Resolving the Near-Infrared Spectrum of Articular Cartilage.

OBJECTIVE: Spectroscopic techniques, such as near-infrared (NIR) spectroscopy, are gaining significant research interest for characterizing connective tissues, particularly articular cartilage, because there is still a largely unmet need for rapid, accurate and objective methods for assessing tissue integrity in real-time during arthroscopic surgery. This study aims to identify the NIR spectral range that is optimal for characterizing cartilage integrity by (a) identifying the contribution of its major constituents (collagen and proteoglycans) to its overall spectrum using proxy constituent models and (b) determining constituent-specific spectral contributions that can be used for assessment of cartilage in its physiological state. DESIGN: The NIR spectra of cartilage matrix constituent models were measured and compared with specific molecular components of organic compounds in the NIR spectral range in order to identify their bands and molecular assignments. To verify the identified bands, spectra of the model compounds were compared with those of native cartilage. Since water obscures some bands in the NIR range, spectral measurements of the native cartilage were conducted under conditions of decreasing water content to amplify features of the solid matrix components. The identified spectral bands were then compared and examined in the resulting spectra of the intact cartilage samples. RESULTS: As water was progressively eliminated from cartilage, the specific contribution of the different matrix components was observed to correspond with those identified from the proxy cartilage component models. CONCLUSION: Spectral peaks in the regions 5500 to 6250 cm-1 and 8100 to 8600 cm-1 were identified to be effective for characterizing cartilage proteoglycan and collagen contents, respectively.

Potential enhancement of articular cartilage histological grading with collagen integrity.

BACKGROUND: Histological evaluation of articular cartilage, such as using the Mankin scoring system, is the gold standard for characterization of tissue integrity. This scoring system takes into account several parameters indicative of the tissue's health; however, the collagen integrity, which is a primary indicator of cartilage health is not taken into consideration. Thus, there is need to enhance histological grading of articular cartilage by incorporating explicit scoring of collagen degeneration into the Modified Mankin grading system. This paper explores a new histological grading parameter for collagen network degradation and how this information can be used to augment a widely used grading scheme like the Modified Mankin grading system. METHODS: Intact and degenerated human cartilage were examined histologically and then subjected to second harmonic generation imaging, leading to qualitative and quantitative description of collagen disruption emanating from the surface to subsurface layers of the tissue. This data was then incorporated into the Modified Mankin grading system. FINDINGS: Second harmonic generation image analysis reveals a relationship between changes in collagen architecture and histologically observed tissue disruption in degenerated articular cartilage. INTERPRETATION: Histological tissue disruption in degenerated human articular cartilage is directly related to the reorganization of collagen fibrils in the form of intense fibril aggregation, either as a result of degeneration or aging. This method of mapping disrupted tissue regions to quantitative collagen fibril damage can be coded into cartilage grading systems and could inform clinical practice and scientific research.

A new mechanical indentation framework for functional assessment of articular cartilage.

The conventional mechanical properties of articular cartilage, such as compressive stiffness, have been shown to have limited capacity to distinguish visually normal from degraded cartilage samples. In this study, a new mechanical indentation framework for assessing functional properties of articular cartilage during loading/unloading, i.e. deformation and recovery, was established. The capacity of a ring-shaped indenter integrated with an ultrasound transducer to distinguish mechanically intact from proteoglycan-depleted tissue was investigated. To achieve this, normal and enzymatically degraded bovine osteochondral samples were subjected to loading/unloading while the response of the tissue at the middle was captured by ultrasound at the same time. The enzymatic degradation model was characterized by amount of proteoglycan content, glycosaminoglycan release and proteomic analysis. The mechanical response of a wider continuum of articular cartilage in the loaded area and its surrounding region was captured in this framework leading to investigate two parameters, L and TS, related to the surrounding tissue of the loaded area for functional assessment of cartilage. L is the distance between the ultrasound transducer and articular cartilage surface and TS is the transient strain of articular cartilage during loading and unloading. Classification Analysis based on Principal Component Analysis was used to investigate the capacity of the new parameters to assess the functionality of the tissue. Multivariate statistics based on Partial Least Squares regression was employed to identify the correlation between the response of the tissue in the indented area and its surrounding cartilage. The results of this study indicate that L during loading (deformation) can differentiate normal and mildly proteoglycan-depleted samples from severely depleted samples and L during unloading (recovery) can distinguish between normal and proteoglycan-depleted tissue. However, TS during deformation and recovery is unable to discriminate normal cartilage samples from proteoglycan-depleted tissue. The results also demonstrate a strong correlation between mechanical properties of the loaded area with the response of its surrounding cartilage during recovery. It is therefore concluded that L in this newly established framework can discriminate between normal and proteoglycan-depleted cartilage samples. However, more samples will be needed to verify the demarcation between samples degraded for varying amount of time.

Monitoring osteoarthritis progression using near infrared (NIR) spectroscopy.

We demonstrate in this study the potential of near infrared (NIR) spectroscopy as a tool for monitoring progression of cartilage degeneration in an animal model. Osteoarthritic degeneration was artificially induced in one joint in laboratory rats, and the animals were sacrificed at four time points: 1, 2, 4, and 6 weeks (3 animals/week). NIR spectra were acquired from both (injured and intact) knees. Subsequently, the joint samples were subjected to histological evaluation and glycosaminoglycan (GAG) content analysis, to assess disease severity based on the Mankin scoring system and to determine proteoglycan loss, respectively. Multivariate spectral techniques were then employed for classification (principal component analysis and support vector machines) and prediction (partial least squares regression) of the samples' Mankin scores and GAG content from their NIR spectra. Our results demonstrate that NIR spectroscopy is sensitive to degenerative changes in articular cartilage, and is capable of distinguishing between mild (weeks 1&2; Mankin <=2) and advanced (weeks 4&6; Mankin =>3) cartilage degeneration. In addition, the spectral data contains information that enables estimation of the tissue's Mankin score (error = 12.6%, R2 = 86.2%) and GAG content (error = 7.6%, R2 = 95%). We conclude that NIR spectroscopy is a viable tool for assessing cartilage degeneration post-injury, such as, post-traumatic osteoarthritis.

Characterization of Articular Cartilage Recovery and Its Correlation with Optical Response in the Near-Infrared Spectral Range.

OBJECTIVES: In this study, we examine the capacity of a new parameter, based on the recovery response of articular cartilage, to distinguish between healthy and damaged tissues. We also investigate whether or not this new parameter correlates with the near-infrared (NIR) optical response of articular cartilage. DESIGN: Normal and artificially degenerated (proteoglycan-depleted) bovine cartilage samples were nondestructively probed using NIR spectroscopy. Subsequently they were subjected to a load and unloading protocol, and the recovery response was logged during unloading. The recovery parameter, elastic rebound ( ER), is based on the strain energy released as the samples underwent instantaneous elastic recovery. RESULTS: Our results reveal positive relationship between the rebound parameter and cartilage proteoglycan content (normal samples: 2.20 ± 0.10 N mm; proteoglycan-depleted samples: 0.50 ± 0.04 N mm for 1 hour of enzymatic treatment and 0.13 ± 0.02 N mm for 4 hours of enzymatic treatment). In addition, multivariate analysis using partial least squares regression was employed to investigate the relationship between ER and NIR spectral data. The results reveal significantly high correlation ( R2cal = 98.35% and R2val = 79.87%; P < 0.0001), with relatively low error (14%), between the recovery and optical response of cartilage in the combined NIR regions 5,450 to 6,100 cm-1 and 7,500 to 12,500 cm-1. CONCLUSION: We conclude that ER can indicate the mechanical condition and state of health of articular cartilage. The correlation of ER with cartilage optical response in the NIR range could facilitate real-time evaluation of the tissue's integrity during arthroscopic surgery and could also provide an important tool for cartilage assessment in tissue engineering and regeneration research.

Bactericidal Effects of Natural Nanotopography of Dragonfly Wing on Escherichia coli.

Nanotextured surfaces (NTSs) are critical to organisms as self-adaptation and survival tools. These NTSs have been actively mimicked in the process of developing bactericidal surfaces for diverse biomedical and hygiene applications. To design and fabricate bactericidal topographies effectively for various applications, understanding the bactericidal mechanism of NTS in nature is essential. The current mechanistic explanations on natural bactericidal activity of nanopillars have not utilized recent advances in microscopy to study the natural interaction. This research reveals the natural bactericidal interaction between E. coli and a dragonfly wing's (Orthetrum villosovittatum) NTS using advanced microscopy techniques and proposes a model. Contrary to the existing mechanistic models, this experimental approach demonstrated that the NTS of Orthetrum villosovittatum dragonfly wings has two prominent nanopillar populations and the resolved interface shows membrane damage occurred without direct contact of the bacterial cell membrane with the nanopillars. We propose that the bacterial membrane damage is initiated by a combination of strong adhesion between nanopillars and bacterium EPS layer as well as shear force when immobilized bacterium attempts to move on the NTS. These findings could help guide the design of novel biomimetic nanomaterials by maximizing the synergies between biochemical and mechanical bactericidal effects.

Investigation of the Effects of Extracellular Osmotic Pressure on Morphology and Mechanical Properties of Individual Chondrocyte.

It has been demonstrated that most cells of the body respond to osmotic pressure in a systematic manner. The disruption of the collagen network in the early stages of osteoarthritis causes an increase in water content of cartilage which leads to a reduction of pericellular osmolality in chondrocytes distributed within the extracellular environment. It is therefore arguable that an insight into the mechanical properties of chondrocytes under varying osmotic pressure would provide a better understanding of chondrocyte mechanotransduction and potentially contribute to knowledge on cartilage degeneration. In this present study, the chondrocyte cells were exposed to solutions with different osmolality. Changes in their dimensions and mechanical properties were measured over time. Atomic force microscopy (AFM) was used to apply load at various strain-rates and the force-time curves were logged. The thin-layer elastic model was used to extract the elastic stiffness of chondrocytes at different strain-rates and at different solution osmolality. In addition, the porohyperelastic (PHE) model was used to investigate the strain-rate-dependent responses under the loading and osmotic pressure conditions. The results revealed that the hypo-osmotic external environment increased chondrocyte dimensions and reduced Young's modulus of the cells at all strain-rates tested. In contrast, the hyper-osmotic external environment reduced dimensions and increased Young's modulus. Moreover, using the PHE model coupled with inverse FEA simulation, we established that the hydraulic permeability of chondrocytes increased with decreasing extracellular osmolality which is consistent with previous work in the literature. This could be due to a higher intracellular fluid volume fraction with lower osmolality.

Tribological efficacy and stability of phospholipid-based membrane lubricants in varying pH chemical conditions.

In this study, the authors examine the influence of joint chemical environment by measuring changes in the tribological properties (friction coefficient and charge density) of contacting surfaces of normal and degenerated cartilage samples in bath solutions of varying pH (2.0-9.0). Bovine articular cartilage samples (n = 54) were subjected to several surface measurements, including interfacial energy, contact angle, and friction coefficient, at varying pH. The samples were delipidized and then subjected to the same measurement protocols. Our results reveal that the interfacial energy and charge density, which have been shown to be related to friction coefficient, decrease with pH in the acidic range and approach constant values at physiological (or synovial fluid) pH of 7.4 and beyond it, i.e., toward basic pH domain. The authors conclude that this rather complex response explains the long-term efficacy with respect to ageing and associated pH changes, of the phospholipid layers that facilitate the almost frictionless, hydration-lubrication involving contact in the mammalian musculoskeletal system.

The relationship between interfragmentary movement and cell differentiation in early fracture healing under locking plate fixation.

Interfragmentary movement (IFM) at the fracture site plays an important role in fracture healing, particularly during its early stage, via influencing the mechanical microenvironment of mesenchymal stem cells within the fracture callus. However, the effect of changes in IFM resulting from the changes in the configuration of locking plate fixation on cell differentiation has not yet been fully understood. In this study, mechanical experiments on surrogate tibia specimens, manufactured from specially formulated polyurethane, were conducted to investigate changes in IFM of fractures under various locking plate fixation configurations and loading magnitudes. The effect of the observed IFM on callus cell differentiation was then further studied using computational simulation. We found that during the early stage, cell differentiation in the fracture callus is highly influenced by fracture gap size and IFM, which in turn, is highly sensitive to locking plate fixation configuration. The computational model predicted that a small gap size (e.g. 1 mm) under a relatively flexible configuration of locking plate fixation (larger bone-plate distances and working lengths) could experience excessive strain and fluid flow within the fracture site, resulting in excessive fibrous tissue differentiation and delayed healing. By contrast, a relatively flexible configuration of locking plate fixation was predicted to improve cartilaginous callus formation and bone healing for a relatively larger gap size (e.g. 3 mm). If further confirmed by animal and human studies, the research outcome of this paper may have implications for orthopaedic surgeons in optimising the application of locking plate fixations for fractures in clinical practice.

A poroviscohyperelastic model for numerical analysis of mechanical behavior of single chondrocyte.

The aim of this paper is to use a poroviscohyperelastic (PVHE) model, which is developed based on the porohyperelastic (PHE) model to explore the mechanical deformation properties of single chondrocytes. Both creep and relaxation responses are investigated by using finite element analysis models of micropipette aspiration and atomic force microscopy experiments, respectively. The newly developed PVHE model is compared thoroughly with the standard neo-Hookean solid and PHE models. It has been found that the PVHE can accurately capture both creep and stress relaxation behaviors of chondrocytes better than other two models. Hence, the PVHE is a promising model to investigate mechanical properties of single chondrocytes.

Investigation of the mechanical behavior of kangaroo humeral head cartilage tissue by a porohyperelastic model based on the strain-rate-dependent permeability.

Solid-interstitial fluid interaction, which depends on tissue permeability, is significant to the strain-rate-dependent mechanical behavior of humeral head (shoulder) cartilage. Due to anatomical and biomechanical similarities to that of the human shoulder, kangaroos present a suitable animal model. Therefore, indentation experiments were conducted on kangaroo shoulder cartilage tissues from low (10(-4)/s) to moderately high (10(-2)/s) strain-rates. A porohyperelastic model was developed based on the experimental characterization; and a permeability function that takes into account the effect of strain-rate on permeability (strain-rate-dependent permeability) was introduced into the model to investigate the effect of rate-dependent fluid flow on tissue response. The prediction of the model with the strain-rate-dependent permeability was compared with those of the models using constant permeability and strain-dependent permeability. Compared to the model with constant permeability, the models with strain-dependent and strain-rate-dependent permeability were able to better capture the experimental variation at all strain-rates (p < 0.05). Significant differences were not identified between models with strain-dependent and strain-rate-dependent permeability at strain-rate of 5 × 10(-3)/s (p = 0.179). However, at strain-rate of 10(-2)/s, the model with strain-rate-dependent permeability was significantly better at capturing the experimental results (p < 0.005). The findings thus revealed the significance of rate-dependent fluid flow on tissue behavior at large strain-rates, which provides insights into the mechanical deformation mechanisms of cartilage tissues.

Optical absorption spectra of human articular cartilage correlate with biomechanical properties, histological score and biochemical composition.

This study investigates the relationship between the optical response of human articular cartilage in the visible (VIS) and near infrared (NIR) spectral range and its matrix properties.Full-thickness osteochondral cores (dia. = 16 mm, n = 50) were extracted from human cadaver knees (N = 13) at four anatomical locations and divided into quadrants. Absorption spectra were acquired in the spectral range 400-1100 nm from one quadrant. Reference biomechanical, biochemical composition, histological, and cartilage thickness measurements were obtained from two other quadrants. A multivariate statistical technique based on partial least squares (PLS) regression was then employed to investigate the correlation between the absorption spectra and tissue properties.Our results demonstrate that cartilage optical response correlates with its function, composition and morphology, as indicated by the significant relationship between spectral predicted and measured biomechanical (79.0% ⩽ R(2) ⩽ 80.3%, p < 0.0001), biochemical (65.1% ⩽ R(2) ⩽ 81.0%, p < 0.0001), and histological scores ([Formula: see text] = 83.3%, p < 0.0001) properties. Significant correlation was also obtained with the non-calcified cartilage thickness ([Formula: see text] = 83.2%, p < 0.0001).We conclude that optical absorption of human cartilage in the VIS and NIR spectral range correlates with the overall tissue properties, thus providing knowledge that could facilitate development of systems for rapid assessment of tissue integrity.

Microscale consolidation analysis of relaxation behavior of single living chondrocytes subjected to varying strain-rates.

Besides the elastic stiffness, the relaxation behavior of single living cells is also of interest of various researchers when studying cell mechanics. It is hypothesized that the relaxation response of the cells is governed by both intrinsic viscoelasticity of the solid phase and fluid-solid interactions mechanisms. There are a number of mechanical models have been developed to investigate the relaxation behavior of single cells. However, there is lack of model enable to accurately capture both of the mechanisms. Therefore, in this study, the porohyperelastic (PHE) model, which is an extension of the consolidation theory, combined with inverse Finite Element Analysis (FEA) technique was used at the first time to investigate the relaxation response of living chondrocytes. This model was also utilized to study the dependence of relaxation behavior of the cells on strain-rates. The stress-relaxation experiments under the various strain-rates were conducted with the Atomic Force Microscopy (AFM). The results have demonstrated that the PHE model could effectively capture the stress-relaxation behavior of the living chondrocytes, especially at intermediate to high strain-rates. Although this model gave some errors at lower strain-rates, its performance was acceptable. Therefore, the PHE model is properly a promising model for single cell mechanics studies. Moreover, it has been found that the hydraulic permeability of living chondrocytes reduced with decreasing of strain-rates. It might be due to the intracellular fluid volume fraction and the fluid pore pressure gradients of chondrocytes were higher when higher strain-rates applied.

Spatial mapping of proteoglycan content in articular cartilage using near-infrared (NIR) spectroscopy.

Diagnosis of articular cartilage pathology in the early disease stages using current clinical diagnostic imaging modalities is challenging, particularly because there is often no visible change in the tissue surface and matrix content, such as proteoglycans (PG). In this study, we propose the use of near infrared (NIR) spectroscopy to spatially map PG content in articular cartilage. The relationship between NIR spectra and reference data (PG content) obtained from histology of normal and artificially induced PG-depleted cartilage samples was investigated using principal component (PC) and partial least squares (PLS) regression analyses. Significant correlation was obtained between both data (R(2) = 91.40%, p<0.0001). The resulting correlation was used to predict PG content from spectra acquired from whole joint sample, this was then employed to spatially map this component of cartilage across the intact sample. We conclude that NIR spectroscopy is a feasible tool for evaluating cartilage contents and mapping their distribution across mammalian joint.

Near infrared spectroscopy for rapid determination of Mankin score components: a potential tool for quantitative characterization of articular cartilage at surgery.

PURPOSE: The purpose of this study was to demonstrate the potential of near infrared (NIR) spectroscopy for characterizing the health and degenerative state of articular cartilage based on the components of the Mankin score. METHODS: Three models of osteoarthritic degeneration induced in laboratory rats by anterior cruciate ligament (ACL) transection, meniscectomy (MSX), and intra-articular injection of monoiodoacetate (1 mg) (MIA) were used in this study. Degeneration was induced in the right knee joint; each model group consisted of 12 rats (N = 36). After 8 weeks, the animals were euthanized and knee joints were collected. A custom-made diffuse reflectance NIR probe of 5-mm diameter was placed on the tibial and femoral surfaces, and spectral data were acquired from each specimen in the wave number range of 4,000 to 12,500 cm(-1). After spectral data acquisition, the specimens were fixed and safranin O staining (SOS) was performed to assess disease severity based on the Mankin scoring system. Using multivariate statistical analysis, with spectral preprocessing and wavelength selection technique, the spectral data were then correlated to the structural integrity (SI), cellularity (CEL), and matrix staining (SOS) components of the Mankin score for all the samples tested. RESULTS: ACL models showed mild cartilage degeneration, MSX models had moderate degeneration, and MIA models showed severe cartilage degenerative changes both morphologically and histologically. Our results reveal significant linear correlations between the NIR absorption spectra and SI (R(2) = 94.78%), CEL (R(2) = 88.03%), and SOS (R(2) = 96.39%) parameters of all samples in the models. In addition, clustering of the samples according to their level of degeneration, with respect to the Mankin components, was also observed. CONCLUSIONS: NIR spectroscopic probing of articular cartilage can potentially provide critical information about the health of articular cartilage matrix in early and advanced stages of osteoarthritis (OA). CLINICAL RELEVANCE: This rapid nondestructive method can facilitate clinical appraisal of articular cartilage integrity during arthroscopic surgery.

Simultaneous magnetic resonance imaging and consolidation measurement of articular cartilage.

Magnetic resonance imaging (MRI) offers the opportunity to study biological tissues and processes in a non-disruptive manner. The technique shows promise for the study of the load-bearing performance (consolidation) of articular cartilage and changes in articular cartilage accompanying osteoarthritis. Consolidation of articular cartilage involves the recording of two transient characteristics: the change over time of strain and the hydrostatic excess pore pressure (HEPP). MRI study of cartilage consolidation under mechanical load is limited by difficulties in measuring the HEPP in the presence of the strong magnetic fields associated with the MRI technique. Here we describe the use of MRI to image and characterize bovine articular cartilage deforming under load in an MRI compatible consolidometer while monitoring pressure with a Fabry-Perot interferometer-based fiber-optic pressure transducer.

Molecular dynamics simulation of mechanical behavior of osteopontin-hydroxyapatite interfaces.

Bone is characterized with an optimized combination of high stiffness and toughness. The understanding of bone nanomechanics is critical to the development of new artificial biological materials with unique properties. In this work, the mechanical characteristics of the interfaces between osteopontin (OPN, a noncollagenous protein in extrafibrillar protein matrix) and hydroxyapatite (HA, a mineral nanoplatelet in mineralized collagen fibrils) were investigated using molecular dynamics method. We found that the interfacial mechanical behavior is governed by the electrostatic attraction between acidic amino acid residues in OPN and calcium in HA. Higher energy dissipation is associated with the OPN peptides with a higher number of acidic amino acid residues. When loading in the interface direction, new bonds between some acidic residues and HA surface are formed, resulting in a stick-slip type motion of OPN peptide on the HA surface and high interfacial energy dissipation. The formation of new bonds during loading is considered to be a key mechanism responsible for high fracture resistance observed in bone and other biological materials.

The effect of high voltage, high frequency pulsed electric field on slain ovine cortical bone.

High power, high frequency pulsed electric fields known as pulsed power (PP) has been applied recently in biology and medicine. However, little attention has been paid to investigate the application of pulse power in musculoskeletal system and its possible effect on functional behavior and biomechanical properties of bone tissue. This paper presents the first research investigating whether or not PP can be applied safely on bone tissue as a stimuli and what will be the possible effect of these signals on the characteristics of cortical bone by comparing the mechanical properties of this type of bone pre and post expose to PP and in comparison with the control samples. A positive buck-boost converter was applied to generate adjustable high voltage, high frequency pulses (up to 500 V and 10 kHz). The functional behavior of bone in response to pulse power excitation was elucidated by applying compressive loading until failure. The stiffness, failure stress (strength) and the total fracture energy (bone toughness) were determined as a measure of the main bone characteristics. Furthermore, an ultrasonic technique was applied to determine and comprise bone elasticity before and after pulse power stimulation. The elastic property of cortical bone samples appeared to remain unchanged following exposure to pulse power excitation for all three orthogonal directions obtained from ultrasonic technique and similarly from the compression test. Nevertheless, the compressive strength and toughness of bone samples were increased when they were exposed to 66 h of high power pulsed electromagnetic field compared to the control samples. As the toughness and the strength of the cortical bone tissue are directly associated with the quality and integrity of the collagen matrix whereas its stiffness is primarily related to bone mineral content these overall results may address that although, the pulse power stimulation can influence the arrangement or the quality of the collagen network causing the bone strength and toughness augmentation, it apparently did not affect the mineral phase of the cortical bone material. The results also confirmed that the indirect application of high power pulsed electric field at 500 V and 10 kHz through capacitive coupling method was safe and did not destroy the bone tissue construction.

Sterilizing tissue-materials using pulsed power plasma.

This paper investigates the potential of pulsed power to sterilize hard and soft tissues and its impact on their physico-mechanical properties. It hypothesizes that pulsed plasma can sterilize both vascular and avascular tissues and the transitive layers in between without deleterious effects on their functional characteristics. Cartilage/bone laminate was chosen as a model to demonstrate the concept, treated at low temperature, at atmospheric pressure, in short durations and in buffered environment using a purposed-built pulsed power unit. Input voltage and time of exposure were assigned as controlling parameters in a full factorial design of experiment to determine physical and mechanical alteration pre- and post-treatment. The results demonstrated that, discharges of 11 kV sterilized samples in 45 s, reducing intrinsic elastic modules from 1.4 ± 0.9 to 0.9 ± 0.6 MPa. There was a decrease of 14.1 % in stiffness and 27.8 % in elastic-strain energy for the top quartile. Mechanical impairment was directly proportional to input voltage (P value < 0.05). Bacterial inactivation was proportional to treatment time for input voltages above 32 V (P < 0.001; R Sq = 0.98). Thermal analysis revealed that helix-coil transition decelerated with exposure time and collagen fibrils were destabilized as denaturation enthalpy reduced by 200 µV. We concluded by presenting a safe operating threshold for pulsed power plasma as a feasible protocol for effective sterilization of connective tissues with varying level of loss in mechanical robustness which we argue to be acceptable in certain medical and tissue engineering application.

Pro-osteogenic topographical cues promote early activation of osteoprogenitor differentiation via enhanced TGFß, Wnt, and Notch signaling.

OBJECTIVES: Titanium implant surfaces with modified topographies have improved osteogenic properties in vivo. However, the molecular mechanisms remain obscure. This study explored the signaling pathways responsible for the pro-osteogenic properties of micro-roughened (SLA) and chemically/nanostructurally (modSLA) modified titanium surfaces on human alveolar bone-derived osteoprogenitor cells (BCs) in vitro. MATERIALS AND METHODS: The activation of stem cell signaling pathways (TGFß/BMP, Wnt, FGF, Hedgehog, Notch) was investigated following early exposure (24 and 72 h) of BCs to SLA and modSLA surfaces in the absence of osteogenic cell culture supplements. RESULTS: Key regulatory genes from the TGFß/BMP (TGFBR2, BMPR2, BMPR1B, ACVR1B, SMAD1, SMAD5), Wnt (Wnt/ß-catenin and Wnt/Ca(2+) ) (FZD1, FZD3, FZD5, LRP5, NFATC1, NFATC2, NFATC4, PYGO2, LEF1) and Notch (NOTCH1, NOTCH2, NOTCH4, PSEN1, PSEN2, PSENEN) pathways were upregulated on the modified surfaces. These findings correlated with a higher expression of osteogenic markers bone sialoprotein (IBSP) and osteocalcin (BGLAP), and bone differentiation factors BMP2, BMP6, and GDF15, as observed on the modified surfaces. CONCLUSIONS: These findings demonstrate that the activation of the pro-osteogenic cell signaling pathways by modSLA and SLA surfaces leads to enhanced osteogenic differentiation as evidenced after 7 and 14 days culture in osteogenic media and provides a mechanistic insight into the superior osseointegration on the modified surfaces observed in vivo.