Deep-Freezing Temperatures During Irradiation Preserves the Compressive Strength of Human Cortical Bone Allografts: A Cadaver Study.

BACKGROUND: Gamma irradiation, which minimizes the risk of infectious disease transmission when human bone allograft is used, has been found to negatively affect its biomechanical properties. However, in those studies, the deep-freezing temperature during irradiation was not necessarily maintained during transportation and sterilization, which may have affected the findings. Prior reports have also suggested that controlled deep freezing may mitigate the detrimental effects of irradiation on the mechanical properties of bone allograft. QUESTION/PURPOSE: Does a controlled deep-freezing temperature during irradiation help preserve the compressive mechanical properties of human femoral cortical bone allografts? METHODS: Cortical bone cube samples, each measuring 64 mm3, were cut from the mid-diaphyseal midshaft of five fresh-frozen cadaver femurs (four male donors, mean [range] age at procurement 42 years [42 to 43]) and were allocated via block randomization into one of three experimental groups (with equal numbers of samples from each donor allocated into each group). Each experimental group consisted of 20 bone cube samples. Samples irradiated in dry ice were subjected to irradiation doses ranging from 26.7 kGy to 27.1 kGy (mean 26.9 kGy) at a deep-freezing temperature below -40°C (the recommended long-term storage temperature for allografts). Samples irradiated in gel ice underwent irradiation doses ranging from 26.2 kGy and 26.4 kGy (mean 26.3 kGy) in a freezing temperature range between -40°C and 0°C. Acting as controls, samples in a third group were not subjected to gamma irradiation. The mechanical properties (0.2% offset yield stress, ultimate compression stress, toughness, and the Young modulus) of samples from each group were subsequently evaluated via axial compression loading to failure along the long axis of the bone. The investigators were blinded to sample group during compression testing. RESULTS: The mean ultimate compression stress (84 ± 27 MPa versus 119 ± 31 MPa, mean difference 35 [95% CI 9 to 60]; p = 0.005) and toughness (3622 ± 1720 kJ/m3 versus 5854 ± 2900 kJ/m3, mean difference 2232 [95% CI 70 to 4394]; p = 0.009) of samples irradiated at a higher temperature range (-40°C to 0°C) were lower than in those irradiated at deep-freezing temperatures (below -40°C). The mean 0.2% offset yield stress (73 ± 28 MPa versus 109 ± 38 MPa, mean difference 36 [95% CI 11 to 60]; p = 0.002) and ultimate compression stress (84 ± 27 MPa versus 128 ± 40 MPa, mean difference 44 [95% CI 17 to 69]; p < 0.001) of samples irradiated at a higher temperature range (-40°C to 0°C) were lower than the nonirradiated control group samples. The mean 0.2% offset yield stress (73 ± 28 MPa versus 101 ± 28 MPa, mean difference 28 [95% CI 3 to 52]; p = 0.02; effect size = 1.0 [95% CI 0.8 to 1.2]) of samples irradiated at higher temperature range (-40°C to 0°C) were no different with the numbers available to those irradiated at deep-freezing temperature. The mean toughness (3622 ± 1720 kJ/m3 versus 6231 ± 3410 kJ/m3, mean difference 2609 [95% CI 447 to 4771]; p = 0.02; effect size = 1.0 [95% CI 0.8 to 1.2]) of samples irradiated at higher temperature range (-40°C to 0°C) were no different with the numbers available to the non-irradiated control group samples. The mean 0.2% offset yield stress, ultimate compression stress, and toughness of samples irradiated in deep-freezing temperatures (below -40°C) were not different with the numbers available to the non-irradiated control group samples. The Young modulus was not different with the numbers available among the three groups. CONCLUSION: In this study, maintenance of a deep-freezing temperature below -40°C, using dry ice as a cooling agent, consistently mitigated the adverse effects of irradiation on the monotonic-compression mechanical properties of human cortical bone tissue. Preserving the mechanical properties of a cortical allograft, when irradiated in a deep-freezing temperature, may have resulted from attenuation of the deleterious, indirect effects of gamma radiation on its collagen architecture in a frozen state. Immobilization of water molecules in this state prevents radiolysis and the subsequent generation of free radicals. This hypothesis was supported by an apparent loss of the protective effect when a range of higher freezing temperatures was used during irradiation. CLINICAL RELEVANCE: Deep-freezing temperatures below -40°C during gamma irradiation may be a promising approach to better retain the native mechanical properties of cortical bone allografts. A further study of the effect of deep-freezing during gamma radiation sterilization on sterility and other important biomechanical properties of cortical bone (such as, tensile strength, fracture toughness, and fatigue) is needed to confirm these findings.

Evaluation of degree of conversion, rate of cure, microhardness, depth of cure, and contraction stress of new nanohybrid composites containing pre-polymerized spherical filler.

The aim of the present study was to characterize nanohybrid and nanofilled composites in terms of degree of conversion (DC), rate of cure (RC), microhardness (Vickers hardness number; VHN), depth of cure, and contraction stress (CS). Ceram.X® universal- A3, duo enamel E2, and duo dentin D3 composites were compared to Tetric EvoCeram® and FiltekTMSupreme XTE composites of equivalent dentin and enamel shades under a 40 s photopolymerization protocol. DC was measured by infrared spectroscopy, calculating RC from the kinetic curve. Top and bottom VHN were determined using a Vickers indenter, and bottom/top surface ratio (Vickers hardness ratio; VHR) calculated. CS vs. time was assessed by a universal testing machine and normalized for the specimen bonding area. All materials showed DC < 60%, Ceram.X® composites reaching higher values than the other composites of corresponding shades. RC at 5 s of photopolymerization was always higher than that at 10 s. All the Ceram.X® composites and the lighter-shaded Tetric EvoCeram® and FiltekTMSupreme XTE composites reached the RC plateau after 25 s, the remaining materials showed a slower kinetic trend. Tetric EvoCeram® and FiltekTMSupreme XTE composites displayed the softest and the hardest surfaces, respectively. Differently from darker-shaded materials, the universal and the three enamel-shaded composites resulted optimally cured (VHR > 80%). The tested composites differed in CS both during and after light cure, Tetric EvoCeram® and FiltekTMSupreme XTE composites displaying the highest and the lowest CS, respectively. Only the Ceram.X® universal-A3 reached a CS plateau value. The tested composites exhibited material-dependent chemo-mechanical properties. Increasing the curing time and/or reducing the composite layer thickness for dentin-shaded composites appears advisable.

Bonding integrity and compressive strength of re-bonded, surface conditioned and Er Cr YSGG laser treated lithium disilicate ceramics.

AIM: The aim of the study was to investigate the shear bond strength (SBS) and compressive strength (CS) of Er Cr YSGG laser (ECL) treated, re-bonded lithium disilicate (LD) ceramic in comparison to standard conventional conditioning (hydrofluoric acid (HFA) and silane). METHODS: One hundred LD ceramic disks were divided equally for SBS and CS testing. Eighty samples were conventionally surface treated and bonded to resin cement followed by de-bonding of the cement build-up. All de-bonded specimens were divided into four groups based on re-bonding surface treatments (HFA, primer, adhesive, and ECL). Resin cement build-ups were performed in 40 specimens for SBS testing (universal testing machine); however, the remaining 40 specimens were tested for CS. Ten specimens each were used as controls (surface treatment was performed once and no primary resin cement bonding) for SBS and CS assessment. Surface topography was assessed using a scanning electron microscope. RESULTS: The maximum and minimum SBS values were shown by groups: control (33.42 ± 3.28 megapascals (MPa)); and ECL (17.50 ± 2.22 MPa) respectively. The maximum and minimum CSs were displayed by specimens in the ECL group (439.45 ± 70.68 MPa) and the control group (237.28 ± 19.96 MPa), respectively. For ECL specimens, SBS was significantly lower and CS was significantly higher as compared to control specimens. CONCLUSIONS: Application of the Er Cr YSGG laser significantly improved the CS of de-bonded ceramic specimens. However, it did not show a positive influence on the bond integrity of re-bonded ceramics in comparison to conventional surface treatment regimes.

Effects of ex vivo ionizing radiation on collagen structure and whole-bone mechanical properties of mouse vertebrae.

Bone can become brittle when exposed to ionizing radiation across a wide range of clinically relevant doses that span from radiotherapy (accumulative 50 Gy) to sterilization (~35,000 Gy). While irradiation-induced embrittlement has been attributed to changes in the collagen molecular structure, the relative role of collagen fragmentation versus non-enzymatic collagen crosslinking remains unclear. To better understand the effects of radiation on the bone material without cellular activity, we conducted an ex vivo x-ray radiation experiment on excised mouse lumbar vertebrae. Spinal tissue from twenty-week old, female, C57BL/6J mice were randomly assigned to a single x-ray radiation dose of either 0 (control), 50, 1000, 17,000, or 35,000 Gy. Measurements were made for collagen fragmentation, non-enzymatic collagen crosslinking, and both monotonic and cyclic-loading compressive mechanical properties. We found that the group differences for mechanical properties were more consistent with those for collagen fragmentation than for non-enzymatic collagen crosslinking. Monotonic strength at 17,000 and 35,000 Gy was lower than that of the control by 50% and 73% respectively, (p < 0.001) but at 50 and 1000 Gy was not different than the control. Consistent with those trends, collagen fragmentation only occurred at 17,000 and 35,000 Gy. By contrast, non-enzymatic collagen crosslinking was greater than control for all radiation doses (p < 0.001). All results were consistent both for monotonic and cyclic loading conditions. We conclude that the reductions in bone compressive monotonic strength and fatigue life due to ex vivo ionizing radiation are more likely caused by fragmentation of the collagen backbone than any increases in non-enzymatic collagen crosslinks.

In vitro degradation and mechanical properties of PLA-PCL copolymer unit cell scaffolds generated by two-photon polymerization.

The manufacture of 3D scaffolds with specific controlled porous architecture, defined microstructure and an adjustable degradation profile was achieved using two-photon polymerization (TPP) with a size of 2 × 4 × 2 mm(3). Scaffolds made from poly(D,L-lactide-co-ɛ-caprolactone) copolymer with varying lactic acid (LA) and ɛ -caprolactone (CL) ratios (LC16:4, 18:2 and 9:1) were generated via ring-opening-polymerization and photoactivation. The reactivity was quantified using photo-DSC, yielding a double bond conversion ranging from 70% to 90%. The pore sizes for all LC scaffolds were see 300 µm and throat sizes varied from 152 to 177 µm. In vitro degradation was conducted at different temperatures; 37, 50 and 65 °C. Change in compressive properties immersed at 37 °C over time was also measured. Variations in thermal, degradation and mechanical properties of the LC scaffolds were related to the LA/CL ratio. Scaffold LC16:4 showed significantly lower glass transition temperature (T g) (4.8 °C) in comparison with the LC 18:2 and 9:1 (see 32 °C). Rates of mass loss for the LC16:4 scaffolds at all temperatures were significantly lower than that for LC18:2 and 9:1. The degradation activation energies for scaffold materials ranged from 82.7 to 94.9 kJ mol(-1). A prediction for degradation time was applied through a correlation between long-term degradation studies at 37 °C and short-term studies at elevated temperatures (50 and 65 °C) using the half-life of mass loss (Time (M1/2)) parameter. However, the initial compressive moduli for LC18:2 and 9:1 scaffolds were 7 to 14 times higher than LC16:4 (see 0.27) which was suggested to be due to its higher CL content (20%). All scaffolds showed a gradual loss in their compressive strength and modulus over time as a result of progressive mass loss over time. The manufacturing process utilized and the scaffolds produced have potential for use in tissue engineering and regenerative medicine applications.

Effect of compressive loading and incubation with clodronate on the RANKL/OPG system of human osteoblasts.

OBJECTIVES: In vivo studies have shown that bisphosphonates result in slow rates of orthodontic tooth movement. This study investigated whether clodronate modifies the impact of mechanical loading on the RANKL/OPG system of human osteoblasts. METHODS: Osteoblasts were cultured in vitro with 0.5 or 5.0 µM clodronate for 48 h and/or subjected to 3 h of compressive loading at 34.9 g/cm(2). Cell viability was determined by MTT assay. Real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and immunocytochemical staining were used to analyze the cells for their production of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) at the transcriptional and protein levels. RESULTS: Compressive loading did not affect osteoblast viability in a significant way. Clodronate (5.0 µM) mildly reduced the viability of both compressed and uncompressed cells. Compressive loading induced a 4.2-fold increase in RANKL gene expression, while clodronate led to a concentration-dependent inhibition of this effect (1.8-fold increase at 5.0 µM). OPG gene expression was decreased by compressive loading both in the presence of 0.5 µM clodronate and in the absence of clodronate, and OPG protein synthesis in the compressed cells was significantly decreased in the presence of clodronate. Immunocytochemical staining revealed an increase of RANKL protein synthesis in compressed cells, while clodronate and cell compression reduced this increase. CONCLUSION: This study demonstrates that clodronate decreases the compression-induced RANKL/OPG ratio expressed by human osteoblasts. Reported in vivo findings of reduced osteoclast numbers on the compression side of orthodontic tooth movement under the action of clodronate-and the associated slow rate of tooth movement-might be attributable not only to a direct impact on osteoclasts but also to changes in osteoblast-osteoclast interaction resulting from the presence of clodronate.

The effect of post-mastectomy radiation therapy on breast implants: Unveiling biomaterial alterations with potential implications on capsular contracture.

Post-mastectomy breast reconstruction with expanders and implants is recognized as an integral part of breast cancer treatment. Its main complication is represented by capsular contracture, which leads to poor expansion, breast deformation, and pain, often requiring additional surgery. In such a scenario, the debate continues as to whether the second stage of breast reconstruction should be performed before or after post-mastectomy radiation therapy, in light of potential alterations induced by irradiation to silicone biomaterial. This work provides a novel, multi-technique approach to unveil the role of radiotherapy in biomaterial alterations, with potential involvement in capsular contracture. Following irradiation, implant shells underwent mechanical, chemical, and microstructural evaluation by means of tensile testing, Attenuated Total Reflectance Fourier Transform InfraRed spectroscopy (ATR/FTIR), Scanning Electron Microscopy (SEM), high resolution stylus profilometry, and Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS). Our findings are consistent with radiation-induced modifications of silicone that, although not detectable at the microscale, can be evidenced by more sophisticated nanoscale surface analyses. In light of these results, biomaterial irradiation cannot be ruled out as one of the possible co-factors underlying capsular contracture.

Time-dependent changes in dynamic mechanical properties of irradiated bone.

The increased use of allograft tissue for musculoskeletal repair has brought more focus to the safety of allogenic tissue and the efficacy of various sterilization techniques. The currently available literature contains few examples of studies on long-lasting strains of bones but no example for irradiated bones. In this study the bovine femurs from a 2-year-old animal were machine cut and irradiated with the doses of 10, 15, 25, 35, 45 and 50 kGy. The dynamic mechanical analysis was performed at 1 Hz at the room temperature in a 3-point bending configuration for 2880 minutes. The final values of E' and E″ were dose independent but they were reached at different periods. For this reason, so called "critical point" was introduced for the further analysis. All the examined sample groups were characterized by statistically significant lower values of the critical point in comparison with the control samples (p<0.05) but the biggest differences were observed between the control samples and the samples irradiated with the doses of 10, 15 and 25 kGy. Current results and literature review suggest that the dose of 35 kGy is the optimal dose for ionizing radiation sterilization.

Selective flow-induced vesicle rupture to sort by membrane mechanical properties.

Vesicle and cell rupture caused by large viscous stresses in ultrasonication is central to biomedical and bioprocessing applications. The flow-induced opening of lipid membranes can be exploited to deliver drugs into cells, or to recover products from cells, provided that it can be obtained in a controlled fashion. Here we demonstrate that differences in lipid membrane and vesicle properties can enable selective flow-induced vesicle break-up. We obtained vesicle populations with different membrane properties by using different lipids (SOPC, DOPC, or POPC) and lipid:cholesterol mixtures (SOPC:chol and DOPC:chol). We subjected vesicles to large deformations in the acoustic microstreaming flow generated by ultrasound-driven microbubbles. By simultaneously deforming vesicles with different properties in the same flow, we determined the conditions in which rupture is selective with respect to the membrane stretching elasticity. We also investigated the effect of vesicle radius and excess area on the threshold for rupture, and identified conditions for robust selectivity based solely on the mechanical properties of the membrane. Our work should enable new sorting mechanisms based on the difference in membrane composition and mechanical properties between different vesicles, capsules, or cells.

Effects study on the thermal stresses in a LEU metal foil annular target.

The effects of fission gas pressure, uranium swelling and thermal contact conductance on the thermal-mechanical behavior of an annular target containing a low-enriched uranium foil (LEU) encapsulated in a nickel foil have been presented in this paper. The draw-plug assembly method is simulated to obtain the residual stresses, which are applied to the irradiation model as initial inputs, and the integrated assembly-irradiation process is simulated as an axisymmetric problem using the commercial finite element code Abaqus FEA. Parametric studies were performed on the LEU heat generation rate and the results indicate satisfactory irradiation performance of the annular target. The temperature and stress margins have been provided along with a discussion of the results.

Meniscal allograft sterilisation: effect on biomechanical and histological properties.

Sterilisation of allografts are a crucial step in ensuring safety and viability. Current sterilisation standards such as 25 kGy gamma irradiation (γ) can have adverse effects on the ultrastructure and biomechanical properties of allograft tissue. Supercritical CO2 (SCCO2) technology, represents an improved sterilisation process that potentially preserves tissue properties. This study aimed to test the effect of SCCO2 sterilisation on the biomechanical and histological properties of the meniscus and compare this to the current standard of γ. Thirty-two 18-month old ovine menisci were randomly assigned into three groups for sterilisation (SCCO2, γ and control). After treatment, biomechanical indentation testing (stiffness and stress relaxation) or histological analysis [percentage of void, cells and extracellular matrix (ECM) per slide] was undertaken. Both SCCO2 and gamma groups displayed an increase in stiffness and stress relaxation as compared to control, however, this difference was lesser in samples treated with SCCO2. No significant histological quantitative differences were detected between SCCO2 and control specimens. Gamma-treated samples demonstrated a significant increase in void and decrease in ECM. Interestingly, both treatment groups demonstrated a decreasing mean void and increasing ECM percentage when analysed from outer to inner zones. No significant differences were detected in all-endpoints when analysed by section. SCCO2 sterilisation represents a potential feasible alternative to existing sterilization techniques such as γ.

Cell encapsulation in a magnetically aligned collagen-GAG copolymer microenvironment.

Engineered tissue microenvironments impart specialized cues that drive distinct cellular phenotypes and function. Microenvironments with defined properties, such as mechanical properties and fibril alignment, can elicit specific cellular responses that emulate those observed in vivo. Collagen- and glycosaminoglycan (GAG)-based tissue matrices have been popularized due to their biological ubiquity in a broad range of tissues and the ability to tune structure and mechanical properties through a variety of processes. Here, we investigate the combined effects of static magnetic fields, and GAG and cell encapsulation, on the structure (e.g. collagen fibril orientation) and material properties of collagen matrices. We found that magnetic fields align the collagen-GAG matrix, alter equilibrium mechanical properties and provide a method for encapsulating cells within a three-dimensional aligned matrix. Cells are encapsulated prior to polymerization, allowing for controlled cell density and eliminating the need for cell seeding. Increased relative GAG concentrations reduced the ability to magnetically align collagen fibrils, in part through a mechanism involving increased viscosity and polymerization time of the collagen-GAG solution. This work provides a functional design space for the development of pure collagen and hybrid collagen-GAG matrices in the presence of magnetic fields. Additionally, this work shows that magnetic fields are effective for the fabrication of collagen constructs with controlled fibril orientation, and can be coupled with GAG incorporation to modulate mechanical properties and the response of embedded cells.

Microstructure, mechanical properties and in vitro bioactivity of akermanite scaffolds fabricated by laser sintering.

Akermanite had attracted great attention due to the favourable mechanical properties and excellent biological performance. In this research, the microstructure and mechanical properties of akermanite scaffolds fabricated via laser sintering under different process conditions were studied and characterized. The results showed that the akermanite particles gradually mixed together, grew up and reached complete densification with the scanning speed decreasing from 450 to 150 mm/min, while micro defects such as air holes occurred at 50mm/min. simultaneously, the compressive strength of the scaffolds went up and then descended, and the optimum value was 5.92 ± 0.41 MPa. The Vickers hardness and fracture toughness increased consistently and then tended to stabilize. X-ray diffraction (XRD) results indicated no new phase appeared under all process conditions. MG-63 cell culture revealed that cell adhesion and proliferation occurred, indicating excellent cytocompatibility of the scaffolds. Moreover, in vitro bioactivity tests showed that the apatite layer formed on the scaffolds and became dense and thick with the increase of soaking time in simulated body fluid (SBF), and this fact was further confirmed by energy-dispersive spectroscopy (EDS).

Effect of ultrasonication on physical properties of mineral trioxide aggregate.

AIM: To evaluate the effect on physical properties of Mineral Trioxide Aggregate (MTA) of using direct hand compaction during placement and when using hand compaction with indirect ultrasonic activation with different application times. METHODS: One hundred acrylic canals were obturated in 3 increments with MTA in sample sizes of 10. One group was obturated by hand with an endodontic plugger and the remainder obturated with indirect ultrasonic application, with times ranging from 2 seconds to 18 seconds per increment. Microhardness values, dye penetration depths, and radiographs of the samples were evaluated. RESULTS: As ultrasonic application time per increment increased, microhardness values fell significantly (P < 0.001) while dye penetration values increased (P < 0.001). Microhardness of MTA ultrasonicated for 2 seconds was significantly higher than hand compaction (P = 0.03). Most radiographic voids were visible in the hand-compacted group (P < 0.001), which also had higher dye penetration depths than the 2-second ultrasonicated samples. Ultrasonication of MTA for 10-18 seconds resulted in significantly more voids than 2-8 seconds of ultrasonication (P = 0.02). CONCLUSION: The use of ultrasonics with MTA improved the compaction and flow of MTA, but excessive ultrasonication adversely affected MTA properties. A time of 2 seconds of ultrasonication per increment presented the best compromise between microhardness values, dye penetration depths, and lack of radiographic voids.

KrF excimer laser precision machining of hard and brittle ceramic biomaterials.

KrF excimer laser precision machining of porous hard-brittle ceramic biomaterials was studied to find a suitable way of machining the materials into various desired shapes and sizes without distorting their intrinsic structure and porosity. Calcium phosphate glass ceramics (CPGs) and hydroxyapatite (HA) were chosen for the study. It was found that KrF excimer laser can cut both CPGs and HA with high efficiency and precision. The ablation rates of CPGs and HA are respectively 0.081 µm/(pulse J cm(-2)) and 0.048 µm/(pulse J cm(-2)), while their threshold fluences are individually 0.72 and 1.5 J cm(-2). The cutting quality (smoothness of the cut surface) is a function of laser repetition rate and cutting speed. The higher the repetition rate and lower the cutting speed, the better the cutting quality. A comparison between the cross sections of CPGs and HA cut using the excimer laser and using a conventional diamond cutting blade indicates that those cut by the excimer laser could retain their intrinsic porosity and geometry without distortion. In contrast, those cut by conventional machining had distorted geometry and most of their surface porosities were lost. Therefore, when cutting hard-brittle ceramic biomaterials to prepare scaffold and implant or when sectioning them for porosity evaluation, it is better to choose KrF excimer laser machining.

PHBV/PAM scaffolds with local oriented structure through UV polymerization for tissue engineering.

Locally oriented tissue engineering scaffolds can provoke cellular orientation and direct cell spread and migration, offering an exciting potential way for the regeneration of the complex tissue. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds with locally oriented hydrophilic polyacrylamide (PAM) inside the macropores of the scaffolds were achieved through UV graft polymerization. The interpenetrating PAM chains enabled good interconnectivity of PHBV/PAM scaffolds that presented a lower porosity and minor diameter of pores than PHBV scaffolds. The pores with diameter below 100 µm increased to 82.15% of PHBV/PAM scaffolds compared with 31.5% of PHBV scaffolds. PHBV/PAM scaffold showed a much higher compressive elastic modulus than PHBV scaffold due to PAM stuffing. At 5 days of culturing, sheep chondrocytes spread along the similar direction in the macropores of PHBV/PAM scaffolds. The locally oriented PAM chains might guide the attachment and spreading of chondrocytes and direct the formation of microfilaments via contact guidance.

Cartilage tissue engineering application of injectable gelatin hydrogel with in situ visible-light-activated gelation capability in both air and aqueous solution.

Chondroprogenitor cells encapsulated in a chondrogenically supportive, three-dimensional hydrogel scaffold represents a promising, regenerative approach to articular cartilage repair. In this study, we have developed an injectable, biodegradable methacrylated gelatin (mGL)-based hydrogel capable of rapid gelation via visible light (VL)-activated crosslinking in air or aqueous solution. The mild photocrosslinking conditions permitted the incorporation of cells during the gelation process. Encapsulated human-bone-marrow-derived mesenchymal stem cells (hBMSCs) showed high, long-term viability (up to 90 days) throughout the scaffold. To assess the applicability of the mGL hydrogel for cartilage tissue engineering, we have evaluated the efficacy of chondrogenesis of the encapsulated hBMSCs, using hBMSCs seeded in agarose as control. The ability of hBMSC-laden mGL constructs to integrate with host tissues after implantation was further investigated utilizing an in vitro cartilage repair model. The results showed that the mGL hydrogel, which could be photopolymerized in air and aqueous solution, supports hBMSC growth and TGF-ß3-induced chondrogenesis. Compared with agarose, mGL constructs laden with hBMSCs are mechanically stronger with time, and integrate well with native cartilage tissue upon implantation based on push-out mechanical testing. VL-photocrosslinked mGL scaffold thus represents a promising scaffold for cell-based repair and resurfacing of articular cartilage defects.

Multi-parametric monitoring and assessment of high-intensity focused ultrasound (HIFU) boiling by harmonic motion imaging for focused ultrasound (HMIFU): an ex vivo feasibility study.

Harmonic motion imaging for focused ultrasound (HMIFU) is a recently developed high-intensity focused ultrasound (HIFU) treatment monitoring method with feasibilities demonstrated in vitro and in vivo. Here, a multi-parametric study is performed to investigate both elastic and acoustics-independent viscoelastic tissue changes using the Harmonic Motion Imaging (HMI) displacement, axial compressive strain and change in relative phase shift during high energy HIFU treatment with tissue boiling. Forty three (n = 43) thermal lesions were formed in ex vivo canine liver specimens (n = 28). Two-dimensional (2D) transverse HMI displacement maps were also obtained before and after lesion formation. The same method was repeated in 10 s, 20 s and 30 s HIFU durations at three different acoustic powers of 8, 10, and 11 W, which were selected and verified as treatment parameters capable of inducing boiling using both thermocouple and passive cavitation detection (PCD) measurements. Although a steady decrease in the displacement, compressive strain, and relative change in the focal phase shift (Δϕ) were obtained in numerous cases, indicating an overall increase in relative stiffness, the study outcomes also showed that during boiling, a reverse lesion-to-background displacement contrast was detected, indicating potential change in tissue absorption, geometrical change and/or, mechanical gelatification or pulverization. Following treatment, corresponding 2D HMI displacement images of the thermal lesions also mapped consistent discrepancy in the lesion-to-background displacement contrast. Despite the expectedly chaotic changes in acoustic properties with boiling, the relative change in phase shift showed a consistent decrease, indicating its robustness to monitor biomechanical properties independent of the acoustic property changes throughout the HIFU treatment. In addition, the 2D HMI displacement images confirmed and indicated the increase in the thermal lesion size with treatment duration, which was validated against pathology. In conclusion, multi-parametric HMIFU was shown capable of monitoring and mapping tissue viscoelastic response changes during and after HIFU boiling, some of which were independent of the acoustic parameter changes.

Bone embrittlement and collagen modifications due to high-dose gamma-irradiation sterilization.

Bone allografts are often used in orthopedic reconstruction of skeletal defects resulting from trauma, bone cancer or revision of joint arthroplasty. γ-Irradiation sterilization is a widely-used biological safety measure; however it is known to embrittle bone. Irradiation has been shown to affect the post-yield properties, which are attributed to the collagen component of bone. In order to find a solution to the loss of toughness in irradiated bone allografts, it is important to fully understand the effects of irradiation on bone collagen. The objective of this study was to evaluate changes in the structure and integrity of bone collagen as a result of γ-irradiation, with the hypothesis that irradiation fragments collagen molecules leading to a loss of collagen network connectivity and therefore loss of toughness. Using cortical bone from bovine tibiae, sample beams irradiated at 33kGy on dry ice were compared to native bone beams (paired controls). All beams were subjected to three-point bend testing to failure followed by characterization of the decalcified bone collagen, using differential scanning calorimetry (DSC), hydrothermal isometric tension testing (HIT), high performance liquid chromatography (HPLC) and gel electrophoresis (SDS-PAGE). The carbonyl content of demineralized bone collagen was also measured chemically to assess oxidative damage. Barium sulfate staining after single edge notch bending (SEN(B)) fracture testing was also performed on bovine tibia bone beams with a machined and sharpened notch to evaluate the fracture toughness and ability of irradiated bone to form micro-damage during fracture. Irradiation resulted in a 62% loss of work-to-fracture (p≤0.001). There was significantly less micro-damage formed during fracture propagation in the irradiated bone. HPLC showed no significant effect on pentosidine, pyridinoline, or hydroxypyridinoline levels suggesting that the loss of toughness is not due to changes in these stable crosslinks. For DSC, there was a 20% decrease in thermal stability (p<0.001) with a 100% increase (p<0.001) in enthalpy of denaturation (melting). HIT testing also showed a decrease in thermal stability (20% lower denaturation temperature, p<0.001) and greatly reduced measures of collagen network connectivity (p<0.001). Interestingly, the increase in enthalpy of denaturation suggests that irradiated collagen requires more energy to denature (melt), perhaps a result of alterations in the hydrogen bonding sites (increased carbonyl content detected in the insoluble collagen) on the irradiated bone collagen. Altogether, this new data strongly indicates that a large loss of overall collagen connectivity due to collagen fragmentation resulting from γ-irradiation sterilization leads to inferior cortical bone toughness. In addition, notable changes in the thermal denaturation of the bone collagen along with chemical indicators of oxidative modification of the bone collagen indicate that the embrittlement may be a function not only of collagen fragmentation but also of changes in bonding.

Hierarchical modelling of in situ elastic deformation of human enamel based on photoelastic and diffraction analysis of stresses and strains.

Human enamel is a typical hierarchical mineralized tissue with a two-level composite structure. To date, few studies have focused on how the mechanical behaviour of this tissue is affected by both the rod orientation at the microscale and the preferred orientation of mineral crystallites at the nanoscale. In this study, wide-angle X-ray scattering was used to determine the internal lattice strain response of human enamel samples (with differing rod directions) as a function of in situ uniaxial compressive loading. Quantitative stress distribution evaluation in the birefringent mounting epoxy was performed in parallel using photoelastic techniques. The resulting experimental data was analysed using an advanced multiscale Eshelby inclusion model that takes into account the two-level hierarchical structure of human enamel, and reflects the differing rod directions and orientation distributions of hydroxyapatite crystals. The achieved satisfactory agreement between the model and the experimental data, in terms of the values of multidirectional strain components under the action of differently orientated loads, suggests that the multiscale approach captures reasonably successfully the structure-property relationship between the hierarchical architecture of human enamel and its response to the applied forces. This novel and systematic approach can be used to improve the interpretation of the mechanical properties of enamel, as well as of the textured hierarchical biomaterials in general.