Modulating bone cells response onto starch-based biomaterials by surface plasma treatment and protein adsorption.

The effect of oxygen-based radio frequency glow discharge (rfGD) on the surface of different starch-based biomaterials (SBB) and the influence of proteins adsorption on modulating bone-cells behavior was studied. Bovine serum albumin, fibronectin and vitronectin were used in single and complex protein systems. RfGD-treated surfaces showed to increase in hydrophilicity and surface energy when compared to non-modified SBB. Biodegradable polymeric blends of cornstarch with cellulose acetate (SCA; 50/50wt%), ethylene vinyl alcohol (SEVA-C; 50/50wt%) and polycaprolactone (SPCL; 30/70wt%) were studied. SCA and SCA reinforced with 10% hydroxyapatite (HA) showed the highest degree of modification as result of the rfGD treatment. Protein and control solutions were used to incubate with the characterized SBB and, following this, MG63 osteoblast-like osteosarcoma cells were seeded over the surfaces. Cell adhesion and proliferation onto SCA was found to be enhanced for non-treated surfaces and on SCA+10%HA no alteration was brought up by the plasma modification. Onto SCA surfaces, BSA, FN and VN single solutions improved cell adhesion, and this same effect was found upscaled for ternary systems. In addition, plasma treated SEVA-C directed an increase in both adhesion and proliferation comparing to non-treated surfaces. Even though adhesion onto treated and untreated SPCL was quite similar, plasma modification clearly promoted MG63 cells proliferation. Regarding MG63 cells morphology it was shown that onto SEVA-C surfaces the variation of cell shape was primarily defined by the protein system, while onto SPCL it was mainly affected by the plasma treatment.

Use of compact sandwich specimen to determine the critical strain energy release rate of bone.

The current methods to measure bone fracture toughness were initially developed for engineering materials and use specimen configurations that demand large samples. However, in many cases it is hard if not impossible to obtain such specimens from limited bone stock. Therefore, a new compact sandwich (CS) test method was formulated to measure the critical strain energy release rate (a measure of fracture toughness) of bone requiring only small samples. This technique may be used to assess bone fracture toughness and subsequently the bone quality.

In vitro anti-bacterial and biological properties of magnetron co-sputtered silver-containing hydroxyapatite coating.

Bacterial infection after implant placement is a significant rising complication. In order to reduce the incidence of implant-associated infections, several biomaterial surface treatments have been proposed. In this study, the effect of in vitro antibacterial activity and in vitro cytotoxicity of co-sputtered silver (Ag)-containing hydroxyapatite (HA) coating was evaluated. Deposition was achieved by a concurrent supply of 10 W to the Ag target and 300 W to the HA target. Heat treatment at 400 degrees C for 4 h was performed after 3 h deposition. X-ray diffraction, contact angles measurements, and surface roughness were used to characterize the coating surfaces. The RP12 strain of Staphylococcus epidermidis (ATCC 35984) and the Cowan I strain of Staphylococcus aureus were used to evaluate the antibacterial activity of the Ag-HA coatings, whereas human embryonic palatal mesenchyme cells, an osteoblast precursor cell line, were used to evaluate the in vitro cytotoxicity of the coatings. X-ray diffraction analysis performed in this study indicated peaks corresponding to Ag and HA on the co-sputtered Ag-HA surfaces. The contact angles for HA and Ag-HA surfaces were observed to be significantly lower when compared to Ti surfaces, whereas no significant difference in surface roughness was observed for all groups. In vitro bacterial adhesion study indicated a significantly reduced number of S. epidermidis and S. aureus on Ag-HA surface when compared to titanium (Ti) and HA surfaces. In addition, no significant difference in the in vitro cytotoxicty was observed between HA and Ag-HA surfaces. Overall, it was concluded that the creation of a multifunctional surface can be achieved by co-sputtering the osteoconductive HA with antibacterial Ag.

Changes in the fracture toughness of bone may not be reflected in its mineral density, porosity, and tensile properties.

Age-related changes in the skeleton often lead to an increase in the susceptibility of bone to fracture. Such changes most likely occur in the constituents of bone, namely, the mineral and organic phases, and in their spatial arrangement manifested as orientation and microstructure. In the past, however, bone loss or decline in bone mineral density has been considered to be the major contributing factor for the increased risk of bone fractures, and elastic modulus and ultimate strength have been commonly used to assess bone quality and strength. However, whether these properties provide sufficient information regarding the likelihood of bone to fracture remains debatable. Using a novel fracture toughness test, which measures the energy or stress intensity required to propagate a crack within a material, the objective of this study was to investigate if the mineral density and mechanical properties of bone can accurately predict bone fragility as measured by fracture toughness. Changes in fracture toughness (K(IC)), bone mineral density (BMD), elastic modulus (E), yield and ultimate strength (sigma y and sigma s), porosity (P0), and microhardness (Hv) of bone were examined as a function of age in a baboon model. With increasing age, the fracture toughness of bone decreased, and its microhardness increased. However, no significant changes were found in BMD, E, P0, sigma y, and sigma s as a function of age. In addition, simple regression analyses revealed no significant correlation between bone fracture toughness and the other parameters, except for microhardness of bone. The results of this study indicate that changes in bone fracture toughness may not be necessarily reflected in its mineral density, porosity, elastic modulus, yield strength, and ultimate strength.

Deformation characteristics of a bioabsorbable intravascular stent.

RATIONALE AND OBJECTIVES: Biodegradable polymeric stents offer an alternative to metallic stents, which have a significant compliance mismatch with blood vessels and have the potential for long-term complications. In the current study, the deformation characteristics of polymeric stents have been investigated. METHODS: Stents were subjected to radial compressive pressure by inserting them in a wrap-around collar, one end of which was loaded in tension using dead-weights. The resulting decrease in stent diameter was measured under an optical microscope. Deformation curves for the stents were plotted, and an effective stiffness, E', was determined for each. RESULTS: The deformation curves exhibited two different regions: an initial linear region, followed by a steep nonlinear region. The transition from the linear region occurred at a fractional decrease in stent diameter of approximately 0.5. CONCLUSION: E' decreases with increasing stent diameter and filament draw-ratio. The relationship between E' and the initial stent diameter is not linear. The deformation curves can be used for choosing the appropriate stents for specific applications.

Protein release kinetics of a biodegradable implant for fracture non-unions.

Non-union of long bone fractures is often a serious complication of fracture healing. It is estimated that 100 000 non-unions occur in the united States annually and result in the loss of function of the involved limb. The present study was performed to develop a microporous polylactic acid-polyglycolic acid (PLA-PGA) implant for the delivery of bone morphogenetic protein (BMP) to sites of fracture non-unions, and to characterize the protein release kinetics of such an implant in vitro. A 50:50 copolymer of PLA-PGA was used to fabricate the implants using a gel formation technique. The implants were subjected to hydrolytic degradation in phosphate-buffered saline at 37 degrees C for up to 72 d. The protein release and the polymer degradation were monitored during this time period. The release kinetics of these implants were studied using a model protein, soybean trypsin inhibitor (TI), as well as BMP. The results indicate that there is a burst release of the proteins in the initial 48 h followed by a lower elution rate. The release of both the proteins followed similar trends. The molecular weight of the polymer decreased at a faster rate compared to its mass.

Sterilization, toxicity, biocompatibility and clinical applications of polylactic acid/polyglycolic acid copolymers.

This is a review of salient studies of sterilization, toxicity, biocompatibility, clinical applications and current work in the field of orthopaedics, using implants made of polylactic acid (PLA), polyglycolic acid (PGA) and their copolymers. The intrinsic nature of these biomaterials renders them suitable for applications where temporally slow releases of bioactive agents in situ may be required. They are also desirable as fixation devices of bone, because they can virtually eliminate osteopenia associated with stress shielding or additional surgery. The majority of currently available sterilization techniques are not suitable for these thermoplastic materials and it may be desirable to develop new sterilization standards, which can account for the special character of PLA-PGA materials. Biocompatibility and toxicity studies suggest that, overall, PLA-PGA biomaterials may be suitable for orthopaedic applications, although certain problems, especially pertaining to reduction in cell proliferation, have been reported. Clinical applications are also promising, albeit not without problems usually associated with transient tissue inflammation. The future of these materials appears bright, especially in soft tissues. They may be used to address the exceedingly complex problem of osteochondral repair, but also as a means to enhance fixation and repair processes in tendons and ligaments.

Effects of fluid flow on the in vitro degradation kinetics of biodegradable scaffolds for tissue engineering.

Scaffolds fabricated from biodegradable polymers are used extensively in the field of tissue engineering. Many of these scaffolds are subjected to fluid flow, either in vivo or in bioreactors ex vivo. The goal of this study was to examine the effects of fluid flow on the degradation characteristics and kinetics of scaffolds in vitro. Scaffolds with different porosity and permeability values were fabricated using a copolymer of polylactic acid and polyglycolic acid. These scaffolds were subjected to degradation in phosphate buffered saline at 37 degrees C for up to 6 weeks under two test conditions: static and flow (250 microl/min). The porosity of the scaffolds decreased up to 2 weeks and then increased, while the elastic modulus first increased and then decreased over the course of the study. The mass and molecular weight of the scaffolds exhibited a steady decrease up to 6 weeks. The results further indicated that lower the porosity and permeability of the scaffolds, the faster their rate of degradation. Additionally, fluid flow decreased the degradation rate significantly. It is possible that the high rates of degradation observed here were due to autocatalysis of the degradation reaction by the acidic degradation products.

Evaluation of poly(L-lactic acid) as a material for intravascular polymeric stents.

Poly(L-lactic acid) (PLLA) monofilaments were evaluated for use as intravascular polymeric stents. The PLLA monofilaments were extruded and drawn to different draw ratios. They were then subjected to different thermal treatments and their mechanical properties characterized. Stents constructed with similar monofilaments were tested under hydrostatic pressure, and the results correlated with the properties of the monofilaments. Stent collapse pressure was a decreasing function of stent diameter and filament draw ratio.

Development of the cytodetachment technique to quantify mechanical adhesiveness of the single cell.

Adhesion of cells to biomaterials or to components of the extracellular matrix is fundamental in many tissue engineering and biotechnological processes, as well as in normal development and tissue maintenance. Many cells on adhesive molecules will spread and form an organized actin cytoskeleton and complex transmembrane signaling regions called focal adhesions. Focal adhesions appear to function as both signaling and stabilizing components of normal adherent cell activity. To better understand adhesion formations between cells and their underlying substrata, we have designed, developed, and utilized a novel 'cytodetachment' methodology to quantify the force required to displace attached cells. We allowed bovine articular chondrocytes to attach and spread on a substratum of either fibronectin, bovine serum albumin, or standard microscope glass. The cytodetacher was then employed to displace the cells from the substratum. Our results demonstrate that a significantly greater force is required to detach cells from fibronectin versus the two other substrata, suggesting that a cell's actin cytoskeleton and perhaps focal adhesions contribute significantly to its mechanical adhesiveness. The cytodetacher allows us to directly measure the force required for cell detachment from a substratum and to indirectly determine the ability of different substrata to support cell adhesion.

The effects of dynamic compressive loading on biodegradable implants of 50-50% polylactic Acid-polyglycolic Acid.

Biodegradable implants that release growth factors or other bioactive agents in a controlled manner are investigated to enhance the repair of musculoskeletal tissues. In this study, the in vitro release characteristics and mechanical properties of a 50:50 polylactic acid/polyglycolic acid two phase implant were examined over a 6-week period under no-load conditions or under a cyclic compressive load, such as that experienced when walking slowly during rehabilitation. The results demonstrated that a cyclic compressive load significantly slows the decrease of molecular chain size during the first week, significantly increases protein release for the first 2-3 weeks, and significantly stiffens the implant for the first 3 weeks. It was also shown that protein release is initially high and steadily decreases with time until the molecular weight declines to about 20% of its original value (approximately 4 weeks). Once this threshold is reached, increased protein release, surface deformation, and mass loss occurs. This study also showed that dynamic loading and the environment in which an implant is placed affect its biodegradation. Therefore, it may be essential that in vitro degradation studies of these or similar implants include a dynamic functional environment.

New technique to extend the useful life of a biodegradable cartilage implant.

Biodegradable implants made of alpha-hydroxypolyesters, such as polylactic acid, polyglycolic acid, or their copolymers, undergo bulk degradation with a concomitant mass loss. Although biocompatibility or toxicity problems, which have occasionally been reported in response to these materials' in vivo behavior, have not been conclusively linked to rapid mass loss, we hypothesized that such implants should degrade and lose mass in a more uniform rate. To this end, we designed a new implant, intended to be used in articular cartilage repair, consisting of a blend of three copolymers of 50:50 poly(d,l)-lactide coglycolide with inherent viscosities of 0.23, 0.58, and 1.37 dL/g. The objective of the blend implant design was to achieve a slower rate of degradation and mass loss in comparison to a previous design, which used a single copolymer of inherent viscosity of 0.58 dL/g. The blend's in vitro degradation characteristics were obtained and compared to those of the control design in terms of mass, molecular weight, pH, mechanical properties, gross morphology, and porosity. Another objective of our study was to design and employ a novel test for assessing the permeability of porous scaffolds, using a custom apparatus under direct permeation conditions. Significant differences in the temporal behavior of the two groups were found. The blend implants maintained their overall structural integrity longer than control specimens (6 weeks versus 3 weeks). This was a surprising finding in light of the fact that losses in molecular weight were similar in the two groups. Extension of structural usefulness to 6 weeks, achieved by the method described in the study, can be expected to enhance the viability of this scaffold in an in vivo application such as cartilage repair. Thus, the blended copolymer implants may be more suitable in orthopedic applications, where a decreased degradation rate would be preferable.

Salient Degradation Features of a 50:50 PLA/PGA Scaffold for Tissue Engineering.

An implant system that undergoes a gradual, time-dependent, nontoxic degradation process may offer an efficacious, safe, and desirable alternative to metallic materials used in the treatment of various musculoskeletal conditions. Such a scaffold may also be a suitable vehicle for growing cells and tissue in the laboratory for tissue engineering applications. We have used a scaffold of this type previously in animal studies for biological resurfacing of large articular cartilage defects.(1) This study examined important in vitro degradation characteristics of a 50:50 polylactic acid/polyglycolic acid (PLG) implant during an 8-week period. It was determined that this particular implant degraded in a biphasic fashion. The initial phase occurred during the first 2 weeks with a decrease in molecular weight and surface axial strain, coupled with an increase in percent porosity. The second phase demonstrated a decline in surface axial strain by 4 weeks and an ongoing decline in molecular weight. Loss of gross structural properties was not evident until the start of the second phase and was complete at 8 weeks. This study demonstrated the potential uses for this implant as a means of providing structural support for cells and tissue ingrowth for up to 8 weeks. Further studies need to be conducted in order to determine the biological effects of the degrading polymer byproducts on host tissues.

Fabrication and Characterization of PLA-PGA Orthopedic Implants.

Fabrication methods and property characterization of polyglycolic acid (PGA), polylactic acid (PLA), and their copolymers are reviewed. Both of these aliphatic polyesters belong to the a-hydroxy group and biodegrade in a physiological environment to monomeric acids, which are readily processed and excreted from the body. The physical and mechanical characteristics discussed include molecular weight, crystallinity, stress-strain behavior, permeability, and melting/glass transition temperatures. The most common methods of fabricating PLA-PGA materials into medical devices are described.

Fundamentals of biomechanics in tissue engineering of bone.

The objective of this review is to provide basic information pertaining to biomechanical aspects of bone as they relate to tissue engineering. The review is written for the general tissue engineering reader, who may not have a biomechanical engineering background. To this end, biomechanical characteristics and properties of normal and repair cortical and cancellous bone are presented. Also, this chapter intends to describe basic structure-function relationships of these two types of bone. Special emphasis is placed on salient classical and modern testing methods, with both material and structural properties described.

The role of collagen in determining bone mechanical properties.

The hypothesis of this study was that collagen denaturation would lead to a significant decrease in the toughness of bone, but has little effect on the stiffness of bone. Using a heating model, effects of collagen denaturation on the biomechanical properties of human cadaveric bone were examined. Prior to testing, bone specimens were heat treated at varied temperatures (37-200 degrees C) to induce different degrees of collagen denaturation. Collagen denaturation and mechanical properties of bone were determined using a selective digestion technique and three-point bending tests, respectively. The densities and weight fractions of the mineral and organic phases in bone also were determined. A repeated measures analysis of variance showed that heating had a significant effect on the biomechanical integrity of bone, corresponding to the degree of collagen denaturation. The results of this study indicate that the toughness and strength of bone decreases significantly with increasing collagen denaturation, whereas the elastic modulus of bone is almost constant irrespective of collagen denaturation. These results suggest that the collagen network plays an important role in the toughness of bone, but has little effect on the stiffness of bone, thereby supporting the hypothesis of this study.

Exercise affects the mechanical properties and histological appearance of equine articular cartilage.

Dorsal carpal osteochondral injury is a major cause of reduced performance in horses undergoing high-intensity training. It was hypothesised that the mechanical behaviour and histology of cartilage are influenced by the intensity of exercise and by location within a joint. Relationships between histology and mechanical behaviour were identified in 2-year-old horses undergoing 19 weeks of high-intensity treadmill training or low-intensity exercise and then compared between groups. Dorsal and palmar test sites were identified on radial, intermediate, and third carpal articular surfaces after euthanasia. The mechanical properties of cartilage were determined with an automated creep indentation apparatus as previously described for equine cartilage. Cartilage morphology was assessed with use of sections stained with haematoxylin and eosin and toluidine blue. Dorsal cartilage was less permeable, thinner, and had a loss of chondrocyte alignment compared with palmar cartilage. Cartilage from strenuously trained horses showed more fibrillation and chondrocyte clusters than did cartilage from gently exercised animals. Dorsal radial carpal cartilage and third carpal cartilage of strenuously trained animals were significantly less stiff than that from gently exercised animals, and the former had reduced superficial toluidine blue staining compared with that from the gently exercised group. These results indicate that topographical and exercise-related differences exist in the morphology and mechanical properties of carpal cartilage and suggest that strenuous training may lead to deterioration of cartilage at sites with a high clinical incidence of lesions.

Ultrahigh molecular weight polyethylene particles have direct effects on proliferation, differentiation, and local factor production of MG63 osteoblast-like cells.

Small particles of ultrahigh molecular weight polyethylene stimulate formation of foreign-body granulomas and bone resorption. Bone formation may also be affected by wear debris. To determine if wear debris directly affects osteoblasts, we characterized a commercial preparation of ultrahigh molecular weight polyethylene (GUR4150) particles and examined their effect on MG63 osteoblast-like cells. In aliquots of the culture medium containing ultrahigh molecular weight polyethylene, 79% of the particles were less than 1 microm in diameter, indicating that the cells were exposed to particles of less than 1 microm. MG63 cell response to the particles was measured by assaying cell number, [3H]thymidine incorporation, alkaline phosphatase specific activity, osteocalcin production, [35S]sulfate incorporation, and production of prostaglandin E2 and transforming growth factor-beta. Cell number and [3H]thymidine incorporation were increased in a dose-dependent manner. Alkaline phosphatase specific activity, a marker of cell differentiation for the cultures, was significantly decreased, but osteocalcin production was not affected. [35S]sulfate incorporation, a measure of extracellular matrix production, was reduced. Prostaglandin E2 release was increased, but transforming growth factor-beta production was decreased in a dose-dependent manner. This shows that ultrahigh molecular weight polyethylene particles affect MG63 proliferation, differentiation, extracellular matrix synthesis, and local factor production. These effects were direct and dose dependent. The findings suggest that ultrahigh molecular weight polyethylene wear debris particles with an average size of approximately 1 microm may inhibit bone formation by inhibiting cell differentiation and reducing transforming growth factor-beta production and matrix synthesis. In addition, increases in prostaglandin E2 production may not only affect osteoblasts by an autocrine pathway but may also stimulate the proliferation and activation of cells in the monocytic lineage. These changes favor decreased bone formation and increased bone resorption as occur in osteolysis.

Influence of post-deposition heating time and the presence of water vapor on sputter-coated calcium phosphate crystallinity.

Extensive research suggested that calcium phosphate (CaP) coatings on titanium implants are essential for early bone response. However, the characterization of CaP crystallinity and the means to control coating crystallinity are not well-established. In this study, the effect of a 400 degrees C heat treatment for 1, 2, or 4 hours, and in the presence or absence of water vapor, on CaP crystallinity was investigated. Scanning electron microscopy indicated dense as-sputtered coatings. Increase in coating crystallinity was observed to be consistent with the increasing number of PO(4) peaks observed as a result of different heat treatments. In addition, x-ray diffraction analyses indicated amorphous as-sputtered coatings, whereas crystalline CaP coatings in the range of 0-85% were observed after different post-deposition heat treatments. It was concluded that the presence of water vapor and post-deposition heat treatment time significantly affect the crystallinity of CaP coatings, which may ultimately affect bone healing.

Analysis of 2-DG autoradiograms using image-averaging and image-differencing procedures for systems-level description of neurobehavioral function.

Computer assisted 2-deoxyglucose (2-DG) autoradiography has been used to provide functional maps of areas of altered neural activity related to changes in an animal's behavior or state. The standard procedure for comparison of autoradiograms between different treatment groups has been to take measurement samples from predefined neuroanatomical regions and to average these across brains to attain statistical sensitivity for detecting treatment effects. Unfortunately, when sampling is restricted to predefined areas, important topographic information is lost along with the ability to reveal an unexpected change in neural activity. To preserve the rich topographical detail of metabolic information and to enhance the capacity to uncover novel areas of altered metabolic activity, we have developed a system for averaging entire images from 2-DG autoradiograms and for comparing the average images from two experimental groups by creating an image of differences. This procedure does not rely on sampling only preselected regions, but still allows statistical comparisons between experimental groups. The procedures we describe can be readily and inexpensively adapted for use in individual laboratories and are based on modifications of preexisting image analysis software. We show that, when average and difference images are created using standardized protocols for sectioning brain tissue and editing section images, they are impressively resolved and realistic and can serve as effective topographic descriptions of group differences in neural activity of functional and behavioral relevance.