Electrospinning is a very powerful method to create cellular scaffolds for regenerative medicine – especially for artificial vascular grafts. Commercially available thermoplastic polyurethane elastomers (TPUs), like Pellethane™ are FDA approved and have already shown excellent biomechanical properties as electrospun vascular grafts. In order to induce the growth of a neo artery and hence increase the long-term patency of the graft, the use of biodegradable TPUs is beneficial. Therefore we aim for the development of degradable TPUs. In preliminary studies the mechanical properties of segmented TPUs were examined. The tendencies of the properties of the compression-molded bulk materials were also found for the electrospun materials. It could also be shown that the substitution of the aromatic 4,4′-methylene diphenyl diisocyanate building blocks in Pellethane™ with the aliphatic hexamethylene diisocyanate – to avoid toxic aromatic amines as degradation products - only causes minor loss of strength. To obtain degradable TPUs, our concept is to incorporate cleavable ester bonds into the polymer chain. For this purpose, lactic- and terephthalic ester-based cleavable chain extenders were used. The expected degradation products showed no cytotoxicity in-vitro. Degradation tests of polymer samples in phosphate buffered saline at elevated temperatures confirmed the degradability of the new polymers.

A bioglass of composition SiO2 (67.12 mol%), CaO (28.5 mol%), and P2O5 (4.38%) was synthesized and stabilized by a novel technique using ethanol. Bioactive glasses have a wide range of application in the field of biomaterials promoting bone bonding as well as bonding to soft tissue. Earlier our lab developed a novel PVA-PCL semi IPN porous and 3D scaffold that was found to favor chondrogenesis. In the present study, a composite of this polymer and bioglass is prepared by an emulsion freeze-drying process, as a porous 3 dimensional scaffold. The scaffolds were characterized for their physiochemical properties and ability to support cartilage tissue regeneration. The composite scaffolds were observed to be non-cytotoxic. The chondrocytes cells cultured in vitro for a month on the composite scaffolds regenerate cartilaginous tissue, secreting GAGs and collagen in amounts nearly comparable to the amounts on the control PVA-PCL scaffold. The composite scaffold is also biomimetic and bioactive and favors mineralization by forming a hydroxycarbonate apatite layer, when immersed in simulated body fluid for a 14 day period. The PVA-PCL-bioglass composite is hence expected to have potential implications as a scaffold for osteochondral tissue engineering.

Bioglass 45S is a promising bone implant material with superior biocompatibility. Past research showed that adhesion of bone cells to titanium is strongly affected by its surface architecture. However, little is known about the role of surface topology of glass on its use as an implant. Thus, we systematically investigated the effect of surface roughness (Ra ∼ 0.01 – 1.2 μm) on cell adhesion and proliferation on 45S Bioglass in vitro. MG63 osteosarcoma and MC3T3 osteoblast precursor cells were seeded on the glass samples, and incubated for up to 6 days. The attachment, morphology and proliferation of cells were investigated using fluorescence microscopy. Our results show that cell attachment (as indicated by cell spreading and number of focal adhesion sites), and proliferation rate decrease with increasing roughness of bioactive glass surface. These findings provide important insight for improving surface characteristics of bioactive glass bone implants.

Additive Manufacturing Technologies (AMTs) have become an appealing method for the fabrication of 3D cellular scaffolds for tissue engineering and regenerative medicine. To circumvent the use of (meth)acrylate based photopolymers, that suffer from skin irritation and sometimes cytotoxicity, new monomers based on vinyl esters, carbonates and carbamates were prepared. The new materials, giving poly(vinyl alcohol) upon hydrolysis, showed similar results compared to (meth)acrylate references concerning the photoreactivity and mechanical properties, yet being significantly less cytotoxic.

To study the kinetics of hydrolytic degradation, the influence of the different polymerizable groups was investigated by hydrolysis of model compounds under alkaline conditions. We were able to show that the ester moiety of a vinyl ester based polymer could be used to immobilize alkaline phosphatase, therefore they exhibit the ability to immobilize enzymes for selective cell adhesion.

Finally, 3D test structures by AMT techniques could be fabricated and in-vivo testing thereof proofed the biocompatibility of vinyl ester-based scaffolds.

Cell fate modification is a critical step in preparing cells and tissues for implantation therapeutics. Novel materials possessing physical, mechanical, and chemical properties similar to those found in vivo provide a potential platform in building artificial microenvironments for therapeutic applications and well-defined biointerfaces for examining differentiation potential in stem cell biology. Poly(glycerol sebacate) (PGS), a novel biocompatible and biodegradable elastomer is one such material. With tunable mechanical properties in the range of common soft tissue, the material provides an invaluable alternative platform for use in cell-to-substrate interaction studies. This paper describes the tunability of PGS mechanical properties based on variations in curing temperatures (130, 140, and 165 °C). We characterized the material by examining properties that include equilibrium Young's modulus (E), glass transition temperature (Tg), loss factor (tan δ), degree of crosslinking, cure kinetics, protein conformational changes, and molecular bonding compositions. Variations in PGS curing temperature provide differences in physical cues presented to the hMSCs, and work is underway to examine the cellular responses of these hMSCs to microstructured PGS. Ultimately, micro- and nanostructured PGS may be useful tools in the maintenance, differentiation, and control of signaling pathways in hMSCs.

Magnesium and its alloys are used as implants because of their biocompatibility and high strength-to-weight ratio. In contrast to other commonly used implantable materials such as stainless steel and Co-Cr-alloys, which may release toxic metallic ions, magnesium belongs to the natural composition of the human body. Our work is focused on engineering a cellular carrier system based on biodegradable magnesium and magnesium alloy substrates, which are decorated with gold nanoparticles to form magnesium-gold (Mg-Au) hybrid materials. Specifically, we deposited gold nanoparticles on MgO single crystals (100 orientation) and on AZ31-Mg alloy substrates using diblock copolymer micelle nanolithography. The gold nanoparticles were arranged in ordered arrays with controllable interparticle distances of 25 to 300 nm and were further functionalized with RGD-thiols or fibronectin. This method allowed the deposition of a protein carpet on top of the surface and facilitated the initial adhesion of cells on the magnesium substrates. In order to determine the stability of the substrates in a physiological environment, their corrosive behavior was studied by comparing the weight of the substrates before and after a 24h-long submersion in water, PBS, or cell culture medium and by studying the post-submersion surface morphology with scanning electron microscopy. Corrosion rates of magnesium substrates in PBS and cell culture medium were significantly higher than in water. The spreading and survival of C2C12 mouse myoblasts and human mesenchymal stem cells cultured on MgO single crystals and AZ31-alloys were investigated with fluorescence and phase contrast microscopy. The spreading and survival of C2C12 myoblasts on the Mg-Au hybrid materials were different than on non-functionalized Mg substrates. Additionally, cells on non-functionalized MgO crystals showed reduced filopodia activity. Our results show that biofunctionalized and engineered magnesium-based substrates can be used as carriers for different cellular systems and promising initial steps have been taken towards an implantable device with a defined biological surface activity made from magnesium materials.

Rapid healing of acute and chronic skin defects is an important objective. In the present work, we report on the design and feasibility of a co-culture system for fibroblasts and keratinocytes by using electrospun polycaprolactone (PCL) scaffolds. Specifically, we quantified the effect of scaffold fiber diameter on keratinocyte attachment, proliferation and differentiation along with collagen secretion by fibroblasts post vacuum seeding with fibroblasts at various depths. The results show that fibroblasts secrete more collagen and keratinocytes differentiate more on 400 nm scaffolds than on 1000 nm scaffolds. Also, fibroblasts co-cultured with keratinocytes provide increased collagen secretion and keratinocyte differentiation. These results suggest that the fiber architecture can be a useful parameter in skin tissue engineering.

The biocompatibility of 6H-SiC (0001) surfaces was increased by more than a factor of six through the covalent grafting of NH2 terminated self-assembled monolayers (SAM) using APDEMS and APTES molecules. Surface functionalization began with a hydroxyl, OH, surface termination. The study included two NH2 terminated surfaces obtained through silanization with APDEMS (aminopropyldiethoxymethylsilane) and APTES (aminopropyltriethoxysilane) molecules (hydrophilic surfaces) and a CH3 terminated surface produced via alkylation with 1-octadecene (hydrophobic surface). H4 human neuroglioma and PC12 rat pheochromocytoma cells were seeded on the functionalized surfaces and the cell morphology was evaluated with atomic force microscopy (AFM). In addition, 96 hour MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were employed to evaluate the cell viability on the SAM modified samples. The biocompatibility was enhanced with a 2 fold (171-240%) increase with 1-octadecene, 3-6 fold (320-670%) increase with APDEMS and 5-8 fold (476-850%) increase with APTES with respect to untreated 6H-SiC surfaces.

A novel direct cell printing technique has been developed to control and manipulate the position of cells on solid surfaces. The method utilizes microfabricated polymeric “quill-pen” cantilevers to transfer living cells onto a wide variety of surfaces. In contrast with existing cell deposition methods, such as ink jet or laser ablation methods, the quill-pen approach imparts minimal thermal and shear stress to cells, preserving cell viability and biological functionality. Deposition of both bacterial and mammalian cells into defined patterns has been demonstrated using this method. The size of printed, cell-containing droplets could be controlled by varying the geometry of the quill-pen stylus and by varying printing conditions such as contact time, relative humidity, and surface hydrophobicity. Initial experiments using 10 μm diameter polymer beads demonstrated that the number of beads per droplet could be controlled by varying spot size and particle concentration in the printing solution. Spots could be printed ranging from 20 μm and 100 μm in diameter with approximate volumes ranging from 1-250 pL. We demonstrated deposition of both cells and beads onto a variety of solid surfaces including agarose gel, polystyrene, polyethylene, and glass. Printed cells have also been immobilized on glass and polymer surfaces using biocompatible hydrogel materials (both alginic acid and hyaluronic acid-based matrices) as well as poly-L-lysine. Similar to polymer beads, the number of cells in printed droplets was shown to be dependent upon the size of the droplet, and could be varied by adjusting the concentration of cells present in the printing fluid. As few as one cell per spot could be achieved by adjusting these parameters. The viability and proliferation of printed cells has been evaluated using live optical imaging to observe cell growth and division. Both bacterial cells (Escherichia coli) and mammalian cells were able to divide and proliferate for at least 96 hr post-printing (experiments were discontinued after 96 hr). Live/dead staining was also used to confirm the viability of printed cells. Rat mammary adenocarcinoma MTLn3 cells and mouse embryonic stem cells were also shown to survive the printing process for at least 24 - 96 hr post-printing. These results demonstrate the feasibility of the printing method and its compatibility with a wide range of cell types. It is especially noteworthy that embryonic stem cells could survive the printing process (and proliferate on the printing substrate). This novel printing method has applications for tissue engineering, cell-to-cell signaling studies, and for directly interfacing cells with nanodevices and biosensors.

Novel biocompatible and biodegradable monomers based on phosphorus-containing vinyl esters and vinyl carbamates for radical photopolymerization were prepared. By photo-Differential Scanning Calorimetry (photo-DSC) the reactivity of the mono-, di- and trifunctional monomers was investigated. Furthermore, their cytotoxicity, mechanical properties and hydrolytic degradation behavior were evaluated, aiming at a future application of our compounds in the biomedical area.

Stromal derived factor-1α (SDF-1α) is an important chemokine in stem cell trafficking and plays a critical role in the homing, osteogenesis as well as angiogenesis of bone marrow stromal (BMS) cells. The objective of this work was to investigate the release characteristics of SDF-1α from the degradable poly(lactide ethylene oxide fumarate) (PLEOF) hydrogels and to determine the effect of sustained release of SDF-1α on migration of BMS cells. Three PLEOF macromers with PLA content of 6, 9, and 24 by weight were synthesized by condensation polymerization. The cumulative amount of biologically-active SDF-1α released from the PLEOF hydrogels after 3 weeks was between 20-25% of the initial loading and was independent of PLA/PEG ratio in the hydrogel. The migration of BMS cells in response to the time-release SDF-1α from PLEOF hydrogels closely followed the release kinetics of SDF-1α from the hydrogels. Results demonstrate that migration of BMS cells was significantly increased by the sustained release of SDF-1α from PLEOF hydrogels.

In the previous studies, we have successfully developed a novel spiral structured nanofibrous scaffolds with improved osteoconductivity for bone tissue engineering. The spiral structure design facilitates the nutrient transport and waste removal, and allows uniform cellular growth and distribution within the scaffolds, thus enhanced the bioactivity of the scaffolds. In this chapter, HAP and BMP-2 were incorporated within the nanofibrous spiral scaffolds in order to enhance the osteoinductivity of the established system. The effect of the blending materials was evaluated through cell proliferation, cell differentiation of human osteoblast cells seeded on the scaffolds and cultured for 4 and 8 days. The results has demonstrated that the functionalization of PCL nanofibrous spiral scaffolds leads to higher ALP expression level and increased amount of mineralization level however lower cell proliferation rate.

Current biomaterials as a scaffold for bone regeneration are limited by the extent of degradation concurrent with bone formation, mechanical strength, and the extent of osteogenic differentiation of marrow stromal cells migrating from the surrounding tissues. In this project, a novel laminated nanocomposite scaffold is fabricated, consisting of poly (L-lactide ethylene oxide fumarate) (PLEOF) hydrogel reinforced with poly (L-lactide acid) (PLLA) electrospun nanofibers and hydroxyapatite (HA) nanoparticles. The laminated nanocomposites were fabricated by dry-hand lay up technique followed by compression molding and thermal crosslinking. The laminated nanocomposites were evaluated with respect to mechanical strength and osteogenic differentiation of marrow stromal (BMS) cells. Laminates showed modulus values much higher than that of hydrogel or fiber. The effect of laminated nanocomposites on osteogenic differentiation of BMS cells was determined in terms of ALPase activity and calcium content. Our results demonstrate that grafting RGD peptide to a PLEOF/HA hydrogel reinforced with PLLA nanofibers synergistically enhances osteogenic differentiation of BMS cells.

Many publications have examined the biodegradable polymer poly(propylene fumerate) (PPF) for use in tissue engineering applications. We have examined a similar crosslinkable polymer system, poly(propylene fumerate)-co-(propylene maleate) (PPFcPM), derived from maleic anhydride (MA) and 1,2-propanediol (PD). Two methods were examined in order to synthesis the copolymer. In the first case, the reaction was carried out at high temperature (250°C) under nitrogen using tosic acid as the catalyst. Only PPF was identified due to the thermal isomerization of the maleate groups to the more stable fumerate group. In the second case, toluene was used as the solvent to azeotropically (85 °C) remove water and drive the acid catalyzed esterification reaction. In the lower temperature case, a small amount of fumerate (<30%) was identified. Both polymer systems had glass transition temperatures (Tg) below room temperature. The PPFcPM copolymer was electrospun and crosslinked in situ to form porous micro- and nano fiber mats. Initial biocompatibility studies have also been preformed.