Structural and nanoindentation studies of stem cell-based tissue-engineered bone

Stem cell-based gene therapy and tissue engineering have been shown to be an efficient method for the regeneration of critical-sized bone defects. Despite being an area of active research over the last decade, no knowledge of the intrinsic ultrastructural and nanomechanical properties of such bone tissue exists. In this study, we report the nanomechanical properties of engineered bone tissue derived from genetically modified mesenchymal stem cells (MSCs) overexpressing the rhBMP2 gene, grown in vivo in the thigh muscle of immunocompetent mice for 4 weeks, compared to femoral bone adjacent to the transplantation site. The two types of bone had similar mineral contents (61 and 65 wt% for engineered and femoral bone, respectively), overall microstructures showing lacunae and canaliculi (both measured by back-scattered electron microscopy), chemical compositions (measured by energy dispersive X-ray analysis), and nanoscale topographical morphologies (measured by tapping-mode atomic force microscopy imaging or TMAFM). Nanoindentation experiments revealed that the small length scale mechanical properties were statistically different with the femoral bone (indented parallel to the bone long axis) being stiffer and harder (apparent elastic modulus, E approximately 27.3+/-10.5 GPa and hardness, H approximately 1.0+/-0.7G Pa) than the genetically engineered bone (E approximately 19.8+/-5.6 GPa, H approximately 0.9+/-0.4G Pa). TMAFM imaging showed clear residual indents characteristic of viscoelastic plastic deformation for both types of bone. However, fine differences in the residual indent area (smaller for the engineered bone), pile up (smaller for the engineered bone), and fracture mechanisms (microcracks for the engineered bone) were observed with the genetically engineered bone behaving more brittle than the femoral control.     

The effect of recovery time and test conditions on viscoelastic measures of tensile damage in cortical bone

Stiffness degradation and strength degradation are often measured to monitor and characterize the effects of damage accumulation in bone. Based on evidence that these properties could be affected by not only damage magnitude but also test conditions, the present study investigated the effect of hold condition and recovery time on measures of tensile damage. Machined human femoral cortical bone specimens were subjected to tensile tests consisting of a pre-damage diagnostic loading cycle, a damage loading cycle and post-damage cycle. Controlled variables were recovery time (1, 10, and 100 min) and hold condition (zero load or zero strain) after the damage cycle. Damage measures were calculated as the ratio of each post-damage cycle to the pre-damage value for loading modulus, secant modulus, unloading modulus, stress relaxation and strain (stress) recovery at 1 min post-diagnostic time. The damage cycle caused reductions in all measures, and some measures varied with recovery time and hold condition. Apparent modulus degradation for both hold conditions decreased with recovery time. Stress relaxation was unaffected by recovery time for both hold conditions. Zero-strain hold conditions resulted in lower values for degradation of modulus and change of relaxation. Stress or strain recovery after the damage cycle was evident through 100 min, but 90% of the recovery occurred within 10 min. The results demonstrate that choice of test conditions can influence the apparent magnitude of damage effects. They also indicate that 10 min recovery time was sufficient to stabilize most measures of the damage state.     

Influence of confluent holding time on UV light mutagenesis in human diploid fibroblasts

We have investigated the induction of mutants resistant to 6-thioguanine (6TG) following 254 nm ultraviolet light exposure of density-inhibited cultures of human diploid fibroblasts. Phenotypic expression of 6TG resistance was maximal within 9 days and remained stable through 19 days after irradiation. In reconstruction studies, complete recovery of 6TG-resistant mutants occurred at cell densities of up to 35 000 cells per 100-mm petri dish. The induced mutation frequency increased linearly with dose over the range of 3–9 J/m²; the D₀ of the survival curve was 4.2 J/m². Delaying subculture to low density for 1.5–24 h after irradiation produced unexpected alterations in induced mutation frequencies. An increase in UV-induced mutations of approximately 3-fold was observed in cultures maintained in confluence for 3 h. This trend was reversed with longer holding times: the mutation frequency declined sharply in cultures held for 6 h compared to the 3-h value, and thereafter showed a steady and gradual diminution to background levels.

These data suggest that the repair of potentíally mutagenic damage is a complex phenomenon which can lead to an increase or decrease in mutation frequency as a function of holding time. Although the decline in mutation frequency observed following longer holding intervals is consistent with the notion of an error-free process, we hypothesize that the increased mutation frequency produced by a short holding period reflects the existence of a cell-mediated process which enhances the mutagenic potential of at least some UV-induced DNA photoproducts.     

Mechanical properties of porcine femoral cortical bone measured by nanoindentation

This study uses a nanoindentation technique to examine variations in the local mechanical properties of porcine femoral cortical bone under hydrated conditions. Bone specimens from three age groups (6, 12 and 42 months), representing developing bone, ranging from young to mature animals, were tested on the longitudinal and transverse cross-sectional surfaces. Elastic modulus and hardness of individual lamellae within bone's microstructure: laminar bone, interstitial bone, and osteons, were measured. Both the elastic modulus and hardness increased with age. However, the magnitudes of these increases were different for each microstructural component. The longitudinal moduli were higher than the transverse moduli. Dehydrated samples were also tested to allow a comparison with hydrated samples and these resulted in higher moduli and hardness than the hydrated samples. Again, the degree of variation was different for each microstructural component. These results indicate that the developmental changes in bone have different rates of mechanical change within each microstructural component.     

Intracellular calcium waves in bone cell networks under single cell nanoindentation

In this study, bone cells were successfully cultured into a micropatterned network with dimensions close to that of in vivo osteocyte networks using microcontact printing and self-assembled monolyers (SAMs). The optimal geometric parameters for the formation of these networks were determined in terms of circle diameters and line widths. Bone cells patterned in these networks were also able to form gap junctions with each other, shown by immunofluorescent staining for the gap junction protein connexin 43, as well as the transfer of gap-junction permeable calcein-AM dye. We have demonstrated for the first time, that the intracellular calcium response of a single bone cell indented in this bone cell network, can be transmitted to neighboring bone cells through multiple calcium waves. Furthermore, the propagation of these calcium waves was diminished with increased cell separation distance. Thus, this study provides new experimental data that support the idea of osteocyte network memory of mechanical loading similar to memory in neural networks.     

The effect of agent treatment time on the induction of sister-chromatid exchanges in mouse bone marrow cells in vivo

The effect of time of agent administration, via intraperitoneal injection, on the yield of SCEs in bone marrow cells of male B6C3F1 mice was determined for cyclophosphamide (CP), 7,12-dimethylbenz[a]anthracene (DMBA) and mitomycin C (MMC). Animals were treated with several doses of each carcinogen/mutagen at 3 different treatment times: -1, +1 and +8 h in relation to the onset of BrdUrd administration. The results of these studies indicate that the optimal treatment time for inducing a maximal SCE response is agent-specific. For CP, the slope of the SCE response was greatest at the +8 h treatment time while the maximal response for DMBA occurred at the -1 h treatment time. For MMC, the slope of the SCE response was independent of treatment time and of the method of bromodeoxyuridine administration (intravenous infusion vs. tablet implantation) but dependent on the laboratory conducting the study (Brookhaven National Laboratory vs. Oak Ridge Associated Universities). Based on the results of these studies, the +1 h acute treatment time is considered optimal for the in vivo cytogenetic evaluation of suspect chemicals for genotoxic activity when bone marrow is used as the target cell population.     

The effect of radiation sensitivity and cell stage on liquid holding response in Schizosaccharomyces pombe

Summary The response of the wild type strain and 20 different radiation sensitive mutants of S. pombe to liquid holding after ultraviolet irradiation was ivestigated. Three of the sensitive mutants tested showed appreciable liquid holding recovery, as opposed to the negative liquid holding effect observed in the wild type cells. One of these mutants is reported to be recombination-deficient while the other two have a normal recombination capability. Further experiments were carried out by using G₁ cells and ascospores to test the possible role of a recombinational type of repair pathway in the failure of wild type S. pombe to show liquid holding recovery. Data from such studies indicated that the negative liquid holding effect observed in the wild type cannot be ascribed to this particular pathway. This conclusion is further supported by the observation that caffeine which is believed to inhibit mainly the recombinational repair in this yeast, did not alter the negative liquid holding effect in the wild type. This observation implies that the caffeine-sensitive repair process occurs only in a rich medium and not in the non-nutrient solution. Data have been discussed as these relate to possible cause(s) of negative liquid holding effect in this organism.     

Results from demineralized bone creep tests suggest that collagen is responsible for the creep behavior of bone

Cortical and trabecular bone have similar creep behaviors that have been described by power-law relationships, with increases in temperature resulting in faster creep damage accumulation according to the usual Arrhenius (damage rate approximately exp (-Temp.-1)) relationship. In an attempt to determine the phase (collagen or hydroxyapatite) responsible for these similar creep behaviors, we investigated the creep behavior of demineralized cortical bone, recognizing that the organic (i.e., demineralized) matrix of both cortical and trabecular bone is composed primarily of type I collagen. We prepared waisted specimens of bovine cortical bone and demineralized them according to an established protocol. Creep tests were conducted on 18 specimens at various normalized stresses sigma/E0 and temperatures using a noninvasive optical technique to measure strain. Denaturation tests were also conducted to investigate the effect of temperature on the structure of demineralized bone. The creep behavior was characterized by the three classical stages of decreasing, constant, and increasing creep rates at all applied normalized stresses and temperatures. Strong (r2 > 0.79) and significant (p < 0.01) power-law relationships were found between the damage accumulation parameters (steady-state creep rate d epsilon/dt and time-to-failure tf) and the applied normalized stress sigma/E0. The creep behavior was also a function of temperature, following an Arrhenius creep relationship with an activation energy Q = 113 kJ/mole, within the range of activation energies for cortical (44 kJ/mole) and trabecular (136 kJ/mole) bone. The denaturation behavior was characterized by axial shrinkage at temperatures greater than approximately 56 degrees C. Lastly an analysis of covariance (ANCOVA) of our demineralized cortical bone regressions with those found in the literature for cortical and trabecular bone indicates than all three tissues creep with the same power-law exponents. These similar creep activation energies and exponents suggest that collagen is the phase responsible for creep in bone.     

An application of nanoindentation technique to measure bone tissue Lamellae properties

Measuring the microscopic mechanical properties of bone tissue is important in support of understanding the etiology and pathogenesis of many bone diseases. Knowledge about these properties provides a context for estimating the local mechanical environment of bone related cells thait coordinate the adaptation to loads experienced at the whole organ level. The objective of this study was to determine the effects of experimental testing parameters on nanoindentation measures of lamellar-level bone mechanical properties. Specifically, we examined the effect of specimen preparation condition, indentation depth, repetitive loading, time delay, and displacement rate. The nanoindentation experiments produced measures of lamellar elastic moduli for human cortical bone (average value of 17.7 +/- 4.0 GPa for osteons and 19.3 +/- 4.7 GPa for interstitial bone tissue). In addition, the hardness measurements produced results consistent with data in the literature (average 0.52 +/- 0.15 GPa for osteons and 0.59 +/- 0.20 GPa for interstitial bone tissue). Consistent modulus values can be obtained from a 500-nm-deep indent. The results also indicated that the moduli and hardnesses of the dry specimens are significantly greater (22.6% and 56.9%, respectively) than those of the wet and wet and embedded specimens. The latter two groups were not different. The moduli obtained at a 5-nm/s loading rate were significantly lower than the values at the 10- and 20-nm/s loading rates while the 10- and 20-nm/s rates were not significantly different. The hardness measurements showed similar rate-dependent results. The preliminary results indicated that interstitial bone tissue has significantly higher modulus and hardness than osteonal bone tissue. In addition, a significant correlation between hardness and elastic modulus was observed.     

Precision of nanoindentation protocols for measurement of viscoelasticity in cortical and trabecular bone

Nanoindentation has recently gained attention as a characterization technique for mechanical properties of biological tissues, such as bone, on the sub-micron level. However, optimal methods to characterize viscoelastic properties of bones are yet to be established. This study aimed to compare the time-dependent viscoelastic properties of bone tissue obtained with different nanoindentation methods. Bovine cortical and trabecular bone samples (n=8) from the distal femur and proximal tibia were dehydrated, embedded and polished. The material properties determined using nanoindentation were hardness and reduced modulus, as well as time-dependent parameters based on creep, loading-rate, dissipated energy and semi-dynamic testing under load control. Each loading protocol was repeated 160 times and the reproducibility was assessed based on the coefficient of variation (CV). Additionally, three well-characterized polymers were tested and CV values were calculated for reference.The employed methods were able to characterize time-dependent viscoelastic properties of bone. However, their reproducibility varied highly (CV 9–40%). The creep constant increased with increasing dwell time. The reproducibility was best with a 30 s creep period (CV 18%). The dissipated energy was stable after three repeated load cycles, and the reproducibility improved with each cycle (CV 23%). The viscoelastic properties determined with semi-dynamic test increased with increase in frequency. These measurements were most reproducible at high frequencies (CV 9–10%). Our results indicate that several methods are feasible for the determination of viscoelastic properties of bone material. The high frequency semi-dynamic test showed the highest precision within the tested nanoindentation protocols.     

The clinical application of serial bone mass measurements

Although the short-term precision of various bone mineral content (BMC) measurements is known, questions about the clinical use of serial BMC measurements remain: how frequently should BMC be measured? When is it appropriate to calculate bone loss rates? How are estimates of loss rate interpreted? This paper discusses both biological and technical sources of uncertainty, and the estimation of confidence limits for measured bone loss rates. For many, possibly most, patients, calculation of bone loss rate may not be necessary; however, repeated measures of BMC can still be useful for re-evaluating fracture risk. Indications for repeating BMC measurements may include low initial BMC (moderate to high fracture risk), anticipation of rapid bone loss (e.g., menopause, estrogen discontinuation), and verification of treatment efficacy.     

The effect of liquid holding on chemical induced lethality and mitotic gene conversion in Saccharomyces cerevisiae

Summary Mitotic gene conversion was induced with a variety of chemical mutagens in a double heteroallelic strain of Saccharomyces cerevisiae. Cells were treated with various mutagens and plated immediately onto selective and nonselective growth medium or else they were subject before plating to liquid holding in buffer for various lengths of time. In respiratory competent cells liquid holding caused a decrease in lethality and in conversion frequencies. Respiratory deficient cells, unable to use a non-fermentable substrate as an energy source, behaved different. Untreated cells started to die in buffer after two days of storage, and moreover, there was a considerable increase in potential convertants i.e. cells giving rise to gene convertants when plated on selective growth media. Respiratory deficient cells treated with various chemical mutagens were still more sensitive to liquid holding. After low, sublethal doses cells started to die after one day of liquid holding already and when plated on media selective for convertants, showed an increasing frequency of gene convertants. Addition of very low concentrations of glucose to the liquid holding buffer post-poned the lethal and convertogenic effects. Higher concentrations of glucose completely abolished sensitivity to liquid holding-induced lethality and genetic alterations. The results are interpreted to mean that in respiratory deficient cells no repair activities are possible to an accumulation of spontaneous lethal damage and genetic alterations which are expressed as gene conversion when an energy source becomes available. Such a repairless condition causes an increased sensitivity to genetically active agents, and provides a useful system to detect genetic effects of slowly reacting agents.     

Simulation of creep in non-homogenous samples of human cortical bone

Characterising the mechanisms causing viscoelastic mechanical properties of human cortical bone, as well as understanding sources of variation, is important in predicting response of the bone to creep and fatigue loads. Any better understanding, when incorporated into simulations including finite element analysis, would assist bioengineers, clinicians and biomedical scientists. In this study, we used an empirically verified model of creep strain accumulation, in a simulation of 10 non-homogeneous samples, which were created from micro-CT scans of human cortical bone of the femur midshaft obtained from a 74-year-old female cadaver. These non-homogeneous samples incorporate the presence of Haversian canals and resorption cavities. The influence of inhomogeneity on the response and variation in the samples in both creep and stress relaxation tests are examined. The relationship between steady-state creep rate, applied loads (stress relaxation and creep tests) and microstructure, that is bone apparent porosity, is obtained. These relations may provide insight into damage accumulation of whole human bones and be relevant to studies on osteoporosis.     

Simulation of creep in non-homogenous samples of human cortical bone

Characterising the mechanisms causing viscoelastic mechanical properties of human cortical bone, as well as understanding sources of variation, is important in predicting response of the bone to creep and fatigue loads. Any better understanding, when incorporated into simulations including finite element analysis, would assist bioengineers, clinicians and biomedical scientists. In this study, we used an empirically verified model of creep strain accumulation, in a simulation of 10 non-homogeneous samples, which were created from micro-CT scans of human cortical bone of the femur midshaft obtained from a 74-year-old female cadaver. These non-homogeneous samples incorporate the presence of Haversian canals and resorption cavities. The influence of inhomogeneity on the response and variation in the samples in both creep and stress relaxation tests are examined. The relationship between steady-state creep rate, applied loads (stress relaxation and creep tests) and microstructure, that is bone apparent porosity, is obtained. These relations may provide insight into damage accumulation of whole human bones and be relevant to studies on osteoporosis.