BMM Factor

Osteogenic maturation of the osteoblast is crucial for bone formation. In this study, multi-walled carbon nanotubes (MWCNTs) and graphite (GP) were pressed as compacts. The greater ability of carbon nanotubes to adsorb proteins, compared with graphite, was shown. Human osteoblast-like SaoS2 cells were cultured and the cell response to the two kinds of compacts was compared in vitro. Meanwhile, we used cell culture on the culture plate as a control. Assays for osteonectin, osteopontin and osteocalcin gene expression, total protein (TP) amount, alkaline phosphatase activity (ALP) and DNA of cells cultured on the samples were done. During the conventional culture, significantly higher osteonectin, osteopontin and osteocalcin gene expression level, ALP/DNA and TP/DNA on carbon nanotubes were found. To confirm the hypothesis that the larger amount of specific proteins adsorbed on the carbon nanotubes was crucial for this, the compacts were pre-soaked in culture medium having additional recombinant human bone morphogenetic protein-2 (rhBMP-2) before cell culture. Compared with GP, osteonectin, osteopontin and osteocalcin gene expression level, ALP/DNA and TP/DNA of the cells tested increased more on the MWCNTs after the compacts were pre-soaked in the culture medium with rhBMP-2. The results indicated that the carbon nanotubes might induce osteogenic maturation of the osteoblast by adsorbing more specific proteins.

In the present paper, we demonstrate the feasibility of depositing a tailored calcium phosphate (Ca–P) coating on a Ti–6Al–4V substrate by using a pulsed Nd:YAG laser system. Different textures were obtained by varying the laser spot overlap with change in laser traverse speed. Surface roughness measurements using laser confocal microscopy indicated a decrease in roughness with increasing laser scan speed. X-ray diffraction studies revealed the formation of α-TCP, TiO₂, Ti and Al as the major phases. An instrumented nanoindenation technique used to study the mechanical properties of the coatings, revealed a very high hardness and Young's modulus of the coating surface as compared to the substrate. This further proved the retainment of the ceramic phase on the surface. Wear studies in a simulated biofluid (SBF) environment demonstrated an increased wear resistance of the coated samples as compared to the bare Ti–6Al–4V. Formation of an apatite-like layer after immersion in SBF for different time periods further demonstrated the bioactivity of the coated samples.

Electrospinning has recently received much attention in biomedical applications, and has shown great potential as a novel scaffold fabrication method for tissue engineering. The nano scale diameter of the fibers produced and the structure of the web resemble certain supramolecular features of extracellular matrix which is favorable for cell attachment, growth and proliferation. There are various parameters that can alter the electrospinning process, and varying one or more of these conditions will result in producing different nanofibrous webs. So the aim of this study was to investigate the effect of material variables and process variables on the morphology of electrospun 50:50 poly(L-lactide-co-ε-caprolactone) (PLCL) nanofibrous structures. The morphology of the nanofibers produced was strongly influenced by parameters such as the flow rate of the polymer solution, the electrospinning voltage and the solution concentration. The diameter was found to increase with solution concentration in a direct linear relationship. Finally, it has been successfully demonstrated that by increasing the rotation speed of the collector mandrel, the alignment of the fibers can be controlled in a preferred direction. These findings contribute to determining the functional conditions to electrospin this biodegradable elastomeric copolymer which has potential as a scaffold material for vascular tissue engineering.

Interest in stimuli-responsive polymers is steadily gaining increasing momentum especially in the fields of controlled and self-regulated drug delivery. Delivery systems based on these polymers are developed to closely resemble the normal physiological process of the diseased state ensuring optimum drug release according to the physiological need. Also termed 'environmental-sensitive' or 'smart', these polymers experience rapid changes in their microstructure from a hydrophilic to a hydrophobic state triggered by small changes in the environment. The changes are reversible; therefore, the polymer is capable of returning to its initial state as soon as the trigger is removed. Stimuli may occur internally (e.g. a change in pH in certain organs or diseased states, a change in temperature or the presence of specific enzymes or antigens). External stimuli include magnetic or electric fields, light, ultrasound, etc. This review will delve into the various internally and externally stimuli-responsive polymers and the drug delivery systems that exploit them.

The aim of this study is to demonstrate the effect of extracellular calcium ion (Ca²⁺) and inorganic phosphate (Pi) concentrations on the growth and differentiation of bone-marrow-derived mesenchymal stem cells (MSCs), which is essential to understand the interaction between calcium phosphate ceramic (CPC) scaffolds and seeded cells during the construction of tissue-engineered bones. MSCs were separated from rabbits and cultured in media with different concentrations of Ca²⁺ and Pi supplements. Their proliferation, apoptosis, mineralization and osteogenic differentiation were determined by the MTT assay, TUNEL assay, Vonkossa stain and RT-PCR examination. A two-way ANOVA calculation with comparisons of estimated marginal means by LSD was used for statistical analysis. Results showed that the optimal extracellular Ca²⁺ and Pi concentrations for the cells to proliferate and differentiate were 1.8 mM and 0.09 mM, respectively, which are the concentrations supplied in many commonly used culture media such as DMEM and α-MEM. Cell proliferation and differentiation decreased significantly with greater or lower concentrations of the Pi supplement. Greater Pi concentrations also led to significant cell apoptosis. Greater Ca²⁺ concentrations did not change cell proliferation but significantly inhibited cell differentiation. In addition, greater Ca²⁺ concentrations could significantly enhance cell mineralization. In conclusion, extracellular Ca²⁺ and Pi significantly influence the growth and osteogenic differentiation of MSCs. It is important to take the cellular effect of Ca²⁺ and Pi into consideration when designing or constructing scaffolds for bone tissue engineering with CPC.

Despite the satisfactory short-term implant survivorship of metal-on-metal hip resurfacing arthroplasty, periprosthetic soft-tissue masses such as pseudotumours are being increasingly reported. Cytotoxic effects of cobalt or chromium have been suggested to play a role in its aetiology. The aim of this study was to investigate the effects of clinically relevant metal nanoparticles and ions on the viability of macrophages in vitro. A RAW 264.7 murine macrophage cell line was cultured in the presence of either: (1) cobalt, chromium and titanium nanoparticles sized 30–35 nm; or (2) cobalt sulphate and chromium chloride. Two methods were used to quantify cell viability: Alamar Blue assay and Live/Dead assay. The cytotoxicity was observed only with cobalt. Cobalt nanoparticles and ions demonstrated dose-dependent cytotoxic effects on macrophages in vitro: the cytotoxic concentrations of nanoparticles and ions were 1 × 10¹² particles ml⁻¹ and 1000 µM, respectively. The high concentration of cobalt nanoparticles required for cytotoxicity of macrophages in vitro suggests that increased production of cobalt nanoparticles in vivo, due to excessive MoM implant wear, may lead to local adverse biological effects. Therefore, cytotoxicity of high concentrations of metal nanoparticles phagocytosed by macrophages located in the periprosthetic tissues may be an important factor in pathogenesis of pseudotumours.

Development of tissue-engineered bone constructs has recently focused on the use of electrospun composite scaffolds seeded with stem cells from various source tissues. In this study, we fabricated electrospun composite scaffolds consisting of β-tricalcium phosphate (TCP) crystals and poly(L-lactic acid) (PLA) at varying loading levels of TCP (0, 5, 10, 20 wt%) and assessed the composite scaffolds' material properties and ability to induce proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) in the presence of osteogenic differentiating medium. The electrospun scaffolds all exhibited a nonwoven structure with an interconnected porous network. With the addition of TCP, the fiber diameter increased with each treatment ranging from 503.39 ± 20.31 nm for 0 wt% TCP to 1267.36 ± 59.03 nm for 20 wt% TCP. Tensile properties of the composite scaffolds were assessed and the overall tensile strength of the neat scaffold (0 wt% TCP) was 847 ± 89.43 kPA; the addition of TCP significantly decreased this value to an average of 350.83 ± 38.57 kPa. As the electrospun composite scaffolds degraded in vitro, TCP was released into the medium with the largest release occurring within the first 6 days. Human ASCs were able to adhere, proliferate and osteogenically differentiate on all scaffold combinations. DNA content increased in a temporal manner for each scaffold over 18 days in culture although for the day 12 timepoint, the 10 wt% TCP scaffold induced the greatest hASC proliferation. Endogenous alkaline phosphatase activity was enhanced on the composite PLA/TCP scaffolds compared to the PLA control particularly by day 18. It was noted that at the highest TCP loading levels of 10 and 20 wt%, there was a dramatic increase in the amount of cell-mediated mineralization compared to the 5 wt% TCP and the neat PLA scaffold. This work suggests that local environment cues provided by the biochemical nature of the scaffold can accelerate the overall osteogenic differentiation of hASCs and encourage rapid ossification.

A bioabsorbable nanofibrous scaffold was developed for early adhesion of mesenchymal stem cells (MSCs). Collagen nanofibers with diameters of 430 ± 170 nm were fabricated by electrospinning. Over 45% of the MSC population adhered to this collagen nanofiber after 30 min at room temperature. Remarkably, collagen-coated P(LLA-CL) electrospun nanofibers were almost as efficient as collagen nanofibers whereas collagen cast film did not enhance early capture when it was applied on cover slips. The adhesive efficiency could be further increased to over 20% at 20 min and over 55% at 30 min when collagen nanofibers were grafted with monoclonal antibodies recognizing CD29 or CD49a. These data demonstrate that the early adhesive behavior is highly dependent on both the surface texture and the surface chemistry of the substrate. These findings have potential applications for early capture of MSCs in an ex vivo setting under time constraints such as in a surgical setting.

Extracellular matrices are arranged with a specific geometry based on tissue type and mechanical stimulus. For blood vessels in the body, preferential alignment of fibers is in the direction of repetitive force. Electrospinning is a controllable process which can result in fiber alignment and randomization depending on the parameters utilized. In this study, arterial grafts composed of polycaprolactone (PCL), polydioxanone (PDO) and silk fibroin in blends of 100:0 and 50:50 for both PCL:silk and PDO:silk were investigated to determine if fibers could be controllably aligned using a mandrel rotational speed ranging from 500 to 8000 revolutions per minute (RPM). Results revealed that large- and small-diameter mandrels produced different degrees of fiber alignment based on a fast Fourier transform of scanning electron microscope images. Uniaxial tensile testing further demonstrated scaffold anisotropy through changes in peak stress, modulus and strain at break at mandrel rotational speeds of 500 and 8000 RPM, causing peak stress and modulus for PCL to increase 5- and 4.5-fold, respectively, as rotational speed increased. Additional mechanical testing was performed on grafts using dynamic compliance, burst strength and longitudinal strength displaying that grafts electrospun at higher rotational rates produced stiffer conduits which had lower compliance and higher burst strength compared to the lower mandrel rotational rate. Scaffold properties were found to depend on several parameters in the electrospinning process: mandrel rotational rate, polymer type, and mandrel size. Vascular scaffold design under anisotropic conditions provided interesting insights and warrants further investigation.

A magnetic field has been applied to accelerate bone healing for a long time. In this study, in order to combine the bone repair capability of calcium phosphate (CaP) ceramics with the magnetic field, a novel CaP ceramic–magnetic nanoparticle (CaP–MNP) composite was fabricated through integrating the superparamagnetic nanoparticles into the CaP ceramics. Two kinds of CaP ceramics were chosen: hydroxyapatite (HA) and HA/tricalcium phosphate (65/35, HT). The samples were cultured with Ros17/2.8 and MG63 cells respectively in vitro to evaluate the cell proliferation and differentiation via MTT and alkaline phosphatase activity tests. In order to find the influence of the magnetic materials on the expression of the bone morphological protein (BMP), the samples composited with BMP-2 were implanted subcutaneously in the fasciae of rat back muscles for 30 days. Compared with ordinary CaP ceramics, the results indicated that the CaP–MNP composite had good biocompatibility and was able to promote cell proliferation and differentiation significantly. The in vivo test showed that the expression of BMP-2 would be accelerated by HT composited with MNPs, and new bone-like tissue formation could be observed. Accordingly, it might be expected that this CaP–MNP composite could become a potential bone substitute or bone tissue engineering scaffold.

Bio-electrospraying (BES) has demonstrated great promise as a rapidly evolving strategy for tissue engineering and regenerative biology/medicine. Since its discovery in 2005, many studies have confirmed that cells (immortalized, primary and stem cells) and whole organisms (Danio rerio, Xenopus tropicalis, Caenorhabditis elegans to Drosophila) remain viable post-bio-electrospraying. Although this bio-protocol has achieved much, it suffers from one crucial problem, namely the ability to precisely control the number of cells within droplets and or encapsulations. If overcome, BES has the potential to become a high-efficiency biotechnique for controlled cell encapsulation, a technique most useful for a wide range of applications in biology and medicine ranging from the forming of three-dimensional cultures to an approach for treating diseases such as type I diabetes. In this communication, we address this issue by demonstrating the coupling of BES with droplet-based microfluidics for controlling live cell numbers within droplets and residues.

The nano-dimensionality of nature has logically given rise to the interest in using nanomaterials in the biomedical field. Currently, a lot of investigations into carbon nanotubes (CNTs), as one of the typical nanomaterials, are being made for biomedical application. In this review, five parts, such as cellular functions induced by CNTs, apatite formation on CNTs, CNT-based tissue engineering scaffold, functionalized CNTs for the delivery of genes and drugs and CNT-based biosensors, are stated, which might indicate that CNTs, with a range of unique properties, appear suited as a biomaterial and may become a useful tool for tissue engineering. However, everything has two parts and CNTs is not an exception. There are still concerns about cytotoxicity and biodegradation of CNTs. Chemical fictionalization may be one of the effective ways to improve the 'disadvantages' and utilize the 'advantages' of CNTs. One of their 'disadvantages', unbiodegradable property, may be utilized by creating monitors in in vivo-engineered tissues or nanosized CNT-based biosensors. Other promising research points, for example proteins adsorbed on CNTs, use of CNTs in combination with other biomaterials to achieve the goals of tissue engineering, mineralization of CNTs and standard toxicological tests for CNTs, are also described in the conclusion and perspectives part.

Research efforts have been devoted to evaluating the application of the chitosan (CS)/glycerol-β-phosphate (GP) disodium salt hydrogel in peripheral nerve regeneration. The gelation time was determined to be 770 s using ultraviolet spectrophotometry. A standard 10 mm long rat sciatic nerve defect model was employed, followed by bridging the proximal and distal stumps with chitosan conduits injected with the Schwann cell-containing hydrogel. Injections of the blank hydrogel, Schwann cell suspension and culture medium were used as controls. Two months later, electrophysiological assessment and fluorogold retrograde tracing showed that compound muscle action potentials (CMAPs) and fluorogold-labeled neurons were only detected in the Schwann cell suspension group and culture medium group. The rats were then killed, and implanted conduits were removed for examination. There were no regenerated nerves found in groups injected with the blank hydrogel or Schwann cell-containing hydrogel, while the other two groups clearly displayed regenerated nerves across the gaps. In the subsequent histological assessment, immunohistochemistry, toluidine blue staining and transmission electron microscopy were performed to evaluate the regenerated nerves. The relative wet weight ratio, Masson trichrome staining and acetylcholinesterase staining were employed for the examination of gastrocnemius muscles in all four groups. The Schwann cell suspension group showed the best results for all these indexes; the culture medium group ranked second and the two hydrogel-injected groups showed the least optimal results. In conclusion, our data revealed that the implanted CS/GP hydrogel actually impeded nerve regeneration, which is inconsistent with former in vitro reports and general supposition. We believe that the application of the CS/GP hydrogel in nerve regeneration requires a further study before a satisfactory result is obtained. In addition, the present study also confirmed that Schwann cell implantation stimulated nerve regeneration.

The clinical suitability of a bone substitute material is determined by the ability to induce a tissue reaction specific to its composition. The aim of this in vivo study was to analyze the tissue reaction to a silica matrix-embedded, nanocrystalline hydroxyapatite bone substitute. The subcutaneous implantation model in Wistar rats was chosen to assess the effect of silica degradation on the vascularization of the biomaterial and its biodegradation within a time period of 6 months. Already at day 10 after implantation, histomorphometrical analysis showed that the vascularization of the implantation bed reached its peak value compared to all other time points. Both vessel density and vascularization significantly decreased until day 90 after implantation. In this time period, the bone substitute underwent a significant degradation initiated by TRAP-positive and TRAP-negative multinucleated giant cells together with macrophages and lymphocytes. Although no specific tissue reaction could be related to the described silica degradation, the biomaterial was close to being fully degraded without a severe inflammatory response. These characteristics are advantageous for bone regeneration and remodeling processes.

Calcium phosphate ceramic coatings, applied on surfaces of metallic and polymeric biomaterials, can improve their performance in bone repair and regeneration. Spider silk is biocompatible, strong and elastic, and hence an attractive biomaterial for applications in connective tissue repair. Recently, artificial spider silk, with mechanical and structural characteristics similar to those of native spider silk, has been produced from recombinant minispidroins. In the present study, supersaturated simulated body fluid was used to deposit calcium phosphate coatings on recombinant spider silk fibres. The mineralization process was followed in time using scanning electron microscopy equipped with an energy dispersive x-ray (EDX) detector and Raman spectroscope. Focused ion beam technology was used to produce a cross section of a coated fibre, which was further analysed by EDX. Preliminary in vitro experiments using a culture of bone marrow-derived human mesenchymal stem cells (hMSCs) on coated fibres were also performed. This study showed that recombinant spider silk fibres were successfully coated with a homogeneous and thick crystalline calcium phosphate layer. In the course of the mineralization process from modified simulated body fluid, sodium chloride crystals were first deposited on the silk surface, followed by the deposition of a calcium phosphate layer. The coated silk fibres supported the attachment and growth of hMSCs.

In this study, we constructed tissue-engineered cartilage using allogeneic cancellous bone matrix gelatin (BMG) as a scaffold. Allogeneic BMG was prepared by sequential defatting, demineralization and denaturation. Isolated rabbit chondrocytes were seeded onto allogeneic cancellous BMG, and cell–BMG constructs were harvested after 1, 3 and 6 weeks for evaluation by hematoxylin and eosin staining for overall morphology, toluidine blue for extracellular matrix (ECM) proteoglycans, immunohistochemical staining for collagen type II and a transmission electron microscope for examining cellular microstructure on BMG. The prepared BMG was highly porous with mechanical strength adjustable by duration of demineralization and was easily trimmed for tissue repair. Cancellous BMG showed favorable porosity for cell habitation and metabolism material exchange with larger pore sizes (100–500 µm) than in cortical BMG (5–15 µm), allowing cell penetration. Cancellous BMG also showed good biocompatibility, which supported chondrocyte proliferation and sustained their differentiated phenotype in culture for up to 6 weeks. Rich and evenly distributed cartilage ECM proteoglycans and collagen type II were observed around chondrocytes on the surface and inside the pores throughout the cancellous BMG. Considering the large supply of banked bone allografts and relatively convenient preparation, our study suggests that allogeneic cancellous BMG is a promising scaffold for cartilage tissue engineering.

Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) fibers containing carbonated hydroxyapatite (CHA) nanoparticles with different CHA amounts (5, 10 and 15 wt%) were electrospun with the aid of ultrasonic power for dispersing the nanoparticles. Scanning electron microscopy and energy-dispersive x-ray spectroscopy results showed that the distribution of CHA within the CHA/PHBV nanocomposite fibers was homogeneous when the CHA content was 10 wt%. Slight particle agglomeration occurred when the CHA content was 15 wt%. The diameters of the electrospun CHA/PHBV nanocomposite fibers and PHBV polymer fibers were around 3 µm. Fourier transform infrared spectroscopic analysis further confirmed the presence of CHA in CHA/PHBV nanocomposite fibers. Both PHBV and CHA/PHBV fibrous membranes exhibited similar tensile properties. Compared with PHBV solvent-cast film, the PHBV fibrous membrane was hydrophobic but the incorporation of CHA nanoparticles dramatically enhanced its wettability. In vitro studies revealed that both types of electrospun fibrous membranes (PHBV and CHA/PHBV) supported the proliferation of human osteoblastic cells (SaOS-2). The alkaline phosphatase activity of SaOS-2 cells seeded on the CHA/PHBV fibrous membranes was higher than that of the cells seeded on the PHBV fibrous membranes after 14 days of cell culture. The electrospun CHA/PHBV nanocomposite fibrous membranes show promises for bone tissue engineering applications.

Bone tissue engineering has been one of the most promising areas of research, providing a potential clinical application to cure bone defects. Recently, various stem cells including embryonic stem cells (ESCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), adipose tissue-derived stem cells (ADSCs), muscle-derived stem cells (MDSCs) and dental pulp stem cells (DPSCs) have received extensive attention in the field of bone tissue engineering due to their distinct biological capability to differentiate into osteogenic lineages. The application of these stem cells to bone tissue engineering requires inducing in vitro differentiation of these cells into bone forming cells, osteoblasts. For this purpose, efficient in vitro differentiation towards osteogenic lineage requires the development of well-defined and proficient protocols. This would reduce the likelihood of spontaneous differentiation into divergent lineages and increase the available cell source for application to bone tissue engineering therapies. This review provides a critical examination of the various experimental strategies that could be used to direct the differentiation of ESC, BM-MSC, UCB-MSC, ADSC, MDSC and DPSC towards osteogenic lineages and their potential applications in tissue engineering, particularly in the regeneration of bone.

Engineering dermal substitutes with electrospun nanofibres have lately been of prime importance for skin tissue regeneration. Simple electrospinning technology served to produce nanofibrous scaffolds morphologically and structurally similar to the extracellular matrix of native tissues. The nanofibrous scaffolds of poly(l-lactic acid)-co-poly(ε-caprolactone) (PLACL) and PLACL/gelatin complexes were fabricated by the electrospinning process. These nanofibres were characterized for fibre morphology, membrane porosity, wettability and chemical properties by FTIR analysis to culture human foreskin fibroblasts for skin tissue engineering. The nanofibre diameter was obtained between 282 and 761 nm for PLACL and PLACL/gelatin scaffolds; expressions of amino and carboxyl groups and porosity up to 87% were obtained for these fibres, while they also exhibited improved hydrophilic properties after plasma treatment. The results showed that fibroblasts proliferation, morphology, CMFDA dye expression and secretion of collagen were significantly increased in plasma-treated PLACL/gelatin scaffolds compared to PLACL nanofibrous scaffolds. The obtained results prove that the plasma-treated PLACL/gelatin nanofibrous scaffold is a potential biocomposite material for skin tissue regeneration.

Stem cell-based tissue engineering has provided an alternative strategy to treat cartilage lesions, and synovium-derived mesenchymal stem cells (SMSCs) are considered as a promising cell source for cartilage repair. In this study, the SMSCs were isolated from rat synovium, and CD105-positive (CD105⁺) cells were enriched using magnetic activated cell sorting. Sorted cells were subsequently seeded onto the chitosan–alginate composite three-dimensional (3D) porous scaffolds and cultured in chondrogenic culture medium in the presence of TGF-β₃ and BMP-2 for 2 weeks in vitro. After 2 weeks in culture, scanning electron microscopy results showed that cells attached and proliferated well on scaffolds, and secreted extracellular matrix were also observed. From day 7 to day 14, the total DNA and glucosaminoglycan content of the cells cultured in scaffolds were found to have increased significantly, and cell cycle analyses revealed that the percentage of cells in the S and G2/M phases increased and the percentage of cells in the G0/G1 phase decreased. Compared with non-sorted cells, the sorted cells cultured in scaffolds underwent more chondrogenic differentiation, as evidenced by higher expression of type II collagen and Sox9 at the protein and mRNA levels. The results suggest that CD105⁺ enriched SMSCs may be a potential cell source for cartilage tissue engineering, and the chitosan–alginate composite 3D porous scaffold could provide a favorable microenvironment for supporting proliferation and chondrogenic differentiation of cells.