Combinatorial use of bone morphogenetic protein 6, noggin and SOST significantly predicts cancer progression.

Emerging evidence has indicated a role of the bone morphogenetic proteins (BMP) in the pathogenesis of certain cancers. The signaling of BMP family members is tightly regulated by their antagonists, including noggin and SOST, which are, in turn, positively regulated by BMP, thereby forming a negative feedback loop. Consequently, the expression of these antagonists should be taken into account in studies on the prognostic significance of BMP. In the present paper, we correlated protein and mRNA expression levels of BMP6, noggin and SOST, alone or in combination, with patient survival in various types of cancer. We found that BMP6 alone was not significantly correlated with esophageal squamous cell carcinoma patient survival. Instead, a high level of inhibitor of differentiation 1, a downstream factor of BMP6, was associated with shorter survival in patients whose tumors stained strongly for BMP6. Knockdown of noggin in esophageal cancer cell line EC109, which expresses BMP6 strongly and SOST weakly, enhanced the non-adherent growth of the cells. Noggin and SOST expression levels, when analyzed alone, were not significantly correlated with patient survival. However, high BMP6 activity, defined by strong BMP6 expression coupled with weak noggin or SOST expression, was significantly associated with shorter survival in esophageal squamous cell carcinoma patients. We further confirmed that BMP6 activity could be used as a prognostic indicator in prostate, bladder and colorectal cancers, using publicly available data on BMP6, noggin and SOST mRNA expression and patient survival. Our results strongly suggest that BMP6, noggin and SOST could be used in combination as a prognostic indicator in cancer progression.

Development and characterisation of a collagen nano-hydroxyapatite composite scaffold for bone tissue engineering.

Bone regeneration requires scaffolds that possess suitable mechanical and biological properties. This study sought to develop a novel collagen-nHA biocomposite scaffold via two new methods. Firstly a stable nHA suspension was produced and added to a collagen slurry (suspension method), and secondly, porous collagen scaffolds were immersed in nHA suspension after freeze-drying (immersion method). Significantly stronger constructs were produced using both methods compared to collagen only scaffolds, with a high porosity maintained (>98.9%). It was found that Coll-nHA composite scaffolds produced by the suspension method were up to 18 times stiffer than the collagen control (5.50 +/- 1.70 kPa vs. 0.30 +/- 0.09 kPa). The suspension method was also more reproducible, and the quantity of nHA incorporated could be varied with greater ease than with the immersion technique. In addition, Coll-nHA composites display excellent biological activity, demonstrating their potential as bone graft substitutes in orthopaedic regenerative medicine.

Orthopaedic tissue engineering and bone regeneration.

Orthopaedic tissue engineering combines the application of scaffold materials, cells and the release of growth factors. It has been described as the science of persuading the body to reconstitute or repair tissues that have failed to regenerate or heal spontaneously. In the case of bone regeneration 3-D scaffolds are used as a framework to guide tissue regeneration. Mesenchymal cells obtained from the patient via biopsy are grown on biomaterials in vitro and then implanted at a desired site in the patient's body. Medical implants that encourage natural tissue regeneration are generally considered more desirable than metallic implants that may need to be removed by subsequent intervention. Numerous polymeric materials, from natural and artificial sources, are under investigation as substitutes for skeletal elements such as cartilage and bone. For bone regeneration, cells (obtained mainly from bone marrow aspirate or as primary cell outgrowths from bone biopsies) can be combined with biodegradable polymeric materials and/or ceramics and absorbed growth factors so that osteoinduction is facilitated together with osteoconduction; through the creation of bioactive rather than bioinert scaffold constructs. Relatively rapid biodegradation enables advantageous filling with natural tissue while loss of polymer strength before mass is disadvantageous. Innovative solutions are required to address this and other issues such as the biocompatibility of material surfaces and the use of appropriate scaffold topography and porosity to influence bone cell gene expression.

Content-Dependent Osteogenic Response of Nanohydroxyapatite: An in Vitro and in Vivo Assessment within Collagen-Based Scaffolds.

The use of collagen-based scaffolds in orthopedic applications has been limited due to poor mechanical properties, but this may be overcome by the introduction of a stiffer supporting phase. Thus, we developed a synthesis technique to produce nonaggregating, stable nanohydroxyapatite (nHA) particles, permitting the fabrication of biomimetic-inspired scaffolds through the combination of nanosized HA with collagen, as found in native bone. This study evaluates the mechanical and biological impact of incorporating increasing concentrations of these nanoparticles into porous collagen scaffolds (1:1 and 5:1 weight ratios of nHA/collagen). Mechanical assessment demonstrated that increasing nHA incorporation correlated with increasing Young's moduli, which could be further amplified using cross-linking treatments. Typically, the porosity of a scaffold is sacrificed to produce a stiffer material; however, through the use of nanosized particles the inclusion of up to 5:1 nHA/collagen content still preserved the high 99% porosity of the composite scaffold, allowing for maximum cell infiltration. Moreover, increasing nHA presence induced significant bioactive responses, achieving superior cellular attachment and enhanced osteogenesis, promoting earlier expression of bone markers and cell-mediated mineralization versus nHA-free collagen controls. Interestingly, these content-dependent results observed in vitro did not directly translate in vivo. Instead, similar levels of bone formation were achieved within critical-sized rat calvarial defects, independent of nHA content, following acellular implantation. The addition of nHA, both 1:1 and 5:1, induced significantly higher levels of mineralization and de novo bone ingrowth versus collagen controls as demonstrated by microcomputed tomography, histological, and histomorphometric analyses. Ultimately, these results demonstrate the immense osteoinductivity of nonaggregated nanoparticles of HA incorporated into collagen-composite scaffolds and emphasize the importance of in vivo-based evaluation of therapies intended for clinical use.

Rapid new bone tissue remodeling during distraction osteogenesis is associated with apoptosis.

During the process of distraction osteogenesis new bone forms and undergoes rapid remodeling. Apoptosis may be one of the regulatory mechanisms governing the removal of the redundant callus during distraction osteogenesis. A rabbit tibial lengthening model was used and lengthened at 0.7 mm/day for 3 weeks. The regenerating tissues from the distraction gap were examined for apoptotic changes by transmission electron microscopy (TEM) and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Osteoclastic bone resorption activities were demonstrated by tartrate resistant acid phosphatase (TRAP) staining. The apoptotic cells were mainly present in the transitional regions between the fibrous tissue and the new bone in the mineralization front, and close to or on the new bone surfaces near the center of the regenerate. The TUNEL labeling was greatly reduced in the mature bone near the osteotomied bone ends. TEM examination confirmed the presence of cells with apoptotic changes at various regions of the regenerate. TRAP staining revealed that osteoclastic bone resorption activities in the regenerate were in a similar pattern of distribution to those of the TUNEL labeling. The localization of apoptotic cells at the different regions of the regenerate, accompanied by the osteoclast activities, suggest that apoptosis is closely related to bone formation and remodeling during distraction osteogenesis.

A reliable externally fixated murine femoral fracture model that accounts for variation in movement between animals.

Fifty-two CFLP mice had an open femoral diaphyseal osteotomy held in compression by a four-pin external fixator. The movement of 34 of the mice in their cages was quantified before and after operation, until sacrifice at 4, 8, 16 or 24 days. Thirty-three specimens underwent histomorphometric analysis and 19 specimens underwent torsional stiffness measurement. The expected combination of intramembranous and endochondral bone formation was observed, and the model was shown to be reliable in that variation in the histological parameters of healing was small between animals at the same time point, compared to the variation between time-points. There was surprisingly large individual variation in the amount of animal movement about the cage, which correlated with both histomorphometric and mechanical measures of healing. Animals that moved more had larger external calluses containing more cartilage and demonstrated lower torsional stiffness at the same time point. Assuming that movement of the whole animal predicts, at least to some extent, movement at the fracture site, this correlation is what would be expected in a model that involves similar processes to those in human fracture healing. Models such as this, employed to determine the effect of experimental interventions, will yield more information if the natural variation in animal motion is measured and included in the analysis.

The limitation of DEXA analysis for bone mass determination in mice.

An increase in femoral and tibio/fibular bone mass following periosteal membrane stimulation by Moloney sarcoma virus inoculation into thigh muscles of mice was measured in situ on formalin fixed excised hind limbs using a Hologic 4500A Fan Beam X-ray bone densitometer adapted for small bone samples. These results were verified by measurements of constant dry bone mass of the same bones liberated from soft limb tissues by NaOH hydrolysis. There was no consistent data correlation found between the DEXA scan and dry bone mass evaluations. It is concluded that the sensitivity of the DEXA measurement is unsuitable when assessing very small bone samples, weighing merely 20-30 mg.

A biomimetic multi-layered collagen-based scaffold for osteochondral repair.

Cartilage and osteochondral defects pose a significant challenge in orthopedics. Tissue engineering has shown promise as a potential method for the treatment of such defects; however, a long-lasting repair strategy has yet to be realized. This study focuses on the development of a layered construct for osteochondral repair, fabricated through a novel "iterative layering" freeze-drying technique. The process involved repeated steps of layer addition followed by freeze-drying, enabling control over material composition, pore size and substrate stiffness in each region of the construct, while also achieving a seamlessly integrated layer structure. The novel construct developed mimics the inherent gradient structure of healthy osteochondral tissue: a bone layer composed of type I collagen and hydroxyapatite (HA), an intermediate layer composed of type I collagen, type II collagen and HA and a cartilaginous region composed of type I collagen, type II collagen and hyaluronic acid. The material properties were designed to provide the biological cues required to encourage infiltration of host cells from the bone marrow while the biomechanical properties were designed to provide an environment optimized to promote differentiation of these cells towards the required lineage in each region. This novel osteochondral graft was shown to have a seamlessly integrated layer structure, high levels of porosity (>97%), a homogeneous pore structure and a high degree of pore interconnectivity. Moreover, homogeneous cellular distribution throughout the entire construct was evident following in vitro culture, demonstrating the potential of this multi-layered scaffold as an advanced strategy for osteochondral defect repair.

Gold nanoparticle cellular uptake, toxicity and radiosensitisation in hypoxic conditions.

BACKGROUND AND PURPOSE: Gold nanoparticles (GNPs) are novel agents that have been shown to cause radiosensitisation in vitro and in vivo. Tumour hypoxia is associated with radiation resistance and reduced survival in cancer patients. The interaction of GNPs with cells in hypoxia is explored. MATERIALS AND METHODS: GNP uptake, localization, toxicity and radiosensitisation were assessed in vitro under oxic and hypoxic conditions. RESULTS: GNP cellular uptake was significantly lower under hypoxic than oxic conditions. A significant reduction in cell proliferation in hypoxic MDA-MB-231 breast cancer cells exposed to GNPs was observed. In these cells significant radiosensitisation occurred in normoxia and moderate hypoxia. However, in near anoxia no significant sensitisation occurred. CONCLUSIONS: GNP uptake occurred in hypoxic conditions, causing radiosensitisation in moderate, but not extreme hypoxia in a breast cancer cell line. These findings may be important for the development of GNPs for cancer therapy.

Innovative collagen nano-hydroxyapatite scaffolds offer a highly efficient non-viral gene delivery platform for stem cell-mediated bone formation.

The ability of nano-hydroxyapatite (nHA) particles developed in-house to act as non-viral delivery vectors is assessed. These nHA particles are combined with collagen to yield bioactive, biodegradable collagen nano-hydroxyapatite (coll-nHA) scaffolds. Their ability to act as gene-activated matrices for BMP2 delivery is demonstrated with successful transfection of mesenchymal stem cells (MSCs) resulting in high calcium production.

The potential of electron beam radiation for simultaneous surface modification and bioresorption control of PLLA.

Bioresorbable polymers have been widely investigated as materials exhibiting significant potential for successful application in the fields of tissue engineering and drug delivery. Further to the ability to control degradation, surface engineering of polymers has been highlighted as a key method central to their development. Previous work has demonstrated the ability of electron beam (e-beam) technology to control the degradation profiles and bioresorption of a number of commercially relevant bioresorbable polymers (poly-l-lactic acid (PLLA), L-lactide/DL-lactide co-polymer (PLDL) and poly(lactic-co-glycolic acid (PLGA)). This work investigates the further potential of e-beam technology to impart added biofunctionality through the manipulation of polymer (PLLA) surface properties. PLLA samples were subjected to e-beam treatments in air, with varying beam energies and doses. Surface characterization was then performed using contact angle analysis, X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and atomic force microscopy. Results demonstrated a significant increase in surface wettability post e-beam treatment. In correlation with this, XPS data showed the introduction of oxygen-containing functional groups to the surface of PLLA. Raman spectroscopy indicated chain scission in the near surface region of PLLA (as predicted). However, e-beam effects on surface properties were not shown to be dependent on beam energy or dose. E-beam irradiation did not seem to affect the surface roughness of PLLA as a direct consequence of the treatment.

Enzymatic mineralization of hydrogels for bone tissue engineering by incorporation of alkaline phosphatase.

Alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, is incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These are collagen type I, a mussel-protein-inspired adhesive consisting of PEG substituted with catechol groups, cPEG, and the PEG/fumaric acid copolymer OPF. After incubation in Ca-GP solution, FTIR, EDS, SEM, XRD, SAED, ICP-OES, and von Kossa staining confirm CaP formation. The amount of mineral formed decreases in the order cPEG > collagen > OPF. The mineral:polymer ratio decreases in the order collagen > cPEG > OPF. Mineralization increases Young's modulus, most profoundly for cPEG. Such enzymatically mineralized hydrogel/CaP composites may find application as bone regeneration materials.

Cell type-dependent uptake, localization, and cytotoxicity of 1.9 nm gold nanoparticles.

BACKGROUND: This follow-up study aims to determine the physical parameters which govern the differential radiosensitization capacity of two tumor cell lines and one immortalized normal cell line to 1.9 nm gold nanoparticles. In addition to comparing the uptake potential, localization, and cytotoxicity of 1.9 nm gold nanoparticles, the current study also draws on comparisons between nanoparticle size and total nanoparticle uptake based on previously published data. METHODS: We quantified gold nanoparticle uptake using atomic emission spectroscopy and imaged intracellular localization by transmission electron microscopy. Cell growth delay and clonogenic assays were used to determine cytotoxicity and radiosensitization potential, respectively. Mechanistic data were obtained by Western blot, flow cytometry, and assays for reactive oxygen species. RESULTS: Gold nanoparticle uptake was preferentially observed in tumor cells, resulting in an increased expression of cleaved caspase proteins and an accumulation of cells in sub G(1) phase. Despite this, gold nanoparticle cytotoxicity remained low, with immortalized normal cells exhibiting an LD(50) concentration approximately 14 times higher than tumor cells. The surviving fraction for gold nanoparticle-treated cells at 3 Gy compared with that of untreated control cells indicated a strong dependence on cell type in respect to radiosensitization potential. CONCLUSION: Gold nanoparticles were most avidly endocytosed and localized within cytoplasmic vesicles during the first 6 hours of exposure. The lack of significant cytotoxicity in the absence of radiation, and the generation of gold nanoparticle-induced reactive oxygen species provide a potential mechanism for previously reported radiosensitization at megavoltage energies.

Through-thickness control of polymer bioresorption via electron beam irradiation.

Predicable and controlled degradation is not only central to the accurate delivery of bioactive agents and drugs, it also plays a vital role in key aspects of bone tissue engineering. The work addressed in this paper investigates the utilisation of e-beam irradiation in order to achieve a controlled (surface) degradation profile. This study focuses on the modification of commercially and clinically relevant materials, namely poly(L-lactic acid) (PLLA), poly(L-lactide-hydroxyapatite) (PLLA-HA), poly(L-lactide-glycolide) co-polymer (PLG) and poly(L-lactide-DL-lactide) co-polymer (PLDL). Samples were subjected to irradiation treatments using a 0.5MeV electron beam with delivered surface doses of 150 and 500 kGy. In addition, an acrylic attenuation shield was used for selected samples to control the penetration of the e-beam. E-beam irradiation induced chain scission in all polymers, as characterized by reduced molecular weights and glass transition temperatures (T(g)). Irradiation not only produced changes in the physical properties of the polymers but also had associated effects on surface erosion of the materials during hydrolytic degradation. Moreover, the extent to which both mechanical and hydrolytic degradation was observed is synonymous with the estimated penetration of the beam (as controlled by the employment of an attenuation shield).

Nanodosimetric effects of gold nanoparticles in megavoltage radiation therapy.

BACKGROUND AND PURPOSE: The addition of gold nanoparticles (GNPs) to tumours leads to an increase in dose due to their high density and energy absorption coefficient, making it a potential radiosensitiser. However, experiments have observed radiosensitisations significantly larger than the increase in dose alone, including at megavoltage energies where gold's relative energy absorption is lowest. This work investigates whether GNPs create dose inhomogeneities on a sub-cellular scale which combine with non-linear dose dependence of cell survival to be the source of radiosensitisation at megavoltage energies. MATERIALS AND METHODS: Monte Carlo simulations were carried out to calculate dose in the vicinity of a single GNP on the nanoscale. The effect of this nanoscale dose distribution was then modelled for MDA-MB-231 cells exposed to 2 nm GNPs, and compared to experimental results. RESULTS: Dramatic dose inhomogeneities occur around GNPs exposed to megavoltage radiation. When analysed using the Local Effect Model, these inhomogeneities lead to significant radiosensitisation, in agreement with experimental results. CONCLUSIONS: This work suggests that GNP radiosensitisation is driven by inhomogeneities in dose on the nanoscale, rather than changes in dose over the entire cell, which may contribute to the similar radiosensitisation observed in megavoltage and kilovoltage experiments. The short range of these inhomogeneities and the variation in enhancement in different cells suggests sub-cellular localisation is important in determining GNP radiosensitisation.

Cell-specific radiosensitization by gold nanoparticles at megavoltage radiation energies.

PURPOSE: Gold nanoparticles (GNPs) have been shown to cause sensitization with kilovoltage (kV) radiation. Differences in the absorption coefficient between gold and soft tissue, as a function of photon energy, predict that maximum enhancement should occur in the kilovoltage (kV) range, with almost no enhancement at megavoltage (MV) energies. Recent studies have shown that GNPs are not biologically inert, causing oxidative stress and even cell death, suggesting a possible biological mechanism for sensitization. The purpose of this study was to assess GNP radiosensitization at clinically relevant MV X-ray energies. METHODS AND MATERIALS: Cellular uptake, intracellular localization, and cytotoxicity of GNPs were assessed in normal L132, prostate cancer DU145, and breast cancer MDA-MB-231 cells. Radiosensitization was measured by clonogenic survival at kV and MV photon energies and MV electron energies. Intracellular DNA double-strand break (DSB) induction and DNA repair were determined and GNP chemosensitization was assessed using the radiomimetic agent bleomycin. RESULTS: GNP uptake occurred in all cell lines and was greatest in MDA-MB-231 cells with nanoparticles accumulating in cytoplasmic lysosomes. In MDA-MB-231 cells, radiation sensitizer enhancement ratios (SERs) of 1.41, 1.29, and 1.16 were achieved using 160 kVp, 6 MV, and 15 MV X-ray energies, respectively. No significant effect was observed in L132 or DU145 cells at kV or MV energies (SER 0.97-1.08). GNP exposure did not increase radiation-induced DSB formation or inhibit DNA repair; however, GNP chemosensitization was observed in MDA-MB-231 cells treated with bleomycin (SER 1.38). CONCLUSIONS: We have demonstrated radiosensitization in MDA-MB-231 cells at MV X-ray energies. The sensitization was cell-specific with comparable effects at kV and MV energies, no increase in DSB formation, and GNP chemopotentiation with bleomycin, suggesting a possible biological mechanism of radiosensitization.

Biological consequences of nanoscale energy deposition near irradiated heavy atom nanoparticles.

Gold nanoparticles (GNPs) are being proposed as contrast agents to enhance X-ray imaging and radiotherapy, seeking to take advantage of the increased X-ray absorption of gold compared to soft tissue. However, there is a great discrepancy between physically predicted increases in X-ray energy deposition and experimentally observed increases in cell killing. In this work, we present the first calculations which take into account the structure of energy deposition in the nanoscale vicinity of GNPs and relate this to biological outcomes, and show for the first time good agreement with experimentally observed cell killing by the combination of X-rays and GNPs. These results are not only relevant to radiotherapy, but also have implications for applications of heavy atom nanoparticles in biological settings or where human exposure is possible because the localised energy deposition high-lighted by these results may cause complex DNA damage, leading to mutation and carcinogenesis.

The synthesis and characterization of nanophase hydroxyapatite using a novel dispersant-aided precipitation method.

The synthesis of nanophase hydroxyapatite (nHA) is of importance in the field of biomaterials and bone tissue engineering. The bioactive and osteoconductive properties of nHA are of much benefit to a wide range of biomedical applications such as producing bone tissue engineered constructs, coating medical implants, or as a carrier for plasmid DNA in gene delivery. This study aimed to develop a novel low-temperature dispersant-aided precipitation reaction to produce nHA particles (<100 nm), which are regarded as being preferable to micron-sized agglomerates of nHA. The variables investigated and optimized include the reaction pH, the rate of reactant mixing, use of sonication, order of addition, and concentration of the primary reactants, in addition, the effect of using poly(vinyl alcohol) (PVA) surfactant and Darvan 821A® dispersing agent during the reaction was also examined. It was found that by fine-tuning the synthesis parameters and incorporating the dispersing agent, monodisperse, phase-pure nano-sized particles under 100 nm were attained, suitable for clinical applications in bone regeneration.

Fabrication and repair of cartilage defects with a novel acellular cartilage matrix scaffold.

The objectives of this study were to develop a three-dimensional acellular cartilage matrix (ACM) and investigate its possibility for use as a scaffold in cartilage tissue engineering. Bovine articular cartilage was decellularized sequentially with trypsin, nuclease solution, hypotonic buffer, and Triton x 100 solution; molded with freeze-drying process; and cross-linked by ultraviolet irradiation. Histological and biochemical analysis showed that the ACM was devoid of cells and still maintained the collagen and glycosaminoglycan components of cartilage. Scanning electronic microscopy and mercury intrusion porosimetry showed that the ACM had a sponge-like structure of high porosity. The ACM scaffold had good biocompatibility with cultured rabbit bone marrow mesenchymal stem cells with no indication of cytotoxicity both in contact and in extraction assays. The cartilage defects repair in rabbit knees with the mesenchymal stem cell-ACM constructs had a significant improvement of histological scores when compared to the control groups at 6 and 12 weeks. In summary, the ACM possessed the characteristics that afford it as a potential scaffold for cartilage tissue engineering.

Surface modification of poly(epsilon-caprolactone) using a dielectric barrier discharge in atmospheric pressure glow discharge mode.

The role of roughening and functionalization processes involved in modifying the wettability of poly(epsilon-caprolactone) (PCL) after treatment by an atmospheric pressure glow discharge plasma is discussed. The change in the ratio of CO/C-O bonds is a significant factor influencing the wettability of PCL. As the contact angle decreases, the level of CO bonds tends to rise. Surface roughness alterations are the driving force for lasting increases in wettability, while the surface functional species are shorter lived. We can approximate from ageing that the increase in wettability for PCL after plasma treatment is 55-60% due to roughening and 40-45% due to surface functionalization for the plasma device investigated.