Current methods for correcting articular cartilage defects are limited by a scarcity of cartilage cells. Here we describe a novel method for the conversion of human dermal fibroblasts to chondrocyte-like cells and the potential application of this methodology to cartilage tissue engineering. Human neonatal foreskin fibroblasts were seeded on two-dimensional, tissue culture polystyrene (TCPS) in high density micromass cultures in the presence of staurosporine (50-200 nM), a protein kinase C (PKC) inhibitor, and lactic acid (40 mM) to induce functional hypoxia. Dermal fibroblasts were similarly cultured on three-dimensional polymer scaffolds composed of a non-woven polyglycolic acid (PGA) fiber mesh reinforced in a dilute solution of poly(L-lactic acid) (PLLA). At 24 hours, northern analysis revealed a staurosporine dose-dependent increase in aggrecan core protein expression in lactate-treated micromass cultures on TCPS, while type I collagen gene expression was virtually abolished in all cultures supplemented with staurosporine. The cells in these cultures displayed a rounded, cobblestone-shaped morphology typical of differentiated chondrocytes (most pronounced at 200 n.M staurosporine and 40 mM lactate), and were organized into nodules which stained positively with Alcian blue. When seeded on PGA/PLLA matrices under identical conditions as described for TCPS, a chondrocyte-like morphology was observed in cultures treated with lactate and staurosporine in contrast to the flattened sheets of fibroblast-like cells seen in untreated controls. Taken together, the above findings suggest that staurosporine treatment coupled with high density micromass culture in the presence of lactate induces chondrogenic differentiation in human dermal fibroblasts, and that these cells may be used in concert with three-dimensional polymer scaffolds for the repair of articular cartilage lesions.

The immunoisolation of xenogeneic hormone-secreting cells is a promising therapy for a wide variety of diseases including diabetes, Parkinson's, and hemophilia. By utilizing microfabrication technology, silicon biocapsules can be fabricated with membranes having precisely controlled and uniform pore sizes, allowing one to optimize parameters specifically for the encapsulation of specific hormone-secreting cell types. This study investigates immunoisolation parameters using microfabricated silicon-based membranes, with uniform membrane pore sizes in the tens of nanometer range. The permeability of IgG was studied in microfabricated biocapsules with various pore sized membranes. In addition, immunoisolative characteristics were monitored by assessing viability and functionality of islets within biocapsules.

A biodegradable hydrogel was prepared by glutaraldehyde crosslinking of acidic gelatin with an isoelectric point (IEP) of 5.0 as a carrier to release basic growth factors on the basis of polyion complexation. Basic fibroblast growth factor (bFGF), transforming growth factor β1 (TGF-β1), and bone morphogenetic protein-2 (BMP-2) were sorbed from their aqueous solution into the dried gelatin hydrogels to prepare respective growth factor-incorporating hydrogels. Under an in vitro non-degradation condition, approximately 20 % of incorporated bFGF and TGF-β1 was released from the hydrogels within initial 40 min, followed by no further release, whereas a large initial release of BMP-2 was observed. After subcutaneous implantation of the gelatin hydrogels incorporating 125I-labeled growth factor in the mouse back, the remaining radioactivity was measured to estimate the in vivo release profile of growth factors. Incorporation into gelatin hydrogels enabled bFGF and TGF-β1 to retain in the body for about 15 days and the retention period well correlated with that of the gelatin hydrogel. Taken together, it is likely that the growth factors ionically complexed with acidic gelatin were released in vivo as a result of hydrogel biodegradation. On the contrary, basic BMP-2 did not ionically interact with acidic gelatin, resulting in no sustained released by the present biodegradable carrier system.

Oxygen supply is expected to be one of major limiting factors that determine survival of islets in microcapsules after transplantation. Fick's law is applied to derive equations describing the oxygen pressure (PO2) profiles. The PO2 profiles calculated demonstrate that the thickness of the microcapsule membrane does not significantly affect oxygen supply to the islet. On the other hand, it drastically decreases as the fibrous tissue surrounding the microcapsule becomes thicker. The islet will be destroyed non-immunologically by limiting oxygen supply, when the microencapsulated islets are surrounded by the thick fibrous tissue.

Silk fibroin is a naturally occurring structural protein with good mechanical properties used in a variety of forms, such as powder, fiber, film, and gel. Although silk fibroin is potentially suitable for use in tissue engineering, it lacks cell regulation functions such as cell adhesion, growth, metabolism, and differentiation. The immobilization of biologically active molecules such as proteins and peptides has been reported as promising in controlling cell behavior. Silk fibroin's phase transition is characterized by a conformational change of protein from a random coil to a beta sheet. During phase transition, biological molecules can be stably entrapped in silk fibroin without the use of chemicals. We designed a novel immobilization using this phase transition mechanism with a chimera fibroin fragment. The chimera fibroin fragment was constructed by linking a bioactive peptide to fibroin fragments including crystal regions. In the first study, a synthetic oligonucleotide encoding Arg-Gly-Asp peptide which promotes cell adhesion, was fused to the fibroin fragment gene through inframe gene fusion, and the chimera fibroin (RGD-fibroin) gene was expressed by E.coli. This paper discusses RGD-fibroin construction, and the results of cell adhesion on fibroin films containing RGD-fibroin.

Lifetime measurement in Positron Annihilation Spectroscopy (PAS) is applied to the study of free-volume collagen characteristics as a function of concentration. The lifetimes of positrons were obtained by a conventional fast-fast coincidence system. All lifetime data are fitted in three components by using the computer program POSITRON FIT and resolved. For each concentration, lifetime distributions were analyzed in order to obtain the different components, thus we have observed three components of which a long component τ3. This long lived component can be associated with a pick-off annihilation of ortho-positronium (o-Ps) trapped in free volumes of amorphous region. This investigation shows the potential of the positron annihilation spectroscopy technique in the study of biopolymer microstructures.

Reconstruction of tissues and organs utilizing cell transplantation offers an attractive approach for the treatment of patients suffering from organ failure or loss. Highly porous synthetic materials are often used to mimic the function of the extracellular matrix (ECM) in tissue engineering, and serve as a cell delivery vehicle for the formation of tissues in vivo. Alginate, a linear copolysaccharide composed of D-mannuronic acid (M) and L-guluronic acid (G) units is widely used as a cell transplantation matrix. Alginate is considered to be biocompatible, and hydrogels are formed in the presence of divalent cations such as Ca2+, Ba2+ and Sr2+. However, ionically cross-linked alginate gels continuously lose their mechanical properties over time with uncontrollable degradation behavior. We have modified alginate via covalent coupling of cross-linking molecules to expand and stabilize the mechanical property ranges of these gels. Several diamino PEG molecules of varying molecular weight (200, 400, 1000, 3400) were synthesized utilizing carbodiimide chemistry. Sodium alginate was covalently cross-linked with these cross-linking molecules, and mechanical properties of the resulting hydrogels were determined. The elastic modulus of the cross-linked alginates depended on the molecular weight of the cross-linking molecules, and ranged from 10-110 kPa. The theoretical cross-link density in the hydrogels was also varied from 3 to 47% (relative to the carboxylic groups in the alginate) and the mechanical properties were measured. The elastic modulus increased gradually and reached a maximum at a cross-link density of 15%. In summary, covalently coupled hydrogels can be synthesized which exhibit a wide range of mechanical properties, and these materials may be useful in a number of tissue engineering applications.

The flow of chemical and mechanical signals among cells, and between cells and their environment plays a crucial role in cell differentiation and morphogenesis. In tissues, type I collagen serves as the template for cell anchorage and migration, and it mediates the flux of regulatory signals via highly specific receptors. Cells respond to mechanical cues by secreting growth factors and remodeling their surrounding matrix in an exquisitely orchestrated spatial and temporal program of matrix turnover and organization. Cellular tractional forces contribute to the organization and orientation of the newly synthesized matrix, establishing the template for subsequent morphogenesis. The junction between cells and collagen plays a key role in cell differentiation, morphogenesis and tissue remodeling. An optimal biomimetic environment would emulate this pathway for the exchange of stimuli. To achieve this goal, we have constructed templates which place cells in apposition to P-15, a synthetic peptide ligand for collagen receptors. These environments prompted 3-D colony formation, induced increased osteogenic differentiation, and the deposition of highly oriented and organized matrix by human dermal and gingival fibroblasts and by osteoblast like HOS cells. These observations support our concept for biomimetic environments for tissue engineering.

Stress levels imparted on a cell have been shown to alter cell organization and function, presumably as a result of morphological cues affecting cytoskeletal organization. Materials with spatially resolved surface chemistry were designed to isolate individual mammalian cells to determine the influence of projected area on cell proliferation and cytoskeletal organization. Surfaces were fabricated using a photolithographic process resulting in islands of cell binding N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) separated by a non-adhesive interpenetrating polymer network [poly acrylamide-co-ethylene glycol; P(AAm-co-EG)]. The surfaces contained over 3800 adhesive islands/cm2, allowing for isolation of single cells with projected areas ranging from 100µm2to 10,000µm2. These surfaces provide a useful tool for researching how cell morphology and mechanical forces affect cell function.

Glass substrates were modified with organic thin films to guide the outgrwoth of cultured neurons dissociated from the central nervous system. Embryonic hippocampal neurons, grown on these modified substrates, developed a morphology by following the surface geometries and they formed contacts with each other. We are now investigating technical procedures for forming physiological synapses using goldfish retinal neurons in culture.

Commercially available and synthesized silica particles were fluorescently labeled with FITC and modified to get a wide variety of particle systems with defined size and surface charge. By a variation of reaction conditions particles with diameters of 10 and 80 nm determined with TEM and with zetapotentials between -50 to +30 mV under physiological conditions (pH: 7.4, PBS-buffer) were available.

A further molecular shell consisiting of avidin was obtained by binding the molecules to negatively charged particle surfaces through electrostatic interactions. The amount of avidin coupled to the silica particles was 1.7 μg per mg particle. Starting with particles with an hydrodynamic diameter determined with PCS of 260 nm, the size increased to 500 nm, while the zeta potential was altered to -8 mV under physiological conditions.

Biotinylated wheat germ agglutinin (bio-WGA) can be bonded to such particles through avidin / biotin complex formation. Up to 2.8 μg lectin per mg particles could be coupled to the particle surface. This leads to a further increase of hydrodynamic diameter to 650 nm. It could be shown by hemagglutination test, that the bonded lectin is still active. No toxic effects of the silica particles were found at 1 wt.-% particle concentration with various cell types (Caco-2, L132). The binding of lectin-particle complexes to cells was increased by a factor of 4.4 in comparison to uncoated particles.

In addition it was found that WGA can directly be coupled to the particle surface. An amount of 1.8 μg Lectin per mg particle was determined. The hydrodynamic diameter increases from 260 nm to 432 rm, while a zetapotential of-28 mV was found under physiological conditions.

It could be shown, that negatively charged silica nanoparticles are suitable systems to couple various biomolecules retaining their biological function.

An ideal surface for many biomaterials applications would resist nonspecific protein adsorption while at the same time providing a means for specifically signaling cells to guide survival, growth, migration, and differentiation. This work was directed towards the investigation of model systems and clinically-applicable materials which provide both of these surface requirements. Model systems were prepared by chemically grafting end-functionalized star poly(ethylene oxide) (PEO) to surfaces. The end-grafted polymers provide a means for cell-signaling through coupling of peptides to the free chain ends. Protein adsorption on star vs. linear grafted layers was compared. To further understand these results, neutron reflectivity studies were carried out in situ for solvated PEO surfaces to determine the concentration profiles of the swollen grafted layers. Surprisingly, grafted PEO layers which resist protein adsorption have low concentrations of polymer segments throughout the swollen layer. We find that dense star architectures which might be expected to impart improved protein resistance in fact allow small proteins to adsorb. For clinical materials, a novel approach to the surface modification of poly(lactide) (PLA) has been taken by surface segregating a comb copolymer containing a PLA backbone and poly(ethylene glycol) teeth. The ends of the teeth provide sites for surface tethering of peptide ligands. Comb surfaces without tethered ligands are cell adhesion resistant, indicating strong protein adsorption resistance. By then incorporating an adhesion ligand, modulation of cell morphology on comb surfaces has been demonstrated. Finally, the surface segregation of the comb to the surface of PLA was shown via cell attachment assays and XPS measurements.

We investigated the crosslinking characteristics of an injectable composite paste of poly(propylene fumarate) (PPF), N-vinyl pyrrolidinone (N-VP), benzoyl peroxide (BP), sodium chloride (NaCl), and β-tricalcium phosphate (β-TCP). We examined the effects of PPF molecular weight, N-VP/PPF ratio, BP/PPF ratio, and NaCl weight percent on the crosslinking temperature, heat release upon crosslinking, gel point, and the composite compressive strength and modulus. The maximum crosslinking temperature did not vary widely between formulations, with the absolute values falling between 38 and 48 °C, and was much lower than that of 94°C for poly(methyl methacrylate) bone cement controls tested under the same conditions. The total heat released upon crosslinking was decreased by an increase in PPF molecular weight and a decrease in N-VP/PPF ratio. The gel point was effected strongly by the PPF molecular weight, with a decrease in PPF molecular weight leading to a more rapid gel point. An increase in initiator concentration had the same effect to a lesser degree. The time frame for curing was varied from 1 -121 minutes, allowing the composite to be tailored to specific applications. The compressive strength and compressive modulus values increased with decreasing N-VP/PPF, increasing NaCl content, and increasing BP/PPF ratio. For all formulations, the compressive strength values fell between 1 and 12 MPa, and the compressive modulus values fell between 23 and 265 MPa. These data suggest that injectable PPF/β-TCP pastes can be prepared with handling characteristics appropriate for clinical orthopaedic applications and that the mechanical properties of the cured composites are suitable for trabecular bone replacement.

A new family of polyanhydrides has been developed which can be cured photochemically to produce degradable networks. These degradable polyanhydride networks can be useful in orthopedics as bone cements and for drug delivery. This system, which is a semi-interpenetrating network (semi-IPN), has been evaluated for tissue compatibility in subcutaneous sites in rats and shown to undergo degradation by surface erosion. It was observed that the inflammatory response to the semi-IPN implants was minimal at both short (3, 6 weeks) and long (28 weeks) time points and the fibrotic response was largely absent throughout the duration of this study. Furthermore, excellent tissue infiltration and integration with good neovascularization was observed around the semi-IPN implants. The osteocompatibility and osteoconductive properties of this system have been evaluated in a longitudinal defect model in the articular surface of the distal femur in adult rabbits. This study compared the semi-IPN implants to control groups consisting of unfilled defects and PLA implants. Histological evaluation (H&E and Trichrome Masson staining) of the implant site revealed that the semi-IPN implant is osteocompatible and well tolerated by the surrounding cancellous bone and marrow constituents. The tissue reaction to the implant is characterized by an ingrowth of vascularized connective tissue and a mild fibrous capsule. Furthermore, partial to complete closure of the articular surface with fibrocartilage and fibrous tissue was observed in all experimental animals containing the semi-IPN polymer implants.

Model biomaterial surfaces were modified with a peptide that contained a -RGD- (Arg-GlyAsp) sequence, unique to bone sialoprotein, to determine its effect on strength of adhesion, spreading, and focal contact formation of primary bone-derived cells. Peptide surfaces were fabricated by using a heterobifunctional crosslinker to graft the peptide to surfaces (quartz and silicon). Contact angle measurements, spectroscopic ellipsometry, and X-ray photoelectron spectroscopy were used to confirm the chemistry and thickness of the overlayers. Furthermore, spectroscopic ellipsometry was used to estimate the density of immobilized peptide on metal oxide surfaces. A radial flow apparatus was used to measure the strength of adhesion on peptide grafted surfaces. Following 20 min of cell incubation, the strength of cell adhesion was significantly (p<0.05) higher on the -RGD- compared to -RGE- (control) surfaces. The mean area of cells contacting the -RGD- surface was significantly (p<0.05) higher than -RGE- surfaces. Vinculin staining revealed formation of focal contact patches on the periphery of bone cells incubated for 4 hr on the -RGD- surfaces; however, cells seeded on the -RGE- grafted surfaces formed little or no focal contacts. The methods of peptide immobilization utilized in this study can be applied to medical devices, biosensors, and diagnostic assays that require specificity in cell adhesion.

Primary human osteoblasts were cultered on biomaterials to develop living bone substitutes by Tissue Engineering. Monolayer cultures were established from iliac crest biopsies. Single cell suspensions were then seeded onto tricalciumphosphate (TCP) BIOBASE®, hydroxyapatitecollagen(HAC), autoclaved cancellous bone (CB) and fibrin glue (FG) Tissucol® The constructs were analyzed by electronmicroscopy, Cell proliferation assay (XTT) and histology to assess adhesion, proliferation and extracellular matrix formation on the biomaterial.

The osteoblastic cells survived in the fibrin glue and established a mesenchymal phenotype. They adhered to the HAC-sponges, TCP and CB. Prolifertion was seen on TCP and CB. HAC had a suppressive effect on the cells. An inmature matrix was found on TCP and a bone neomatrix on CB after 4 weeks in vitro.

Titanium has been successfully used for decades in dental and orthopedic implants, but the mechanism for this metal's biocompatible properties have not been determined. Our hypothesis is that this biocompatibility involves interaction between the surface layer of titanium dioxide on the metal implant and reactive oxygen mediators of the inflammatory response. The affect of different titanium surface oxide layers on the reactive oxygen mediators produced during the inflammatory response has never been examined. Peroxynitrite is a highly reactive and unstable compound produced in vivo by the reaction of nitric oxide with superoxide. We investigated if titanium oxides affect the stability of peroxynitrite by promoting its breakdown. Peroxynitrite levels can be measured by its absorbance at 302 nm. At pH= 13.2, we found a 100% increase in the rate of degradation of peroxynitrite in the presence of titanium particles. Peroxynitrite is capable of nitrating 4-hydroxyphenolacetic acid (4-HPA). The nitrated form of 4-HPA can be measured by its absorbance at 432nm. 3-morpholinosydnonimine (SIN-1), a nitric oxide donor, has been shown to produce superoxide during its breakdown resulting in the formation of peroxynitrite. At physiological pH (7.4), a solution of 0.5mM 4-HPA was exposed to 5mM SIN- 1 on passivated titanium surfaces. There was a decrease of 58% nitrated 4-HPA in the solution exposed to passivated titanium compared to controls. Unpassivated titanium surfaces resulted in only a 10% decrease of nitrated 4-HPA while titanium treated with hydrogen peroxide resulted in a 70% decrease in nitrated 4-HPA concentrations compared to controls. Zirconium and palladium were also tested. These experiments suggest that titanium is capable of enhancing the breakdown of the inflammatory compound peroxynitrite which may account for the metal's biocompatible properties.