For regenerated tissue to be fully functional, it must exhibit the mechanical integrity of normal tissue. Because of the composite nature of tissues, regional variation in properties, and the fact that macroscopic mechanical testing is not always feasible with regenerated tissue, microscopic approaches are needed to better delineate structure-function relations. We have been utilizing an atomic force microscope (AFM) in conjunction with a picoindentation attachment to determine local, sub-micron level mechanical properties of normal and regenerated tissues. As representative data to establish the validity of this approach, regional hardness and stiffness values for dentin are presented. Repeated measures ANOVA indicated that moduli were site-specific. However, local variations at a given point (e.g. properties at < lpm spatial displacement) were less than site to site variations, validating the efficacy of the technique. Hardness and elasticity may be considered quantitative measures of degree of mineralization, enabling linkage between mechanical, structural, and biological measures of tissue regeneration.

Synthetic copolymers poly(E-caprolactone-co-vinylphosphonic acid) (P(MDOVPA) and poly(E-caprolactone-co-dimethylvinylphosphoester) (P(MDOVPE)) were used to prepare composites with polylactide (PLac) and hydroxyapatite (HAp). The P(MDOVPA) was used as filler in PLac films, as it has pendant functional groups P(O)(OH)2, providing nucleation sites for the deposition of HAp in simulated body fluid. HAp growth on PLac-P(MDOVPA) film was observed by XRD. The incorporation of hydrophilic P(MDOVPE) into PLac increased the hydrophilicity of the blend. Synthetic HAp was also used to make multi-layered, alternating organic-inorganic composites with porous PLac-P(MDOVPE) blends.

Porous polymeric media are used in several applications such as solid supports for separations and catalysis, as well as biomedical applications such as vascular grafts and wound dressings. We have developed a novel versatile process to produce polymeric cellular solids. This process which is based on a phase extraction-co-polymer precipitation is applicable to a wide range of polymer systems including water soluble polymers. It is capable of yielding polymer foams of high porosity (> 90%) and excellent mechanical characteristics in a very short time (less than 2 hours) without limitations in foam thickness. Polymer foam with such characteristics have great utility in tissue engineering applications. We have successfully explored polymer foams of biocompatible polymers produced by the presented approach for bone and cartilage engineering using bone marrow stromal cells.

We have constructed osteogenic scaffolds using solid freeform fabrication techniques. Blends of biodegradable polymers, polycaprolactone and poly(D,L-lactic-co-glycolic acid), have been examined as scaffolds for applications in bone tissue engineering. Hydroxyapatite granules were incorporated into the blends and porous discs were prepared. Mechanical properties and degradation rates of the composites were determined. The discs were seeded with rabbit bone marrow or cultured bone marrow stromal cells and in vitro studies were conducted. Electron microscopy and histological analysis revealed an osteogenic composite that supports bone cell growth not only on the surface but throughout the 1 mm thick scaffold as well. Seeded and unseeded discs were mechanically assembled in layers and implanted in a rabbit rectus abdominis muscle. Bone growth was evident after eight weeks in vivo. Electron microscopy and histological analyses indicate vascularization and primitive bone formation throughout the seeded composite, and also a “fusion” of the layers to form a single, solid construct. Finally, we have begun to incorporate the growth factor IGF-I into the scaffold to enhance osteogenicity and/or as an alternative to cell seeding.

A major concern in the development and implementation of metal implants for the clinical use is the assessment of material-induced mutagenesis. In this study we used synchrotron microspectroscopy in the mid-infrared region (4000-400 cm1 ) to non-invasively assess the in situ human cell responses to metal surfaces. Specifically we examined the subtle genetic aberrations of cells as they responded to a range of metals commonly used in metal prosthetic devices. Relative band intensities and band intensity ratios for functional groups of biomolecules that are inherent to the experimental system were examined. The molecular components of the biomolecules as they were perturbed by the interactions with metals were investigated. These results demonstrate the potential use of synchrotron FTIR microspectroscopy to screen the mutagenicity of metal implants.

Proton magnetic resonance imaging (MRI) is applied to determine non–destructively the open porosity of biodegradable scaffolds of poly(3–hydroxybutyrate-–3–hydroxyvalerate) in vitro and tissue infiltration in vivo. Porous matrices with copolymers having 12% and 24% 3–hydroxyvalerate (HV) are prepared using a solvent casting technique. The results of open porosity P(%) measured by MRI (PMRI) show good agreement with those obtained by gravimetry analysis (PGA). Thus, for scaffolds with 12% HV, PMRI = 91.0 ±1.0 (mean ± s.d.) and PGA = 88.0 ± 0.75; and for those with 24% HV, PMRI = 89.4 ±1.3 and PGA = 88.8 ± 1.0. Also, the extent of tissue infiltration in these scaffolds determined by MRI in vivo was similar to that observed by histology. These results illustrate the potential of MRI methods for investigating the behavior of biodegradable scaffolds non-invasively in applications for tissue engineering.

We have applied the Quartz Crystal Microbalance (QCM) technique to continuously record the processes of endothelial cell (EC) adhesion, spreading and cellular mass distribution changes during initial cell to surface contact and homeostatic attachment. ECs (50,000) were layered onto a set volume of media in the QCM device and simultaneously in a mock cell used for photomicroscopy. As cells were observed in the mock cell device to contact and attach to the surface over 45-55 min, we measured in the QCM device a continuous decrease in frequency and continuous increase in resistance, achieving a maximum at about one hr (1400 Ω frequency change and 1400 Ω motional resistance change). These frequency and resistance values stabilized over the next 24 hrs and were unchanged out to 72 hr by QCM measurement (to ∼700 Hz, ∼700 Ω), as the cells were observed to spread in the mock device. Both bovine aortic (BAE) and bovine capillary (BCE) endothelial cells were studied and found to exhibit similar behavior. These studies demonstrate that QCM can be used to detect continuous changes in cell mass and viscoelastic behavior.

A new approach to the vesico-ureteral reflux could be a local regeneration of the defective vesicoureteral junction by transplanting living cells to the target site. The aim of this work is to provide a long-term effective treatment by producing bioresorbable microspheres which can act as support matrix for those cells, with the goal of an in vivo transfer of the in vitro cultured cells with a minimal surgical procedure. After microsphere degradation, the cells should be integrated into the muscular structure of the junction. Most innovative is that these are cultured muscle and urothelial cells from the bladder of the same patient.

A new method for surface functionalization of inherently reactive polymers such as polyesters has been developed. It is based on the direct nucleophilic attack on the ester group in the polyethyleneterephtalate backbone by the acid moiety of polyacrylic acid (PAA) catalyzed by titaniumisopropoxide. The PAA grafted surfaces were characterized by X-ray photoelectron spectroscopy to demonstrated the presence of a thin grafted layer rendering the substrates hydrophilic. Subsequent acid group activation using N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride allowed for in-situ collagen immobilization rendering the surfaces cell adhesive. Human smooth muscle and urothelial cells successfully seeded these surfaces, reaching confluence after 7 days.

Cells react to topography on the micro and nanoscale with a variety of consequences. These reactions can be used to advantage in controlling cell behavior on the surfaces of prostheses. This short paper examines two of the possible causes of these reactions, namely diffusion effects caused by the shape of the substratum and stretch reception by the cells.

Cells recognize and interact with the extracellular matrix (ECM) through heterodimeric receptors known as integrins. The objective of our work is to immobilize integrin-stimulating peptides to bone-contacting implants in order to control cellular activity and response.

We have previously demonstrated that cysteine (C) containing peptides self-assemble onto gold-coated substrates. Investigations have focused on the RGD (Arg-Gly-Asp) peptide sequence since it is found in several bone ECM proteins. Gold was first coated onto glass coverslips by evaporation and the peptide was applied in a 0.22 mM solution. Contact angle and surface plasmon resonance verified RGDC peptide attachment and formation of a monolayer.

Rat calvarial osteoblasts isolated from six-day-old rat pups were used from passages one to three. Cell attachment at 20 minutes is 100% greater on RGDC than on CG (control peptides) or plain gold surfaces. Cells on RGDC also stain positively for vinculin, a protein which is present in focal adhesions (functional structures into which integrins assemble) whereas surfaces without integrin stimulating peptides do not. Scanning electron micrographs show cells to be more spread and have more processes at 20 minutes, 1, 3, and 24 hours on RGDC. Live video images of these surfaces from zero to three hours after plating confirmed earlier and greater cell spreading on RGDC.

Ongoing in vitro experiments are investigating the long-term response of osteoblasts to RGDC and other immobilized peptides in terms of differentiation, matrix production, and integrin expression.

The role of electromagnetism in the physiology of the human body and in the healing process is well accepted. Electroactive polymers, such as electrically conducting polymers, by virtue of their susceptibility to either electrical, mechanical, optical or thermal phenomena, could be utilized as an interface between the external world and the physiological environment1. In this study we have evaluated the utility of polypyrrole (PPy)-a conductive polymer, as an interactive substrate for inducing differentiation in bovine bone marrow stromal cells (BMSC). It was observed, the PPy is an excellent substrate for BMSC attachment, proliferation and differentiation. Furthermore, an application of an electrical stimulus (ES) to BMSC via PPy substrate was found to enhance the osteogenic commitment of these cells.

α-BSM™ is an injectable endothermically setting calcium phosphate bone substitute. α-BSM™ has been shown to be effective in promoting the healing of surgically created critical size defects and restoring bone biomechanical strength in several animal models. It has received regulatory clearance for orthopedic indications in both Canada and Europe and for dental and craniofacial indications in the United States.

α-BSM™ is distinguished as a bone substitute by its prolonged working time at room temperature, and rapid hardening at body temperature. These properties, as well as its compatibility with a variety of aqueous hydrating agents, make α-BSM™ an attractive vehicle for the in situ delivery of therapeutic agents. In vitro studies have shown that antibiotics, such as gentamicin, can be stably incorporated into α-BSM™ prior to and during hardening, and that delivery kinetics can be controlled with the appropriate formulation and preparative procedures. The setting reaction is also compatible with biologically active proteins. rhBMP-2 has been incorporated into α-BSM™ and was demonstrated to be effective in stimulating ectopic bone formation in soft tissue and accelerating the restoration of a differentiated phenotype in a rabbit osteotomy model.

Human bone derived osteoblasts were cultured on collagen-calcium phosphate composites. The ability of the substrates to support cell attachment, proliferation and bone formation was assessed using histochemical staining for alkaline phosphatase activity and immunolocalisation of transforming growth factor- β1and type 1 collagen. The effect of calcium phosphate phase and crystal size was investigated and the calcified samples compared with uncalcified collagen. Osteoblasts adhere to the collagen-calcium phosphate composites and express a mature osteoblast phenotype in vitro. Cell adhesion was greater on unmineralised collagen than on the mineralised composites, however, these cells were less differentiated. The presence of larger crystals seemed to have a detrimental effect on the cells, reducing proliferation and alkaline phosphatase activity. There was no discernible difference between the effect of hydroxyapatite and octacalcium phosphate on the cells.

Hydrolytic polycondensation of silicon alkoxides and organically modified alkoxides shows promise as a route to new materials for medicine. Mathematical models of this polymerization accelerate the development of these materials and processes for their production. With a reliable model, one can rapidly explore a wide variety of options for controlling material properties. Here we describe a model for kinetics of sol-gel copolymerization of a simple pair of ethoxysilanes: (CH3)3Si(OC2H5) and (CH3)2Si(OC2H5)2. We then describe how reactor configuration alone can be used to control of polymer structure by choosing how to mix the reactants. An example is shown of maximizing homogeneity at any reactor residence time of interest in the model copolymer system by using the time of addition of the faster-reacting monomer.

During Electrosurgery, Radio Frequency (RF) electrical power is applied to a cutting surface which cauterizes the incision. The desiccation of surrounding tissue results in hemostasis and surgery without bleeding. In the case of localized heavy bleeding, a coagulation process is employed using modulated RF power. However, substantial amounts of desiccated tissue may stick to the blade, undoing the achieved hemostasis, and possibly worsening the situation. To prevent this, various electrosurgical blade coatings have been developed to both permit the flow of RF power through the coating, and prevent desiccated tissue from sticking to it.

PolyTetraFluoroEthylene (PTFE) is a commonly used electro- surgical blade coating that undergoes substantial surface morphology changes during use, eventually leading to failure. Highly conductive Diamond-Like Nanocomposite (DLN) electrosurgical coatings have been developed with the requisite non-stick characteristics and enhanced durability.

After a brief review of electrosurgical fundamentals, the underlying mechanisms of operation of these two coating materials are compared. In the case of PTFE, establishment and evolution of porosity in the coating is essential to the flow of RF power, yet ultimately leads to long term degradation. Alternate conduction mechanisms for DLN coatings are considered.

Hydroxyapatite (HA) has been universally used because of good biocompatibility. There are varieties of coating methods, however, they still have problems. The most significant problem is layer thickness. We introduced a new coating procedure to eliminate this difficulty.

The characterization of the coating was studied by X-ray diffractometry, infrared spectroscopy, SEM and chemical analysis. Said examination indicated that the double layer consisted of carbonate HA and CaTiO3 and the thickness of the layer was 2-4 μm. The coating was performed on the inner surfaces of 50-200 μm sized pores and was also consistent in the smallest of the pores even those of 50 μm. Our results indicated that the coating layer was homogeneous and thin. In addition, both layers consisted of Ca ions. The consistency of coating into even the smallest pore size suggested that our method promises to have widespread clinical applications in artificial implants because of increased surface contact area.

The elaboration of composite materials has been considered one possibility to improve mechanical properties of bioactive phases based on hydroxyapatite used for medical implants. Ceramic and polymeric materials have been tested as the other phase. The aim of this study is to evaluate the sintering behavior and the mechanical properties (compressive strength, modulus of resiliency and microhardness) of hydroxyapatite-zirconia (ZH) composites prepared by precipitation method. Powders of hydroxyapatite-zirconia (ZH) composites, with 40 vol % of zirconia content, and pure hydroxyapatite (HA) as well as pure ZrO2- CaO (ZO) were sintered at 900, 1100 and 1200°C in air at a pressure of 700 MPa for 3 hours, using uniaxial pressing. The products obtained at 1100 and 12000 C are dense, with density values over 91% of the theoretical ones. Experimental results indicated that the sinterization process can be described as admitting a bi-modal structure for ZH composites consisting of a sphere of ZO embedded into a matrix of HA. In this structure the matrix is consider to sinter faster. Intermediate values (between those of pure phases of HA and ZO) of compressive strength and modulus of resiliency were found for ZH composites, suggesting that these composites have potential applications as implant materials.

Non-reinforced and self-reinforced bioactive-bioabsorbable composite rods were produced by extrusion and solid state die-drawing using P(L/DL)LA matrix and spherical glass particles. The structure and mechanical properties of composites were measured before and after die-drawing. The structure examination of composites by SEM showed the formation of fibrous reinforced structure of the polymer with spindle-shaped pores around the glass spheres during and oriented in the direction of die-drawing. Incorporation of glass into the polymer matrix turns the composite more brittle compared to neat polymer. By die-drawing the brittleness of the composite can be eliminated.

Not only the bioactivity and controlled degradation rate of composite but also structure and mechanical properties achieved during die-drawing make the self-reinforced bioactive glass- bioabsorbable polymer composites a novel implant material for temporary support of healing tissues.

A composite for use as a bone substitute has been produced by the precipitation of calcium phosphate onto a collagen matrix. A collagen sheet is used as a membrane separating reservoirs of calcium and phosphate ions. The ions diffuse through the membrane and calcium phosphate precipitation occurs on and within the collagen matrix.

Composites containing octacalcium phosphate or hydroxyapatite have been produced by varying the precipitation pH. If the correct balance of phosphate and calcium ion concentrations is achieved, precipitation occurs through the entire cross-section of the collagen sheet, giving a composite microstructure analogous to bone. The composite stiffness increases with increasing mineral content, reaching 2.8 GPa with 39 wt % mineral, when measured dry.