Long-term stability of self-assembled monolayers on 316L stainless steel.

316L stainless steel (316L SS) has been extensively used for making orthopedic, dental and cardiovascular implants. The use of phosphonic acid self-assembled monolayers (SAMs) on 316L SS has been previously explored for potential biomedical applications. In this study, we have investigated the long-term stability of methyl (-CH(3)) and carboxylic acid (-COOH)-terminated phosphonic acid SAMs on 316L under physiological conditions. The stability of SAMs on mechanically polished and electropolished 316L SS was also investigated as a part of this study. Well-ordered and uniform -CH(3)- and -COOH-terminated SAMs were coated on mechanically polished and electropolished 316L SS surfaces. The long-term stability of SAMs on 316L SS was investigated for up to 28 days in Tris-buffered saline (TBS) at 37 degrees C using x-ray photoelectron spectroscopy, atomic force microscopy and contact angle goniometry. A significant amount of phosphonic acid molecules was desorbed from the 316L SS surfaces within 1 to 7 days of TBS immersion followed by a slow desorption of molecules over the remaining days. The -COOH-terminated SAM was found to be more stable than the -CH(3)-terminated SAM on both mechanically and electropolished surfaces. No significant differences in the desorption behavior of SAMs were observed between mechanically and electropolished 316L SS surfaces.

Plasma surface modification of poly(D,L-lactic acid) as a tool to enhance protein adsorption and the attachment of different cell types.

We have studied the influence of oxygen radio frequency glow discharge (RfGD) on the surface and bulk properties of poly(D,L-lactic acid) (PDLLA) and the effect of this surface modification on both protein adsorption and bone cell behavior. PDLLA films were characterized before and after plasma surface modification by water contact angle, surface energy, and adhesion tension of water as well as by scanning electron microscopy (SEM), X-ray electron spectroscopy (XPS), and Fourier transform infra-red (FTIR) spectroscopy. RfGD-films showed an increase in hydrophilicity and surface energy when compared with untreated films. Surface morphological changes were observed by SEM. Chemical analysis indicated significant differences in both atomic percentages and oxygen functional group. Protein adsorption was evaluated by combining solute depletion and spectroscopic techniques. Bovine serum albumin (BSA), fibronectin (FN), vitronectin (VN), and fetal bovine serum (FBS) were used in this study. RfGD-treated surfaces adsorbed more BSA and FN from single specie solutions than FBS that is a more complex, multi-specie solution. MG63 osteoblast-like cells and primary cultures of fetal rat calvarial (FRC) cells were used to assess both the effect of RfGD treatment and protein adsorption on cell attachment and proliferation. In the absence of preadsorbed proteins, cells could not distinguish between treated and untreated surfaces, with the exception of MG63 cells cultured for longer periods of time. In contrast, the adsorption of proteins increased the cells' preference for treated surfaces, thus indicating a crucial role for adsorbed proteins in mediating the response of osteogenic cells to the RfGD-treated PDLLA surface.

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.

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.

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.

Intraspecies and interspecies comparison of the compressive properties of the medial meniscus.

Quantification of the compressive material properties of the meniscus is of paramount importance, creating a "gold-standard" reference for future research. The purpose of this study was to determine compressive properties in six animal models (baboon, bovine, canine, human, lapine, and porcine) at six topographical locations. It was hypothesized that topographical variation of the compressive properties would be found in each animal model and that interspecies variations would also be exhibited. To test these hypotheses, creep and recovery indentation experiments were performed on the meniscus using a creep indentation apparatus and analyzed via a finite element optimization method to determine the material properties. Results show significant intraspecies and interspecies variation in the compressive properties among the six topographical locations, with the moduli exhibiting the highest values in the anterior portion. For example, the anterior location of the human meniscus has an aggregate modulus of 160 +/- 40 kPa, whereas the central and posterior portions exhibit aggregate moduli of 100 +/- 30 kPa. Interspecies comparison of the aggregate moduli identifies the lapine anterior location having the highest value (450 +/- 120 kPa) and the human posterior location having the lowest (100 +/- 30 kPa). These baseline values of compressive properties will be of help in future meniscal repair efforts.

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.

Age dependence of in situ termostability of collagen in human bone.

The hypothesis of this study is that the in situ thermostability of collagen in bone changes with age and such changes relate to the structural properties of collagen and/or the interaction between the collagen and mineral phases. To test the hypothesis, the effect of age on the in situ thermostability of collagen in human bone and its correlation with the mineral and collagen phases were investigated. In this study, 30 human cadaveric femurs were collected and divided into three age groups: young adults (20-45 years), middle aged (46-69 years), and elderly (over 70 years of age). The in situ thermostability of collagen was assessed using high performance liquid chromatography (HPLC) in terms of the amount of heat-induced collagen denaturation at eight temperatures between 25 degrees C and 140 degrees C. In addition to the density and weight fraction of the mineral and organic phases, the concentration of collagen crosslinks was measured to assess the structural integrity of collagen. The results of this study indicate that the in situ thermostability of collagen increases with increasing age, and such age-related changes correlate with the following: collagen molecular structure, amount of noncalcified collagen, and the fraction and density of the mineral phase in bone. These results suggest that the age-related changes in the in situ thermostability of collagen most likely relate to the collagen structure and its interaction with the mineral phase. In addition, the bone remodeling process may play a role in the age-related changes in collagen thermostability because noncalcified collagen is commonly associated with this process.

Characterization and dissolution behavior of sputtered calcium phosphate coatings after different postdeposition heat treatment temperatures.

There is a lack of correlation between specific properties of hydroxyapatite coating surfaces, osseointegration processes, and implant success. The aim of this study was to evaluate the relationship between well-characterized structural and chemical properties of radio-frequency sputtered calcium phosphate (CaP) coatings and their dissolution behavior. Sputtered CaP coatings were evaluated as-sputtered (non-heat treated) or after 1 hour of postsputter heat treatments at 400 degrees C or 600 degrees C. All coatings were characterized by X-ray diffraction, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and contact angle measurement. The dissolution behavior of CaP coatings in the presence and absence of proteins was also investigated. It was observed from this study that as-sputtered CaP coatings were amorphous. The 400 degrees C heat-treated CaP coatings exhibited low crystallinity (1.9% +/- 0.4%), whereas the 600 degrees C heat-treated CaP coatings were highly crystalline (67.0% +/- 2.4%). The increase of Ca/P ratio, PO4/HPO4 ratio, and the number of PO4 peaks were observed to be consistent with the increase in heating temperature and the degrees of coating crystallinity. Phosphorus ions released from CaP coatings decreased with the increase of crystallinity of CaP coatings. In addition, immersion of CaP coatings in media containing proteins resulted in an increase in P ions released as compared with coatings immersed in media without proteins. It was concluded that the degree of CaP coating crystallinity can be controlled by varying the postdeposition heat-treatment temperature. It was also concluded that, aside from coating crystallinity, dissolution and reprecipitation of the coatings can be controlled by knowing the presence of proteins in the media and PO4/HPO4 ratio within the coatings.

Effects of collagen unwinding and cleavage on the mechanical integrity of the collagen network in bone.

The objective of this study was to investigate how molecular level changes in the collagen network affect its mechanical integrity. Our hypothesis is that the cleavage and unwinding of triple helices of collagen molecules significantly reduce the mechanical integrity of the collagen network in bone, whereas collagen crosslinks play a major role in sustaining the structural integrity of the collagen network. To test this hypothesis, the collagen molecular structure was altered in demineralized human cadaveric bone samples in the following two ways: heat induced unwinding and pancreas elastase induced cleavage of collagen molecules. Along with control specimens, the treated specimens were mechanically tested in tension to determine their strength, elastic modulus, toughness, and strain to failure. Also, the percentage of denatured collagen molecules and amounts of two major collagen crosslinks (hydroxylysylpyridinoline and lysylpyridinoline) were determined using high-performance liquid chromatography techniques. It was found that unwinding of collagen molecules may cause more reduction in stiffness (E) but less strain to failure (ef) than cleavage. Both collagen denaturation types cause similar changes in the strength (ss) and work to fracture (Wf) of the collagen network with no significant changes in hydroxylysylpyridinoline and lysylpyridinoline crosslinks. The results of this study indicate that the integrity of collagen molecules significantly affect the mechanical properties of the collagen network in bone, and that collagen crosslinks may play an important role in maintaining the mechanical integrity of the collagen network after collagen denaturation occurs.

Biodegradable polymeric scaffolds for musculoskeletal tissue engineering.

Biodegradable scaffolds have played an important role in a number of tissue engineering attempts over the past decade. The goal of this review article is to provide a brief overview of some of the important issues related to scaffolds fabricated from synthetic biodegradable polymers. Various types of such materials are available; some are commercialized and others are still in the laboratories. The properties of the most common of these polymers are discussed here. A variety of fabrication techniques were developed to fashion polymeric materials into porous scaffolds, and a selection of these is presented. The very important issue of scaffold architecture, including the topic of porosity and permeability, is discussed. Other areas such as cell growth on scaffolds, surface modification, scaffold mechanics, and the release of growths factors are also reviewed. A summary outlining the common themes in scaffold-related science that are found in the literature is presented.

The use of dynamic mechanical analysis to assess the viscoelastic properties of human cortical bone.

The purpose of this study was to examine the use of a dynamic mechanical analyzer (DMA) system to study the viscoelastic nature of bone. Cortical bone specimens from human femora were tested isothermally for 150 min at 37 degrees C and the loss factor (tan delta) and storage modulus (E') were measured. To explore the effects of test conditions on tan delta and E', different levels of applied stress, two specimen sizes, and two hydration conditions (wet and vacuum-dried) were evaluated. Finally, nonisothermal tests were performed, wherein specimens were heated up to 70 degrees C at different heating rates: 1 degrees C/min, 3 degrees C/min, and 5 degrees C/min. The results indicated that a threshold level of minimum applied stress was required to obtain repeatable and relatively constant values of tan delta. Specimen size did not significantly affect tan delta although it influenced E'. Moisture content had a significant effect on tan delta; vacuum-dried specimens exhibited a lower tan delta compared to wet specimens. Lastly, heating rates influenced tan delta values with lower rates producing more consistent results. The study demonstrated that DMA can be used as an effective tool to test bone.

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.

A mixed mode fracture toughness test of bone-biomaterial interfaces.

Tissue-biomaterial interfacial bonding plays a significant role in the success of biomaterials used for load-bearing orthopedic and dental prostheses. The objective of this study was to develop a physically sound and practically effective technique for assessment of the strength of bone-biomaterial interfaces under mixed mode loading. A single-edge notched sandwich specimen was developed for this purpose, wherein a bilayer specimen comprising the interface between tissue and biomaterial was sandwiched between two holders and loaded under mixed modes. First, a closed form solution was derived for the sandwich specimen under the assumption of linear elasticity, based on a general solution for sandwich structures reported in the literature. Then, a correction factor was determined for the solution using finite element models to compensate for errors induced by finite interlayer thickness. Moreover, using the same FEA models, it was found that crack closure may occur when the shear component is dominant at the crack. However, its effects were estimated to be limited and negligible. Furthermore, as an example, the strength of a bone/dental cement interface under different loading modes was tested using this sandwich technique. It is expected that the mixed mode technique can provide an effective means for investigators to study the mechanical integrity of bone-biomaterial interfaces under complex loading conditions.

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.

Setscrew distal locking for intramedullary nails: a biomechanical study.

OBJECTIVE: This biomechanical study was undertaken to examine the effectiveness of setscrew distal locking in a static intramedullary (IM) femoral nail on the stability of fixation of femoral shaft fractures. DESIGN: Fifteen fresh-frozen cadeveric femora were randomly separated into three groups of five bones and transversely sectioned immediately distal to the isthmus. After the insertion of the large-diameter nails, distal locking was obtained by conventional method in the first group. In the second group, set-screw design was used in which two transverse screws penetrated only the lateral cortex of the femur and compressed the nail in the intramedullary canal. No distal locking was used in the third group. INTERVENTION: All instrumented femurs were mounted on a servohydraulic testing machine and tested in both rotations (20 degrees) and axial compression (amplitude: 1,000 Newton). MAIN OUTCOME MEASUREMENT: Loading-versus-displacement data, acquired at a ten-Hertz sampling rate, were calibrated and used to calculate maximum torque, stiffness, and energy capacity to failure. Maximum displacement and axial stiffness also were determined. RESULTS: Mean maximum torque at 10 degrees for each group were 15.3+/-4.8 newton-meters for the interlocking group, 8.5 +/-1.2 newton-meters for the setscrew group, and 3.6+/-2.7 newton-meters for the nonlocked femora. At 20 degrees of rotational displacement, the torque measured 37.4+/-2.6 newton-meters; 15.0+/-4.0 newton-meters; and 5.3+/-3.1 newton-meters, respectively (p < 0.05). Mean torsional stiffness was 1.17+/-0.76 newton-meters per degree for the setscrew group and 1.34+/-0.83 newton-meters per degree for the interlocking group (p > 0.05). The setscrew design provided 87 percent of the torsional rigidity of the interlocking group. In the axial compression test, mean maximum shortening was 1.1+/-0.3 millimeters in the interlocking group and 1.4+/-0.6 millimeters in the setscrew group (p > 0.05). The mean stiffness on longitudinal compression provided by the interlocking screws and the setscrews was 918 and 860 newton-meters per millimeter, respectively. CONCLUSION: The distal setscrew design provides adequate distal fixation of intramedullary nail for patients in the postoperative rehabilitation period of the femoral shaft fractures treated with intramedullary nailing.

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.

Microstructural heterogeneity and the fracture toughness of bone.

Age-related changes in the skeleton often lead to an increase in the susceptibility of bone to fracture. The purpose of this study was to determine whether differences in material properties between the osteonal and interstitial regions of bone have an effect on bone fracture properties. Parameters such as longitudinal fracture toughness, transverse fracture toughness, porosity, interstitial microhardness, osteonal microhardness, bone density, and weight fractions of the mineral and organic phases of bone were examined as a function of age using female baboon femurs. With increasing age, the longitudinal fracture toughness decreased significantly as did transverse fracture toughness, whereas the interstitial microhardness increased. However, no significant differences in the other parameters were observed as a function of age. Using the ratio of interstitial microhardness to osteonal microhardness as a measure of the differences in the material properties in these two regions, correlation analysis revealed that the longitudinal fracture toughness of bone has a significant correlation with the microhardness ratio. Localized differences in material properties between osteonal and interstitial regions of bone increase with age; such differences may result in high stress concentrations at cement lines and facilitate longitudinal crack propagation.

Effects of aging and dietary restriction on the structural integrity of rat articular cartilage.

The objectives of this study were to investigate the effects of aging and diet restriction on the biomechanical properties of articular cartilage, using a well-controlled rat model (Fischer 344). This animal model is recommended by the National Institute of Aging specifically to study aging and diet issues. The intrinsic biomechanical properties of articular cartilage were obtained using a creep indentation approach. The ages chosen (6, 12, 18, 24 months of age) correspond to approximate human ages of 20 to 80 years old. The diet regimen employed in this study used either an ad libitum fed group or a group fed 60% of the mean food intake of the ad libitum group. The results demonstrate that, unlike bone, rat articular cartilage biomechanical properties are not affected in a discernible manner by diet restriction, despite the fact that diet-restricted animals were significantly lighter in terms of body weight. Age effects on biomechanical properties are found only at 6 and 12 months probably due to developmental reasons, but not at later ages. It appears that aging and diet restriction have profoundly different effects on articular cartilage and bone. Another significant result of this study was to establish the rat as a suitable animal model to study cartilage biomechanical properties. Thus, the rat can be added to the list of animals that can be used to study structure-function and pathophysiological relationships in articular cartilage.