Diaphyseal bone defect represents a significant problem for orthopaedic surgeons and patients. In order to improve and fasten bone regenerating process we implanted HA biodegradable magnetized scaffolds in a large animal model critical bone defect. A critical long bone defect was created in 6 sheep metatarsus diaphysis; then we implanted a novel porous ceramic composite scaffold (20.0 mm in length; 6.00 mm inner diameter and 17.00 mm outer diameter), made of Hydroxyapatite that incorporates magnetite (HA/Mgn 90/10), proximally fixated by two small cylindrical permanent parylene coated NdFeB magnets (one 6.00 mm diameter magnetic rod firmly incorporated into the scaffold and one 8.00 mm diameter magnetic rods fitted into proximal medullary canal, both 10.00 mm long); to give stability to the complex bone-scaffold-bone, screws and plate was used as a bridge. Scaffolds biocompatibility was previously assessed in vitro using human osteoblast-like cells. Magnetic forces through scaffold were calculated by finite element software (COMSOL Multiphysics, AC/DC Model). One week after surgery, magnetic nanoparticles functionalized with vascular endothelial growth factor (VEGF) were injected at the mid portion of the scaffold using a cutaneous marker positioned during surgery as reference point. After sixteen weeks, sheep were sacrificed to analyze metatarsi. Macroscopical, radiological and microCT examinations were performed. Macroscopical examination shows bone tissue formation inside scaffold pores and with complete coverage of scaffolds, in particular at magnetized bone-scaffold interface. X-rays show a good integration of the scaffold with a good healing process of critical bone defect, and without scaffolds mobilization. These datas were confirmed by the microCT that shown new formation of bone inside the scaffolds, in particular at magnetized bone-scaffold interface. These preliminary results lead our research to exploiting magnetic forces to stimulate bone formation, as attested in both in vitro and in vivo models and to improve fixation at bone scaffold interface, as calculated by finite element software, and moreover to guide targeted drug delivery without functionalized magnetic nanoparticles dissemination in all body. Histological analysis will be performed to confirm and quantify bone tissue regeneration at both interfaces.

Cartilage defects, which are caused by a variety of reasons such as traumatic injuries, osteoarthritis, or osteoporosis, represent common and severe clinical problems. Each year, over 6 million people visit hospitals in the U.S. for various knee, wrist, and ankle problems. As modern medicine advances, new and novel methodologies have been explored and developed in order to solve and improve current medical problems. One of the areas of investigation that has thus far proven to be very promising is tissue engineering [1, 2]. Since cartilage matrix is nanocomposite, the goal of the current work is to use nanomaterials and nanofabrication methods to create novel biologically inspired tissue engineered cartilage scaffolds for facilitating human bone marrow mesenchymal stem cell (MSC) chondrogenesis. For this purpose, through electrospinning techniques, we designed a series of novel 3D biomimetic nanostructured scaffolds based on carbon nanotubes and biocompatible poly(L-lactic acid) (PLLA) polymers. Specifically, a series of electrospun fibrous PLLA scaffolds with controlled fiber dimension were fabricated in this study. In vitro hMSC studies showed that stem cells prefer to attach in the scaffolds with smaller fiber diameter. More importantly, our in vitro differentiation results demonstrated that incorporation of the biomimetic carbon nanotubes and poly L-lysine coating can induce more chondrogenic differentiations of MSCs than controls, which make them promising for cartilage tissue engineering applications.

Nowadays, magnetic materials are receiving special attention due to their potential applications in different fields and in particular in medicine. Magnetic micro-nano-particles have been progressively employed as support materials for enzyme immobilization, and have been used as drug-delivery vehicles, contrast agents for magnetic resonance imaging as well as heat mediators for hyperthermia-based anti-cancer treatments and many other exciting biomedical applications. Magnetic materials have also attracted a big interest in the field of bone tissue regeneration because it has been demonstrated that magnetic nanoparticles have effect of osteoinduction even without external magnetic force. Therefore, one of the most big challenge in this field is the production of magnetic materials with good biocompatibility and biodegradability. In fact, the long-term effects in the human body of iron oxide (maghemite or magnetite), the most popular magnetic phase used in medicine and biotechnology, are not yet completely assessed. To this aim, in this work we developed an innovative biocompatible and bioresorbable superparamagnetic-like phase by doping nano-hydroxyapatite with Fe 2+ /Fe 3+ ions (FeHA). Moreover the same magnetic nanoparticles were used as nano-particulate emulsifier for the preparation of hollow hybrid Fe-HA-poly(L-lactic) acid (PLLA) micro-nano-spheres. PLLA has been used because poly(α-hydroxy-esters) are the most frequently used synthetic polymers for biomedical applications owing to their biocompatibility, hydrolytic degradation process and proper mechanical properties. These micro-nanospheres could be used as new type of scaffold for hard tissue regeneration. In fact, spherical scaffold display several advantages respect to the monolithic counterpart e.g., (i) improving control over sustained delivery of therapeutic agents, signalling biomolecules and even pluripotent stem cells, (ii) serving as stimulus-sensitive delivery vehicles for triggered release, (iii) introducing porosity and/or improve the mechanical properties of bulk scaffolds by acting as porogen or reinforcement phase, (iv) supplying compartmentalized micro-reactors for dedicated biochemical processes, (v) functioning as cell delivery vehicle, and, finally, (vi) giving possibility of preparing injectable and/or mouldable formulations to be applied by using minimally invasive surgery. Moreover, the same magnetic materials could find applications in nanomedicine as a multifunctional carrier. Their magnetic functionality could be utilized to move them into the body towards target organs by an external magnetic field. Furthermore, the superparamagnetic feature of the nanoparticles could allow to tailor the release of the therapeutic agent by switching (on-off) the external magnetic field and/or to treat cancer cells by hyperthermia.

Hierarchical designs of biological structures have remarkable physical, chemical mechanical and biological properties and functionalities over the wide range of length scales [1–4]. Man-made nanocomposites have dramatic improvement of the structural and mechanical properties but however they have very limited hierarchy [5]. Fish scales are bone-like tissues, which form a protective layer on the body of the fish and enable the fish to swim efficiently. Bones and bone-like parts in living organism are formed as tissues by self-assembly of bio-minerals and protein matrix. These tissues are bio-nanocomposites and have hierarchical structure ranging from nanoscale to macroscale [2–4]. Bio-hierarchy contains different bio-macromolecules, bio-minerals, interfacial bonds and porosity which result in gradient mechanical properties at multiple length scales [1–6]. Fish scale consists of inorganic bio-minerals and organic collagens [3,4]. Multilevel hierarchy influences elasticity, hardness and fracture toughness of fish scale. They have additional functions related to movement including reduction or increase of drag [7] and rapid manoeuvre while they are hunting or avoiding predators. In this article we report comparison studies of hierarchical nanocomposite of sardina pilchardus(sp) fish scale and man-made SiO 2 nanoparticles filled nanocomposites.

Anterior cruciate ligament (ACL) reconstructive surgery is a major health concern world-wide because of a large aging population and increased occurrence of sport-related injuries. Tissue engineering is a rapidly growing interdisciplinary field that offers a promising new approach for ACL repair. The aim of this project is to explore novel “smart” surgical fixation devices that not only secure a graft in place without strength failure, but also incorporate and release bioactive materials, intended to promote bone tissue growth. In order to facilitate bioactive reagent release, biopolymeric scaffolds with continuous micro-porous structure were developed. The morphology of the porous structures in the poly-L-lactic acid (PLLA) matrix reflects the differential influence of the concentration of sacrificial material (PS-polystyrene), phase separation levels of the immiscible polymers (PLLA and PS), and melt-blending conditions (Fig. 1) [1]. During removal of the sacrificial material, the chemical solvent might introduce some chemical reactant into the scaffolds. In order to assess the feasibility of using the micro-porous structures for medical applications, 7F2 osteoblasts were cultured on these scaffolds for 7 days. The attachment and proliferation of 7F2 cells on all scaffolds were assessed by fluorescent nuclear staining with Hoechst 33258 and phalloidin. The morphology of 7F2 osteoblasts on solid PLLA and PLLA/HA with 40% porous structure scaffolds till the 7 days pos-seeding was observed under confocal microscopy (Fig. 2A and B). The results showed that removal of the sacrificial material does not influence cell growth and the composites are biocompatible. Besides in vitro cytotoxicity test, in vivo test of all the micro-porous structural scaffolds was performed through rat subcutaneous surgery. Histological analysis (H&E staining) of the porous PLLA/HA with 40% pores retrieved from rat subcutaneous tissue 4 weeks postimplantation show that cells start to grow inside the porous scaffold (Fig. 3A). The morphology of surrounding extracellular matrix (ECM) growing on the scaffolds was observed under SEM. Figure 3B shows soft tissue attached onto PLLA/HA porous scaffold after 1 month post implantation time point, which indicates the good biocompatibility of the scaffolds. Based on these data we predict that these scaffolds will be suitable for inducing and sustaining bone tissue regeneration, and will be feasible for ACL repair.

Tissue engineering (TE) is evolving as a potential solution for repair and reconstruction of diseased or damaged tissues. TE faces major manufacturing challenges: manufacturing techniques are needed to mimic tissue and extra cellular matrix (ECM) architecture, with high resolution (less than 10 μm) for tissues such as myocardium (heart muscle), blood vessels, bone or nerves; innovative methods are needed for delivery of cells and growth factors into scaffolds; and manufacturing techniques are needed to create vascular structure in tissue construct; lack of nutrient transport currently limits the size and cellular content of implants.

Indentation, in addition to the traditional tensile testing, has been widely used for evaluating mechanical properties of hard materials such as metals and bone as well as soft materials like polymer and soft tissues. However, it is difficult to measure the contact area and surface deformation in conventional indentation tests of soft tissue which will bring large errors to the evaluation of the material properties. Also the assumption of isotropic property limited the usage of indentation test in characterizing the nonlinear, anisotropic properties of soft tissue thin film. In this project, 2D and 3D finite element analyses has been carried out to predict hyperelastic material response under indentation and punch tests. A novel indentation test system was developed, which made the direct measurement of local deformation and contact area possible. The apparatus consists of a transparent indenter, a digital microscope, and a computer based control and data acquisition system. The proposed testing system and associated finite element analysis are used to characterize the mechanical properties of multiscale (bulk and thin film) biological tissues.

Collagen is a crucial structural protein material, formed through a hierarchical assembly of tropocollagen molecules, arranged in collagen fibrils that constitute the basis for larger-scale fibrils and fibers. Osteogenesis imperfecta is a genetic disorder in collagen characterized by mechanically weakened tendon, fragile bones, skeletal deformities and in severe cases prenatal death. Even though many studies have attempted to associate specific mutation types with phenotypic severity, the mechanisms by which a single point mutation influences the mechanical behavior of tissues at multiple length-scales remain unknown. In this study, we report a series of systematic molecular scale based bottom-up computational experiments focused on pure collagenous tissue, carried out using atomistic-level molecular dynamics (MD), adaptive Poison-Boltzmann solver (APBS) calculations, and a mesoscale molecular model of collagen fibrils.

Knowledge of mechanical properties of bones is important for the designing of bone replacements and implants as well as the research of bone diseases such as osteoporosis. However, bone, especially trabecular bone, is a highly anisotropic and heterogeneous living tissue. Micro-computed-tomography (micro-CT) and three-dimensional ultrasound imaging techniques are valuable tools for nondestructive investigation of three-dimensional trabecular bone architecture. From a reconstruction of trabecular bone, a numerical model such as finite element (FE) model can be generated. Using this FE model to simulate compression test, and comparing the simulation results to the results from real mechanical test of the same specimen, the relationship between the observed mechanical behaviors and the microstructure can be established.

Here, we incorporate the results of our new “altered phase theory” (Sikdar et al. 2008a) into design of new polymer clay nanocomposites (PCNs) for bone biomaterials applications. Montmorillonite (MMT) clay was modified using unnatural amino acids as potentially new biocompatible modifiers. The longer carbon chain structures of the unnatural amino acids are expected to enhance non bonded interactions with clay as well as maintaining the usefulness of functional groups of natural amino acids. The specific choice of amino acids is based on both the antibacterial activity reported in literature and also our previous studies on role of chain length, functional groups etc of modifiers in influencing mechanical behavior in PCNs. Biocompatibility studies using cell culture experiments as well as mechanical behavior is evaluated for the PCNs. FTIR spectroscopy is used to compare changes to molecular structure. The increase in d 001 spacing of modified clay compared to pure clay obtained from XRD experiments confirms successful intercalation of modifier. The osteoblast cells were found to grow and proliferate over the substrates. The major contribution of this work is the design of novel amino acid biopolymer-clay nanocomposites for biomaterials applications. Porous scaffold structures were also designed and fabricated.

We present a numerical model to mechanically characterise real and Monte-Carlo microstructures of human cortical bone. Femoral mid-shaft human bone microstructures are specific to each individual and exhibit unique morphological and mechanical properties. Real microstructures of female bone samples are explicitly reconstructed using a finite element model. Patient-dependent statistically equivalent models are built from natural morphological parameters measured under reflected light microscopy (RLM) and back-scattered electron microscopy (BSEM). The micro mechanical properties are provided by nanoindentations and microextensometry. The morphological and mechanical characteristics are incorporated into the model to mimic bone micro-scale heterogeneity. The overall stiffness of the microstructure is calculated for a distribution of samples. The effects of the variations of local parameters on the global stiffness are studied. The evolution of these parameters indicates various aging signs of bone. These results are validated through comparison to experiments to assess fracture risk.

Bone is an important multifunctional biological tissue with remarkable mechanical properties: high strength and stiffness, high fracture toughness, and light weight. These superior properties are due, in part, to the hierarchical structure of bone ranging from molecular to macroscopic levels, Fig. 1. Nevertheless, it is not clearly understood how the microstructure and mechanical properties of various hierarchies at different length scales affect the overall behavior of bone. Such understanding is essential in orthopedics for designing implant materials and fabricating synthetic bone substitutes and also for assessing the effect of bone diseases and their medications on bone’s properties. It can, moreover, serve as a guide in design of advanced synthetic bio-inspired materials for a wide range of engineering applications.

In this article we present the value of nanoparticles alloys made out of Ag:Cu as nanocidals for possible treatment of infected bone cells.

The necessity of new clinical approaches regarding musculoskeletal system regeneration is evident. Nowadays different strategies such as autografts, allografts also used in synergy with cell therapy are already used in clinical treatments for moderate defects, but they face significant limitations due to limited supply, and risk of immune rejection. Currently, the treatments of extended osteochondral and bone defects involve invasive permanent metallic prosthesis, challenging reconstructive procedures and long rehabilitation period. Despite that, the gold standard seems to be far to obtain.

Nanodiamond (ND) is an attractive nanomaterial for reinforcement of polymers [1] due to the ND’s superior mechanical and chemical properties, and low biotoxicity. A novel composite material has been produced for bone scaffolds utilizing the biodegradable polymer, poly(L-lactic acid) (PLLA), and octadecylamine-functionalized nanodiamond (ND-ODA) [2]. Composites were prepared by admixing to a PLLA/chloroform solution chloroform suspensions of ND-ODA at concentrations of 0, 1, 3, 5, 7, and 10 (w/w). Dispersion of ND-ODA in the composites was studied by transmission electron microscopy (TEM). The lower-resolution TEM micrograph of 1% wt ND-ODA/PLLA film (Fig. 1a) shows nanodiamond particles dispersed in PLLA film on amorphous carbon support. Due to long hydrocarbon chains of ODA the ND-ODA particles have good wettability with the PLLA so there is no segregation of ND-ODA and PLLA, and the polymer completely surrounds the particles. The high-resolution TEM image (Fig. 1b) shows ND crystals with attached organic material that can be ODA or PLLA. Nanoindentation tests show that the mechanical strength of ND-ODA/PLLA composites improves upon addition of ND (Table 1). Even at low concentrations (1% wt) the ND-ODA increased the hardness of the composite by 60% and Young’s modulus by 20% over neat PLLA. Based on our preliminary observations, we conclude that further additions of ND-ODA resulted in smaller changes with subsequent saturation in the mechanical properties at ∼7% wt (see Table 1). ND is relatively novel nanomaterial. Establishing its biocompatibility requires further studies, especially for modified ND. We studied the biocompatibility of 5–10nm ND and ND-ODA in experiments with a murine osteoblast cell line (7F2, from ATCC). Incubation of a cultured osteoblasts with 1–100μg/ml of ND or ND-ODA particles for 4 hours did not show much influence on the cell viability (Fig. 2), as inferred from an alamarBlue™ assay. To test the feasibility of ND-ODA/PLLA as a matrix material supporting cell growth, osteoblasts were cultured on the composites for 6 days. The attactment and proliferation of 7F2 cells on the scaffolds were assessed, respectively, by fluorescent nuclear staining with Hoechst 33258 and the alamarBlueTM assay. Our results showed that the addition of ND-ODA had only a negligibly small effect on cell proliferation, which is indicative of good biocompatibility of the composites (Fig. 3). The morphology of 7F2 cells growing on all ND-ODA/PLLA composite scaffolds was assessed by SEM. The data (not shown) confirm that the osteoblasts spread on the scaffolds similar to their spreading on TCP (tissue culture plastic). To summarize, the improved mechanical properties of the PLLA/ND-ODA composites and their good biocompatibility suggest that these materials may be suitable for applications in musculoskeletal tissue engineering.

Every year, there are roughly 8 million bone fractures in United States [1]. In addition, approximately 2300 new cases of primary bone cancer are diagnosed each year [2]. Yet, the number of people suffering from bone disease is significantly greater; about 10 million people in the U.S. alone suffer from osteoporosis [3]. Consequently, surgeons perform nearly 500,000 bone graft operations annually making bone grafts the second most frequently transplanted materials [4]. Although there is an extremely high demand for treatment of bone abnormalities, the current grafting methods fail to meet these demands due to several limitations. Autografting has the fewest problems with rejection and pathogen transmission, however in some cases the availability may be limited or not be possible (e.g., genetic diseases). With other methods of transplantation such as allogafting and xenografting where tissue is acquired from other humans or species, respectively, the receptor’s immune system causes an increased risk of chronic rejection [5]. Notably, the major drawback with all these methods is that they often require multiple painful and invasive surgeries.