow osteo-progenitor cells with prostaglandin E2.

An alternative to bone grafting is engineered osteo-conductive material that carries osteo-progenitor cells with osteo-stimulant factors impregnated on a malleable osteo-conductive material. We used bone marrow stem cells as the source of osteo-progenitor cells and stimulated them with prostaglandin E2 using demineralised bone matrix as a carrier. We treated 35 skeletally mature male Wistar albino rats with segmentary radial bone defects using five different treatment groups. Group I received no treatment; the remaining four groups all received a mixture of bone marrow and demineralised bone matrix. In group III, a copolymer was added. In group IV, prostaglandin E2 and in group V prostaglandin E2 within a copolymer was added to the mixture. Eight weeks after the surgical procedure, the rats were sacrificed. Radiological and histological evaluation of the radial bone showed that while there was no significant healing in groups I, II and III, there was a significant healing response in groups IV and V.

Gene delivery: intelligent but just at the beginning.

Gene therapy is used to treat genetic disorders, which may be achieved both ex vivo and in vivo. Gene-delivery systems usually include a carrier system which both protects the gene expression plasmid and allows its extracellular and intracellular trafficking. Viruses are used in most of the clinical trials today; however, they do have important drawbacks. Non-viral vectors based on lipids, water-soluble polycations, other non-condensing polymers and nano- or microparticles/capsules have been proposed. Cationic polymers, especially carrying novel targeting ligands. are receiving increasing attention. Intelligent polymers with temperature, pH, and light sensitivities for a controllable and effective non-viral transfection have recently been introduced but are just at the beginning. Our preliminary studies showed that block copolymers of N-isopropylacrylamide-acrylic acid with poly(ethylene imine) could be one example of these novel non-viral vectors.

Pulsatile roller pump perfusion is safe in high risk patients.

In this study, controllability, safety, blood cell depletion, and hemolysis of a pulsatile roller pump in high-risk patients was evaluated. Sarns 8000 roller pump (Sams, Terumo CVS, Ann Arbor, MI, USA) with a pulsatile control module was used as arterial pump in a clinical setting. Forty patients undergoing elective open heart surgery with high-risk either having chronically obstructive pulmonary disease or chronic renal failure were randomly included in the study to be operated on using pulsatile perfusion or non-pulsatile perfusion. Blood samples were withdrawn at induction of anesthesia, at the time of aortic clamping and de-clamping and at 1 hour and 24 hours following cessation of the bypass. Hematocrit and plasma free hemoglobin values were measured. We observed that the pulsatile roller pump perfusion and the extracorporeal circuit used in the clinical study is safe in high-risk patients undergoing cardiopulmonary bypass. We did not face any emboli, hemolysis, or technical problems. Pulsatile roller pump perfusion with Sarns 8000 heart-lung machine is a simple and reliable technique and can be easily applied during open heart surgery.

A novel biodegradable PCL film for tendon reconstruction: Achilles tendon defect model in rats.

This study aims to investigate applicability of poly(epsilon-caprolactone) (PCL) biodegradable films for repair of gaps in Achilles tendons in a rat model, also comparing surgical repair versus no repair approaches. PCL was synthesized with tailor-made properties, then, PCL films were prepared by solvent casting. Seventy-five outbred Sprague-Dawley rats were randomly allocated into five groups: (i) sham operated (skin incision only); (ii) no repair (complete division of the Achilles tendon and plantaris tendon without repair); (iii) Achilles repair (with a modified Kessler type suture); and (iv) plasty of Achilles tendon defects with the biodegradable PCL films, and (v) animals subjected to 1 cm mid-substance defect with no repair. Functional performance was determined from the measurements of hindpaw prints utilizing the Achilles functional index. The animals were killed 8 weeks after surgery and histological and biomechanical evaluations were made. All groups subjected to Achilles tendon division had a significant functional impairment that gradually improved so that by day 28 there were no functional impairments in any group whereas animals with a defect remained impaired. The magnitude of the biomechanical and morphological changes at postoperative 8 weeks were similar for no repair group (conservative), Achilles repair group and tendonplasty group (biodegradable PCL film group). The initial rate of functional recovery was significantly different for primary suture, Achilles repair group and PCL film group (p>0.01). But, at the 28th day, functional recovery was quite similar to the other groups. In summary, our results suggest that the PCL film can be an alternative biomaterial for tendon replacement.