Controlling whole blood activation and resultant clot properties on various material surfaces : a possible therapeutic approach for enhancing bone healing

Injured bone initiates the healing process by forming a blood clot at the damaged site. However, in severe damage, synthetic bone implants are used to provide structural integrity and restore the healing process. The implant unavoidably comes into direct contact with whole blood, leading to a blood clot formation on its surface. Despite this, most research in bone tissue engineering virtually ignores the important role of a blood clot in supporting healing. Surface chemistry of a biomaterial is a crucial property in mediating blood-biomaterials interactions, and hence the formation of the resultant blood clot. Surfaces presenting mixtures of functional groups carboxyl (–COOH) and methyl (–CH3) have been shown to enhance platelet response and coagulation activation, leading to the formation of fibrin fibres. In addition, it has been shown that varying the compositions of these functional groups and the length of alkyl groups further modulate the immune complement response. In this study, we hypothesised that a biomaterial surface with mixture of –COOH/–CH3(methyl), –CH2CH3 (ethyl) or –(CH2)3CH3 (butyl) groups at different ratios would modulate blood coagulation and complement activation, and eventually tailor the structural and functional properties of the blood clot formed on the surface, which subsequently impacts new bone formation. Firstly, we synthesised a series of materials composed of acrylic acid (AA), and methyl (MMA), ethyl (EMA) or butyl methacrylates (BMA) at different ratios and coated on the inner surfaces of incubation vials. Our surface analysis showed that the amount of –COOH groups on the surface coatings was lower than the ratios of AA prepared in the materials even though the surface content of –COOH groups increased with increasing in AA ratios. It was indicated that the surface hydrophobicity increased with increasing alkyl chain length: –CH 3 > –CH2CH3 > –(CH2)3CH3, and decreased with increasing –COOH groups. No significant differences in surface hydrophobicity was found on surfaces with –CH3 and –CH2CH3 groups in the presence of –COOH groups. The material coating was as smooth as uncoated glass and without any major flaws. The average roughness of material-coated surface (3.99 ± 0.54 nm) was slightly higher than that of uncoated glass surface (2.22 ± 0.29 nm). However, no significant differences in surface average roughness was found among surfaces with the same functionalities at different –COOH ratios nor among surfaces with different alkyl groups but the same –COOH ratios. These suggested that the surface functional groups and their compositions had a combined effect on modulating surface hydrophobicity but not surface roughness. The second part of our study was to investigate the effect of surface functional groups and their compositions on blood cascade activation and structural properties of the formed clots. It was found that surfaces with –COOH/–(CH2)3CH3 induced a faster coagulation activation than those with –COOH/–CH3 and –CH2CH3, regardless of the –COOH ratios. An increase in –COOH ratios on –COOH/–CH3 and –CH2CH3 surfaces decreased the rate of activation. Moreover, all material-coated surfaces markedly reduced the complement activation compared to uncoated glass surfaces, and the pattern of complement activation was entirely similar to that of surface-induced coagulation, suggesting there is an interaction between two cascades. The clots formed on material-coated surfaces had thicker fibrin with a tighter network at the exterior when compared to uncoated glass surfaces. Compared to the clot exteriors, thicker fibrins with a loose network were found in clot interiors. Coated surfaces resulted in more rigid clots with a significantly slower fibrinolysis after 1 h of lysis when compared to uncoated glass surfaces. Significant differences in fibrinolysis after 1 h of lysis among clots on material-coated surfaces correlated well with the differences in fibrin thickness and density at clot exterior. In addition, more growth factors were released during clot formation than during clot lysis. From an intact clot, there was a correlation between the amount of PDGF-AB release and fibrin density. Highest amount of PDGF-AB was released from clots formed on surfaces with 40% –COOH/60% –CH 3 (i.e. 65MMA). During clot lysis, the release of PDGF-AB also correlated with the fibrinolytic rate while the release of TGF-â1 was influenced by the fibrin thickness. This suggested that different clot structures led to different release profiles of growth factors in clot intact and degrading stages. We further validated whether the clots formed on material-coatings provide the microenvironment for improved bone healing by using a rabbit femoral defect model. In this pilot study, the implantation of clots formed on 65MMA coatings significantly increased new bone formation with enhanced chondrogenesis, osteoblasts activity and vascularisation, but decreased inflammatory macrophage number at the defects after 4 weeks when compared to commercial bone grafts ChronOSTM â-TCP granules. Empty defects were observed when blood clot formation was inhibited. In summary, our study demonstrated that surface functional groups and their relative ratios on material coatings synergistically modulate activation of blood cascades, resultant fibrin architecture, rigidity, susceptibility to fibrinolysis as well as growth factor release of the formed clots, which ultimately alter the healing microenvironment of injured bones.

Orthopaedics and trauma Queensland annual report 2012

Orthopaedics and Trauma Queensland, the Centre for Research and Education in Musculoskeletal Disorders, is an internationally recognised research group that continues to develop its reputation as an international leader in research and education. It provides a stimulus for research, education and clinical application within the international orthopaedic and trauma communities. Orthopaedics and Trauma Queensland develops and promotes the innovative use of engineering and technology, in collaboration with surgeons, to provide new techniques, materials, procedures and medical devices. Its integration with clinical practice and strong links with hospitals ensure that the research will be translated into practical outcomes for patients. The group undertakes clinical practice in orthopaedics and trauma and applies core engineering skills to challenges in medicine. The research is built on a strong foundation of knowledge in biomedical engineering, and incorporates expertise in cell biology, mathematical modelling, human anatomy and physiology and clinical medicine in orthopaedics and trauma. New knowledge is being developed and applied to the full range of orthopaedic diseases and injuries, such as knee and hip replacements, fractures and spinal deformities.

Osteogenic differentiation of bone marrow MSCs by β-tricalcium phosphate stimulating macrophages via BMP2 signalling pathway

Immune reactions play important roles in determining the in vivo fate of bone substitute materials, either in new bone formation or inflammatory fibrous tissue encapsulation. The paradigm for the development of bone substitute materials has been shifted from inert to immunomodulatory materials, emphasizing the importance of immune cells in the material evaluation. Macrophages, the major effector cells in the immune reaction to implants, are indispensable for osteogenesis and their heterogeneity and plasticity render macrophages a primer target for immune system modulation. However, there are very few reports about the effects of macrophages on biomaterial-regulated osteogenesis. In this study, we used b-tricalcium phosphate (b-TCP) as a model biomaterial to investigate the role of macrophages on the material stimulated osteogenesis. The macrophage phenotype switched to M2 extreme in response to b-TCP extracts, which was related to the activation of calcium-sensing receptor (CaSR) pathway. Bone morphogenetic protein 2 (BMP2) was also significantly upregulated by the b-TCP stimulation, indicating that macrophage may participate in the b-TCP stimulated osteogenesis. Interestingly, when macrophageconditioned b-TCP extracts were applied to bone marrow mesenchymal stem cells (BMSCs), the osteogenic differentiation of BMSCs was significantly enhanced, indicating the important role of macrophages in biomaterial-induced osteogenesis. These findings provided valuable insights into the mechanism of material-stimulated osteogenesis, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of bone substitute materials.

Mussel-inspired bioceramics with self-assembled Ca-P/polydopamine composite nanolayer : preparation, formation mechanism, improved cellular bioactivity and osteogenic differentiation of bone marrow stromal cells

The nanostructured surface of biomaterials plays an important role in improving their in vitro cellular bioactivity as well as stimulating in vivo tissue regeneration. Inspired by the mussel’s adhesive versatility, which is thought to be due to the plaque–substrate interface being rich in 3,4-dihydroxy-L-phenylalamine (DOPA) and lysine amino acids, in this study we developed a self-assembly method to prepare a uniform calcium phosphate (Ca-P)/polydopamine composite nanolayer on the surface of b-tricalcium phosphate (b-TCP) bioceramics by soaking b-TCP bioceramics in Tris–dopamine solution. It was found that the addition of dopamine, reaction temperature and reaction time are three key factors inducing the formation of a uniform Ca-P/polydopamine composite nanolayer. The formation mechanism of a Ca-P/polydopamine composite nanolayer involved two important steps: (i) the addition of dopamine to Tris–HCl solution decreases the pH value and accelerates Ca and P ionic dissolution from the crystal boundaries of b-TCP ceramics; (ii) dopamine is polymerized to form self-assembled polydopamine film and, at the same time, nanosized Ca-P particles are mineralized with the assistance of polydopamine, in which the formation of polydopamine occurs simultaneously with Ca-P mineralization (formation of nanosized microparticles composed of calcium phosphate-based materials), and finally a self-assembled Ca-P/polydopamine composite nanolayer forms on the surface of the b-TCP ceramics. Furthermore, the formed self-assembled Ca-P/polydopamine composite nanolayer significantly enhances the surface roughness and hydrophilicity of b-TCP ceramics, and stimulates the attachment, proliferation, alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, COL1 and Runx2) of human bone marrow stromal cells. Our results suggest that the preparation of self-assembled Ca-P/polydopamine composite nanolayers is a viable method to modify the surface of biomaterials by significantly improving their surface physicochemical properties and cellular bioactivity for bone regeneration application.

A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin, hyaluronic acid and chondroitin sulfate

The development of hydrogels tailored for cartilage tissue engineering has been a research and clinical goal for over a decade. Directing cells towards a chondrogenic phenotype and promoting new matrix formation are significant challenges that must be overcome for the successful application of hydrogels in cartilage tissue therapies. Gelatin-methacrylamide (Gel-MA) hydrogels have shown promise for the repair of some tissues, but they have not been extensively investigated for cartilage tissue engineering. We encapsulated human chondrocytes in gel-MA based hydrogels, and show that with the incorporation of small quantities of photo-crosslinkable hyaluronic acid methacrylate (HA-MA), and to a lesser extent chondroitin sulfate methacrylate (CS-MA), chondrogenesis and mechanical properties can be enhanced. The addition of HA-MA to Gel-MA constructs resulted in more rounded cell morphologies, enhanced chondrogenesis as assessed by gene expression and immunofluorescence, and increased quantity and distribution of the newly synthesised ECM throughout the construct. Consequently, while the compressive moduli of control Gel-MA constructs increased by 26 kPa after 8 weeks culture, constructs with HA-MA and CS-MA increased by 96 kPa. The enhanced chondrogenic differentiation, distribution of ECM, and improved mechanical properties make these materials potential candidates for cartilage tissue engineering applications.

Technological Advances in Geotextiles

The role of polymer chemistry (pure and applied sciences) is very prominent in the world of science today, but it is heading away from polymers and polymer blends towards composites and nanocomposites. This allows for the creation of new materials with unique properties and new possibilities which is the subject of this new book.

Hyperelastic constitutive relationship for the strain-rate dependent behavior of shoulder and other joint cartilages

Non-linear finite deformations of articular cartilages under physiological loading conditions can be attributed to hyperelastic behavior. This paper contains experimental results of indentation tests in finite deformation and proposes an empirical based new generalized hyperelastic constitutive model to account for strain-rate dependency for humeral head cartilage tissues. The generalized model is based on existing hyperelastic constitutive relationships that are extensively used to represent biological tissues in biomechanical literature. The experimental results were obtained for three loading velocities, corresponding to low (1x10-3 s-1), moderate and high strain-rates (1x10-1 s-1), which represent physiological loading rates that are experienced in daily activities such as lifting, holding objects and sporting activities. Hyperelastic material parameters were identified by non linear curve fitting procedure. Analysis demonstrated that the material behavior of cartilage can be effectively decoupled into strain-rate independent(elastic) and dependent parts. Further, experiments conducted using different indenters indicated that the parameters obtained are significantly affected by the indenter size, potentially due to structural inhomogeneity of the tissue. The hyperelastic constitutive model developed in this paper opens a new avenue for the exploration of material properties of cartilage tissues.

A stimulatory effect of Ca3ZrSi2O9 bioceramics on cementogenic/osteogenic differentiation of periodontal ligament cells

The regeneration of periodontal tissues to cure periodontitis remains a medical challenge. Therefore, it is of great importance to develop a novel biomaterial that could induce cementogenesis and osteogenesis in periodontal tissue engineering. Calcium silicate (Ca–Si) based ceramics have been found to be potential bioactive materials due to their osteostimulatory effect. Recently, it is reported that zirconium modified calcium-silicate-based (Ca3ZrSi2O9) ceramics stimulate cell proliferation and osteogenic differentiation of osteoblasts. However, it is unknown whether Ca3ZrSi2O9 ceramics possess specific cementogenic stimulation for human periodontal ligament cells (hPDLCs) in periodontal tissue regeneration in vitro. The purpose of this study was to investigate whether Ca3ZrSi2O9 ceramic disks and their ionic extracts could stimulate cell growth and cementogenic/osteogenic differentiation of hPDLCs; the possible molecular mechanism involved in this process was also explored by investigating the Wnt/β-catenin signalling pathway of hPDLCs. Our results showed that Ca3ZrSi2O9 ceramic disks supported cell adhesion, proliferation and significantly up-regulated relative alkaline phosphatase (ALP) activity, cementogenic/osteogenic gene expression (CEMP1, CAP, ALP and OPN) and Wnt/β-catenin signalling pathway-related genes (AXIN2 and CTNNB) for hPDLCs, compared to that of β-tricalcium phosphate (β-TCP) bioceramic disks and blank controls. The ionic extracts from Ca3ZrSi2O9 powders also significantly enhanced relative ALP activity, cementogenic/osteogenic and Wnt/β-catenin-related gene expression of hPDLCs. The present results demonstrate that Ca3ZrSi2O9 ceramics are capable of stimulating cementogenic/osteogenic differentiation of hPDLCs possibly via activation of the Wnt/β-catenin signalling pathway, suggesting that Ca3ZrSi2O9 ceramics have the potential to be used for periodontal tissue regeneration.

In vitro assessment of surface congruency and integration of chondrocytes at adjacent edges of micro-fabricated clefts on cartilage

Osteochondral grafts are common treatment options for joint focal defects due to their excellent functionality. However, the difficulty is matching the topography of host and graft(s) surfaces flush to one another. Incongruence could lead to disintegration particularly when the gap reaches subchondoral region. The aim of this study is therefore to investigate cell response to gap geometry when forming cartilage-cartilage bridge at the interface. The question is what would be the characteristics of such a gap if the cells could bridge across to fuse the edges? To answer this, osteochondral plugs devoid of host cells were prepared through enzymatic decellularization and artificial clefts of different sizes were created on the cartilage surface using laser ablation. High density pellets of heterologous chondrocytes were seeded on the defects and cultured with chondrogenic differentiation media for 35 days. The results showed that the behavior of chondrocytes was a function of gap topography. Depending on the distance of the edges two types of responses were generated. Resident cells surrounding distant edges demonstrated superficial attachment to one side whereas clefts of 150 to 250 µm width experienced cell migration and anchorage across the interface. The infiltration of chondrocytes into the gaps provided extra space for their proliferation and laying matrix; as the result faster filling of the initial void space was observed. On the other hand, distant and fit edges created an incomplete healing response due to the limited ability of differentiated chondrocytes to migrate and incorporate within the interface. It seems that the initial condition of the defects and the curvature profile of the adjacent edges were the prime determinants of the quality of repair; however, further studies to reveal the underlying mechanisms of cells adapting to and modifying the new environment would be of particular interest.

Correlating flow induced shear stress and chondrocytes activity in micro-porous scaffold using computational fluid dynamics and rapid prototyping

Flow induced shear stress plays an important role in regulating cell growth and distribution in scaffolds. This study sought to correlate wall shear stress and chondrocytes activity for engineering design of micro-porous osteochondral grafts based on the hypothesis that it is possible to capture and discriminate between the transmitted force and cell response at the inner irregularities. Unlike common tissue engineering therapies with perfusion bioreactors in which flow-mediated stress is the controlling parameter, this work assigned the associated stress as a function of porosity to influence in vitro proliferation of chondrocytes. D-optimality criterion was used to accommodate three pore characteristics for appraisal in a mixed level fractional design of experiment (DOE); namely, pore size (4 levels), distribution pattern (2 levels) and density (3 levels). Micro-porous scaffolds (n=12) were fabricated according to the DOE using rapid prototyping of an acrylic-based bio-photopolymer. Computational fluid dynamics (CFD) models were created correspondingly and used on an idealized boundary condition with a Newtonian fluid domain to simulate the dynamic microenvironment inside the pores. In vitro condition was reproduced for the 3D printed constructs seeded by high pellet densities of human chondrocytes and cultured for 72 hours. The results showed that cell proliferation was significantly different in the constructs (p<0.05). Inlet fluid velocity of 3×10-2mms-1 and average shear stress of 5.65×10-2 Pa corresponded with increased cell proliferation for scaffolds with smaller pores in hexagonal pattern and lower densities. Although the analytical solution of a Poiseuille flow inside the pores was found insufficient for the description of the flow profile probably due to the outside flow induced turbulence, it showed that the shear stress would increase with cell growth and decrease with pore size. This correlation demonstrated the basis for determining the relation between the induced stress and chondrocyte activity to optimize microfabrication of engineered cartilaginous constructs.

Fine tuning of elasticity via crosslinking collagen-based materials to mediate mechanotransduction and stability using corona treatment

The common goal of tissue engineering is to develop substitutes that can closely mimic the structure of extracellular matrix (ECM). However, similarly important is the intensive material properties which have often been overlooked, in particular, for soft tissues that are not to bear load assumingly. The mechanostructural properties determine not only the structural stability of biomaterials but also their physiological functionality by directing cellular activity and regulating cell fate decision. The aim here is to emphasize that cells could sense intensive material properties like elasticity and reside, proliferate, migrate and differentiate accordinglyno matter if the construct is from a natural source like cartilage, skin etc. or of synthetic one. Meanwhile, the very objective of this work is to provide a tunable scheme for manipulating the elasticity of collagen-based constructs to be used to demonstrate how to engineer cell behavior and regulate mechanotransduction. Articular cartilage was chosen as it represents one of the most complex hierarchical arrangements of collagen meshwork in both connective tissues and ECM-like biomaterials. Corona discharge treatment was used to produce constructs with varying density of crosslinked collagen and stiffness accordingly. The results demonstrated that elastic modulus increased up to 33% for samples treated up to one minute as crosslink density was found to increase with exposure time. According to the thermal analysis, longer exposure to corona increased crosslink density as the denaturation enthalpy increased. However the spectroscopy results suggested that despite the stabilization of the collagen structure the integrity of the triple helical structure remained intact. The in vitro superficial culture of heterologous chondrocytes also determined that the corona treatment can modulate migration with increased focal adhesion of cells due to enhanced stiffness, without cytotoxicity effects, and providing the basis for reinforcing three-dimensional collagen-based biomaterials in order to direct cell function and mediate mechanotransduction.

Adhesive characteristics of low dimensional carbon nanomaterial on actin

The biosafety of carbon nanomaterial needs to be critically evaluated with both experimental and theoretical validations before extensive biomedical applications. In this letter, we present an analysis of the binding ability of two dimensional monolayer carbon nanomaterial on actin by molecular simulation to understand their adhesive characteristics on F-actin cytoskeleton. The modelling results indicate that the positively charged carbon nanomaterial has higher binding stability on actin. Compared to crystalline graphene, graphene oxide shows higher binding influence on actin when carrying positive surface charge. This theoretical investigation provides insights into the sensitivity of actin-related cellular activities on carbon nanomaterial.

Local stress-strain distribution and load transfer across cartilage matrix at micro-scale using combined microscopy-based finite element method

Articular cartilage is the load-bearing tissue that consists of proteoglycan macromolecules entrapped between collagen fibrils in a three-dimensional architecture. To date, the drudgery of searching for mathematical models to represent the biomechanics of such a system continues without providing a fitting description of its functional response to load at micro-scale level. We believe that the major complication arose when cartilage was first envisaged as a multiphasic model with distinguishable components and that quantifying those and searching for the laws that govern their interaction is inadequate. To the thesis of this paper, cartilage as a bulk is as much continuum as is the response of its components to the external stimuli. For this reason, we framed the fundamental question as to what would be the mechano-structural functionality of such a system in the total absence of one of its key constituents-proteoglycans. To answer this, hydrated normal and proteoglycan depleted samples were tested under confined compression while finite element models were reproduced, for the first time, based on the structural microarchitecture of the cross-sectional profile of the matrices. These micro-porous in silico models served as virtual transducers to produce an internal noninvasive probing mechanism beyond experimental capabilities to render the matrices micromechanics and several others properties like permeability, orientation etc. The results demonstrated that load transfer was closely related to the microarchitecture of the hyperelastic models that represent solid skeleton stress and fluid response based on the state of the collagen network with and without the swollen proteoglycans. In other words, the stress gradient during deformation was a function of the structural pattern of the network and acted in concert with the position-dependent compositional state of the matrix. This reveals that the interaction between indistinguishable components in real cartilage is superimposed by its microarchitectural state which directly influences macromechanical behavior.

A bioengineered 3D ovarian cancer model for the assessment of peptidase-mediated enhancement of spheroid growth and intraperitoneal spread

Cancer-associated proteases promote peritoneal dissemination and chemoresistance in malignant progression. In this study, kallikrein-related peptidases 4, 5, 6, and 7 (KLK4-7)-cotransfected OV-MZ-6 ovarian cancer cells were embedded in a bioengineered three-dimensional (3D) microenvironment that contains RGD motifs for integrin engagement to analyze their spheroid growth and survival after chemotreatment. KLK4-7-cotransfected cells formed larger spheroids and proliferated more than controls in 3D, particularly within RGD-functionalized matrices, which was reduced upon integrin inhibition. In contrast, KLK4-7-expressing cell monolayers proliferated less than controls, emphasizing the relevance of the 3D microenvironment and integrin engagement. In a spheroid-based animal model, KLK4-7-overexpression induced tumor growth after 4 weeks and intraperitoneal spread after 8 weeks. Upon paclitaxel administration, KLK4-7-expressing tumors declined in size by 91% (controls: 87%) and showed 90% less metastatic outgrowth (controls: 33%, P<0.001). KLK4-7-expressing spheroids showed 53% survival upon paclitaxel treatment (controls: 51%), accompanied by enhanced chemoresistance-related factors, and their survival was further reduced by combination treatment of paclitaxel with KLK4/5/7 (22%, P=0.007) or MAPK (6%, P=0.006) inhibition. The concomitant presence of KLK4-7 in ovarian cancer cells together with integrin activation drives spheroid formation and proliferation. Combinatorial approaches of paclitaxel and KLK/MAPK inhibition may be more efficient for late-stage disease than chemotherapeutics alone as these inhibitory regimens reduced cancer spheroid growth to a greater extent than paclitaxel alone.

Molecular dynamics investigation on shearing between osteopontin and hydroxyapatite in biological materials

Bone, a hard biological material, possesses a combination of high stiffness and toughness, even though the main basic building blocks of bone are simply mineral platelets and protein molecules. Bone has a very complex microstructure with at least seven hierachical levels. This unique material characteristic attracts great attention, but the deformation mechanisms in bone have not been well understood. Simulation at nano-length scale such as molecular dynamics (MD) is proven to be a powerful tool to investigate bone nanomechanics for developing new artificial biological materials. This study focuses on the ultra large and thin layer of extrafibrillar protein matrix (thickness = ~ 1 nm) located between mineralized collagen fibrils (MCF). Non-collagenous proteins such as osteopontin (OPN) can be found in this protein matrix, while MCF consists mainly of hydroxyapatite (HA) nanoplatelets (thickness = 1.5 – 4.5 nm). By using molecular dynamics method, an OPN peptide was pulled between two HA mineral platelets with water in presence. Periodic boundary condition (PBC) was applied. The results indicate that the mechanical response of OPN peptide greatly depends on the attractive electrostatics interaction between the acidic residues in OPN peptide and HA mineral surfaces. These bonds restrict the movement of OPN peptide, leading to a high energy dissipation under shear loading.