Injectable remodeling hydrogels derived from alendronate-tethered alginate calcium complex for enhanced osteogenesis.

A combination of hydrogel materials, and therapeutic agents have been actively reported to facilitate bone defect healing. However, conventionally hydrogels using cross-linker would result in low stability of the hydrogel itself, loss of agents during cross-linking, and complexity of use. In this study, alendronate was tethered to an AlA to improve its bone healing and drug-loading stability. AlA was further functionalized with Ca2+ (AlACa). A mixture of AlACa and alginate formed AlAA hydrogel. The gelation time of AlAA was sufficient for injecting into the defect site. The hydrogel stiffness was controlled, while the stress-relaxation time was fixed. In vitro cell tests demonstrated that the AlAA promoted proliferation and differentiation behaviors. In particular, AlAA showed the best mechanical stiffness with appropriate stress-relaxation and cellular behavior, indicating that it would be beneficial as a scaffold in the bone tissue engineering field.

Stable sol-gel hydroxyapatite coating on zirconia dental implant for improved osseointegration.

Aside from being known for its excellent mechanical properties and aesthetic effect, zirconia has recently attracted attention as a new dental implant material. Many studies have focused on hydroxyapatite (HA) coating for obtaining improved biocompatibility, however the coating stability was reduced by a byproduct produced during the high-temperature sintering process. In this study, to overcome this problem, we simply coated the zirconia surface with a sol-gel-derived hydroxyapatite (HA) layer and then sintered it at a varied temperature (<1000 °C). The surface showed a nanoporous structure, and there was no crystalline phase other than HA and zirconia when the sintering temperature was 800 °C. The adhesion strength of the HA layer (>40 MPa) was also appropriate as a dental implant application. In addition, in vitro cell experiments using a preosteoblast cell line revealed that the HA-coated zirconia surface acts as a preferable surface for cell attachment and proliferation than bare zirconia surface. In vivo animal experiments also demonstrated that the osteoconductivity of zirconia were dramatically enhanced by HA coating, which was comparable to that of Ti implant. These results suggest that the sol-gel-based HA-coated zirconia has a great potential for use as a dental implant material.

PLLA Membrane with Embedded Hydroxyapatite Patterns for Improved Bioactivity and Efficient Delivery of Growth Factor.

Poly(l-lactic) acid (PLLA) is widely used in guided bone regeneration membranes due to its mechanical properties and biodegradability. However, the lack of biocompatibility is a serious disadvantage. Herein, the biocompatibility of PLLA is improved by patterning hydroxyapatite (HA) loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) under it. The HA is obtained by preparing a magnesium pattern via photolithography and hydrothermal converting. After loading rhBMP-2, the pattern is transferred to PLLA. The pattern is tightly embedded in the PLLA and retained its original position after mechanical stimuli. Fluorescence images allow to assess the protein adsorption and gradual release in a controlled manner. The amount of released rhBMP-2 is overwhelmingly large when loaded under HA because of its large surface area. Osteogenic differentiation supports the synergistic effect of HA and rhBMP-2 to improve the biocompatibility. Moreover, in vivo experiments demonstrate that the synergistic effect positively affects the healing rate of bone.

Tantalum-coated polylactic acid fibrous membranes for guided bone regeneration.

Guided bone regeneration (GBR) membrane is necessary to reconstruct the defect bone tissue by defending penetration of soft tissues. Polylactic acid (PLA) attracts much attention to utilize as a GBR membrane because it has relatively high mechanical strength and biodegradability. However, the poor osteoconductivity of PLA is a major concern. The aim of this study is to improve the osteoconductivity of fibrous, electrospun, PLA guided bone regeneration membranes by coating the fiber surface with highly biocompatible tantalum (Ta). Ta coating of electrospun PLA membrane was created through sputtered Ta ions surrounding the PLA fibers. The Ta-coated PLA (Ta-PLA) membranes remain a randomly aligned fibrous structure with no defects caused by sputtering. The chemical composition of Ta-PLA membrane indicates Ta coating was well deposited on PLA fibers. Although the mechanical strength of Ta-PLA was reduced compared with bare PLA membrane, the Ta coating layer does not readily delaminate from the single PLA fiber surface due to its cladded structure which indicates that the Ta coating has high mechanical stability on PLA fibers. In vitro cell tests demonstrate that the attachment, proliferation, and differentiation of preosteoblasts are significantly promoted on the Ta-PLA membranes compared to bare PLA. In an in vivo animal test, most calvarial defects in the Ta-PLA group are covered with newly formed bone within six weeks, while the defects in the bare PLA group are rarely covered. Furthermore, the degree of bone healing in the Ta-PLA group is comparable to healing observed on collagen membranes, which are highly bioactive materials. These results indicate the superior osteoconductivity of Ta-PLA will make it particularly useful as a guided bone regeneration membrane.

Bioactive and mechanically stable hydroxyapatite patterning for rapid endothelialization of artificial vascular graft.

Polymeric vascular grafts have been widely used in the vascular regeneration field because of their ease of application. However, synthetic polymer grafts have the severe problem of low biocompatibility, which may cause delayed endothelialization and hyperplasia. In this study, we fabricated a linear hydroxyapatite (HA) pattern on a silicon wafer and then transferred the pattern to a poly(L-lactic)-acid (PLLA) film for use as a tubular vascular graft. The HA pattern with its characteristic needle-like shape was successfully embedded into the PLLA. The HA-patterned PLLA film exhibited superior mechanical stability compared with that of a HA-coated PLLA film under bending, elongation, and in vitro circulation conditions, suggesting its suitability for use as a tubular vascular graft. In addition, the HA pattern guided rapid endothelialization by promoting proliferation of endothelial cells and their migration along the pattern. The hemocompatibility of the HA-patterned PLLA was also confirmed, with substantially fewer platelets adhered on its surface. Overall, in addition to good mechanical stability, the HA-patterned PLLA exhibited enhanced biocompatibility and hemocompatibility compared with pure PLLA.

Evaluations of miniscrew type-dependent mechanical stability.

BACKGROUND: Miniscrew has been widely used as an absolute anchorage in orthodontic treatment. Types of miniscrew with different diameter, length, shape, and thread dimensions may have a substantial effect on mechanical stability of the miniscrew system. Thus, the objective of this study was to evaluate miniscrew type-dependent mechanical stability to assess mechanical properties of miniscrew systems in various thickness of artificial bone block using different measurement tools. METHODS: Two types of miniscrews (15 Tomas and 15 AbsoAnchor) were placed in artificial bone block with different thickness of 1.5, 2.0, 3.0 mm. Values of maximum insertion torque, removal torque, Periotest, implant stability quotient, static stiffness, dynamic stiffness, and energy dissipation ability were assessed for each miniscrew system. FINDINGS: The maximum insertion torque, removal torque, implant stability quotient, static and dynamic stiffness values significantly increased when the miniscrews were placed in thicker bone block while Periotest values decreased. The static stiffness, Periotest and implant stability quotient values were significantly correlated each other and also with other mechanical properties (p < 0.001) except tan δ (p > 0.35). However, the slopes of some correlations and absolute values of measurement were significantly different dependent on the miniscrew types (p < 0.025). INTERPRETATION: The current findings suggest that miniscrew type-dependent calibrations are required to estimate mechanical stability of the miniscrew systems despite the utilization of same measurement tool.

Synergistically enhanced osteoconductivity and anti-inflammation of PLGA/ß-TCP/Mg(OH)2 composite for orthopedic applications.

Synthetic biodegradable polymers including poly(lactide-co-glycolide) (PLGA) have been widely used as alternatives to metallic implantable materials in the orthopedic field due to their superior biocompatibility and biodegradability. However, weak mechanical properties of the biodegradable polymers and inflammatory reaction caused by the acidic degradation products have limited their biomedical applications. In this study, we have developed a PLGA composite containing beta-tricalcium phosphate (ß-TCP) and magnesium hydroxide (Mg(OH)2) as additives to improve mechanical, osteoconductivity, and anti-inflammation property of the biopolymer composite simultaneously. The ß-TCP has an osteoconductive effect and the Mg(OH)2 has a pH neutralizing effect. The PLGA/inorganic composites were uniformly blended via a twin extrusion process. The mechanical property of the PLGA/ß-TCP/Mg(OH)2 composite was improved compared to the pure PLGA. In particular, the addition of Mg(OH)2 suppressed the inflammatory reaction of normal human osteoblast (NHOst) cells and also inhibited the differentiation of pre-osteoclastic cells into osteoclasts. Moreover, synergistically upregulated late osteogenic differentiation of NHOst cells was observed on the PLGA/ß-TCP/Mg(OH)2 composite. Taken all together, we believe that the use of ß-TCP and Mg(OH)2 as additives with synthetic biodegradable polymers has great potential by the synergistic effect in orthopedic applications.

The RhoGAP Myo9b Promotes Bone Growth by Mediating Osteoblastic Responsiveness to IGF-1.

The Ras homolog A (RhoA) subfamily of Rho guanosine triphosphatases (GTPases) regulates actin-based cellular functions in bone such as differentiation, migration, and mechanotransduction. Polymorphisms or genetic ablation of RHOA and some of its regulatory guanine exchange factors (GEFs) have been linked to poor bone health in humans and mice, but the effects of RhoA-specific GTPase-activating proteins (GAPs) on bone quality have not yet been identified. Therefore, we examined the consequences of RhoGAP Myo9b gene knockout on bone growth, phenotype, and cellular activity. Male and female mice lacking both alleles demonstrated growth retardation and decreased bone formation rates during early puberty. These mice had smaller, weaker bones by 4 weeks of age, but only female KOs had altered cellular numbers, with fewer osteoblasts and more osteoclasts. By 12 weeks of age, bone quality in KOs worsened. In contrast, 4-week-old heterozygotes demonstrated bone defects that resolved by 12 weeks of age. Throughout, Myo9b ablation affected females more than males. Osteoclast activity appeared unaffected. In primary osteogenic cells, Myo9b was distributed in stress fibers and focal adhesions, and its absence resulted in poor spreading and eventual detachment from culture dishes. Similarly, MC3T3-E1 preosteoblasts with transiently suppressed Myo9b levels spread poorly and contained decreased numbers of focal adhesions. These cells also demonstrated reduced ability to undergo IGF-1-induced spreading or chemotaxis toward IGF-1, though responses to PDGF and BMP-2 were unaffected. IGF-1 receptor (IGF1R) activation was normal in cells with diminished Myo9b levels, but the activated receptor was redistributed from stress fibers and focal adhesions into nuclei, potentially affecting receptor accessibility and gene expression. These results demonstrate that Myo9b regulates a subset of RhoA-activated processes necessary for IGF-1 responsiveness in osteogenic cells, and is critical for normal bone formation in growing mice. © 2017 American Society for Bone and Mineral Research.

Facile in situ formation of hybrid gels for direct-forming tissue engineering.

Development of bioactive hydrogel as extracellular matrix (ECM) is a very important field for cell-based therapy. In this study, we provided a facile method based on sol-gel process for fabricating bioactive composite hydrogels. The composite hydrogels were composed of sol-gel derived silica and biopolymer. Different amounts of silica solution (20-80wt%) were mixed with 2% polymer sol (alginate) followed by aging and gelation to form a network so that the alginate-silica hybrid mixture could form a gel without any additional crosslinking process. The self-gelation time of the hybrid hydrogel measured by rheometer was reduced as the content of silica was increased. Such hydrogels had highly porous and interconnected structures. Their strut showed uniform surface texture. Under physiological conditions (PBS, 37°C), these hybrid hydrogels exhibited long-term stability compared to alginate hydrogels as control. The mechanical properties of these hydrogels such as compressive strength, compressive modulus, and work of fracture were significantly enhanced by hybridization with sol-gel derived silica. In vitro cell tests revealed that these hybrid hydrogels exhibited improved cell adhesion and proliferation behaviors compared to pure alginate hydrogel cross-linked by CaCl2 solution. Furthermore, cell encapsulation within these hydrogels revealed that their alginate-silica composite provided suitable microenvironment for cell survival.

Fibronectin immobilization on to robotic-dispensed nanobioactive glass/polycaprolactone scaffolds for bone tissue engineering.

Bioactive nanocomposite scaffolds with cell-adhesive surface have excellent bone regeneration capacities. Fibronectin (FN)-immobilized nanobioactive glass (nBG)/polycaprolactone (PCL) (FN-nBG/PCL) scaffolds with an open pore architecture were generated by a robotic-dispensing technique. The surface immobilization level of FN was significantly higher on the nBG/PCL scaffolds than on the PCL scaffolds, mainly due to the incorporated nBG that provided hydrophilic chemical-linking sites. FN-nBG/PCL scaffolds significantly improved cell responses, including initial anchorage and subsequent cell proliferation. Although further in-depth studies on cell differentiation and the in vivo animal responses are required, bioactive nanocomposite scaffolds with cell-favoring surface are considered to provide promising three-dimensional substrate for bone regeneration.

Nanostructured biointerfacing of metals with carbon nanotube/chitosan hybrids by electrodeposition for cell stimulation and therapeutics delivery.

Exploring the biological interfaces of metallic implants has been an important issue in achieving biofunctional success. Here we develop a biointerface with nanotopological features and bioactive composition, comprising a carbon nanotube (CNT) and chitosan (Chi) hybrid, via an electrophoretic deposition (EPD). The physicochemical properties, in vitro biocompatibility, and protein delivering capacity of the decorated nanohybrid layer were investigated, to address its potential usefulness as bone regenerating implants. Over a wide compositional range, the nanostructured hybrid interfaces were successfully formed with varying thicknesses, depending on the electrodeposition parameters. CNT-Chi hybrid interfaces showed a time-sequenced degradation in saline water, and a rapid induction of hydroxyapatite mineral in a simulated body fluid. The nanostructured hybrid substrates stimulated the initial adhesion events of the osteoblastic cells, including cell adhesion rate, spreading behaviors, and expression of adhesive proteins. The nanostructured hybrid interfaces significantly improved the adsorption of protein molecules, which was enabled by the surface charge interaction, and increased surface area of the nanotopology. Furthermore, the incorporated protein was released at a highly sustained rate, profiling a diffusion-controlled pattern over a couple of weeks, suggesting the possible usefulness as a protein delivery device. Collectively, the nanostructured hybrid CNT-Chi layer, implemented by an electrodeposition, is considered a biocompatible, cell-stimulating, and protein-delivering biointerface of metallic implants.

Biointerface control of electrospun fiber scaffolds for bone regeneration: engineered protein link to mineralized surface.

Control over the interface of biomaterials that favors the initial adhesion and subsequent differentiation of stem cells is one of the key strategies in bone tissue engineering. Here we engineer the interface of biopolymer electrospun fiber matrices with a fusion protein of fibronectin 9-10 domain (FNIII9-10) and osteocalcin (OCN), aiming to stimulate mesenchymal stem cell (MSC) functions, including initial adhesion, growth and osteogenic differentiation. In particular, a specific tethering of FNIII9-10-OCN protein was facilitated by the hydroxyapatite (HA) mineralization of the biopolymer surface through a molecular recognition of OCN to the HA crystal lattice. The FNIII9-10-OCN anchorage to the HA-mineralized fiber was observed to be highly specific and tightly bound to preserve stability over a long period. Initial cell adhesion levels, as well as the spreading shape and process, of MSCs within 24h were strikingly different between the fibers linked with and without fusion protein. Significant up-regulations in the mRNA expression of adhesion signaling molecules occurred with the fusion protein link, as analyzed by the reverse transcriptase polymerase chain reaction. The expression of a series of osteogenic-related genes at later stages, over 2-3weeks, was significantly improved in the fusion protein-tailored fiber, and the osteogenic protein levels were highly stimulated, as confirmed by immunofluorescence imaging and fluorescence-activated cell sorting analyses. In vivo study in a rat calvarium model confirmed a higher quantity of new bone formation in the fiber linked with fusion protein, and a further increase was noticed when the MSCs were tissue-engineered with the fusion protein-linked fiber. Collectively, these results indicate that FN-OCN fusion protein links via HA mineralization is a facile tool to generate a biointerface with cell-attractive and osteogenic potential, and that the engineered fibrous matrix is a potential bone regenerative scaffold.

Creation of nanoporous TiO2 surface onto polyetheretherketone for effective immobilization and delivery of bone morphogenetic protein.

This study evaluated the utility of the creation of a nanoporous TiO2 surface to enhance the in vitro biocompatibility and in vivo osseoconductivity of polyetheretherketone (PEEK) implants by providing favorable sites for the effective immobilization of bone morphogenetic protein-2 (BMP-2). A uniform nanoporous TiO2 layer with a pore diameter of ∼70 nm was successfully created by anodizing a Ti film, which had been deposited onto a PEEK substrate via electron beam (e-beam) evaporation technique. This nanoporous, hydrophilic TiO2 surface enabled the efficient immobilization of BMP-2, resulting in a remarkable enhancement in in vitro biocompatibility that was assessed in terms of cell attachment, proliferation, and differentiation. The in vivo animal tests also confirmed that the nanoporous TiO2 surface immobilized with BMP-2 could significantly enhance the osseoconductivity of PEEK implants. The BMP-immobilized PEEK implant with the nanoporous TiO2 surface showed much higher bone-to-implant contact (BIC) ratio (60%) than the bare PEEK (30%), PEEK with the nanoporous TiO2 surface (50%) and even BMP-immobilized PEEK without the nanoporous TiO2 surface (32%).

Gelatin-apatite bone mimetic co-precipitates incorporated within biopolymer matrix to improve mechanical and biological properties useful for hard tissue repair.

Synthetic biopolymers are commonly used for the repair and regeneration of damaged tissues. Specifically targeting bone, the composite approach of utilizing inorganic components is considered promising in terms of improving mechanical and biological properties. We developed gelatin-apatite co-precipitates which mimic the native bone matrix composition within poly(lactide-co-caprolactone) (PLCL). Ionic reaction of calcium and phosphate with gelatin molecules enabled the co-precipitate formation of gelatin-apatite nanocrystals at varying ratios. The gelatin-apatite precipitates formed were carbonated apatite in nature, and were homogeneously distributed within the gelatin matrix. The incorporation of gelatin-apatite significantly improved the mechanical properties, including tensile strength, elastic modulus and elongation at break, and the improvement was more pronounced as the apatite content increased. Of note, the tensile strength increased to as high as 45 MPa (a four-fold increase vs. PLCL), the elastic modulus was increased up to 1500 MPa (a five-fold increase vs. PLCL), and the elongation rate was ~240% (twice vs. PLCL). These results support the strengthening role of the gelatin-apatite precipitates within PLCL. The gelatin-apatite addition considerably enhanced the water affinity and the acellular mineral-forming ability in vitro in simulated body fluid; moreover, it stimulated cell proliferation and osteogenic differentiation. Taken together, the GAp-PLCL nanocomposite composition is considered to have excellent mechanical and biological properties, which hold great potential for use as bone regenerative matrices.

Highly aligned porous Ti scaffold coated with bone morphogenetic protein-loaded silica/chitosan hybrid for enhanced bone regeneration.

Porous Ti has been widely investigated for orthopedic and dental applications on account of their ability to promote implant fixation via bone ingrowth into pores. In this study, highly aligned porous Ti scaffolds coated with a bone morphogenetic protein (BMP)-loaded silica/chitosan hybrid were produced, and their bone regeneration ability was evaluated by in vivo animal experiments. Reverse freeze casting allowed for the creation of highly aligned pores, resulting in a high compressive strength of 254 ± 21 MPa of the scaffolds at a porosity level of ∼51 vol %. In addition, a BMP-loaded silica/chitosan hybrid coating layer with a thickness of ∼1 µm was uniformly deposited on the porous Ti scaffold, which enabled the sustained release of the BMP over a prolonged period of time up to 26 days. The cumulative amount of the BMP released was ∼4 µg, which was much higher than that released from the specimen without a hybrid coating layer. In addition, the bone regeneration ability of the porous Ti scaffold with a BMP-loaded silica/chitosan coating layer was examined by in vivo animal testing using a rabbit calvarial defect model and compared with those of the as-produced porous Ti scaffold and porous Ti scaffold with a silica/chitosan coating layer. After 4 weeks of healing, the specimen coated with a BMP-loaded silica/chitosan hybrid showed a much higher bone regeneration volume (∼36%) than the as-produced specimen (∼15%) (p < 0.005) and even the specimen coated with a silica/chitosan hybrid (∼25%) (p < 0.05).

The electron beam deposition of titanium on polyetheretherketone (PEEK) and the resulting enhanced biological properties.

The surface of polyetheretherketone (PEEK) was coated with a pure titanium (Ti) layer using an electron beam (e-beam) deposition method in order to enhance its biocompatibility and adhesion to bone tissue. The e-beam deposition method was a low-temperature coating process that formed a dense, uniform and well crystallized Ti layer without deteriorating the characteristics of the PEEK implant. The Ti coating layer strongly adhered to the substrate and remarkably enhanced its wettability. The Ti-coated samples were evaluated in terms of their in vitro cellular behaviors and in vivo osteointegration, and the results were compared to a pure PEEK substrate. The level of proliferation of the cells (MC3T3-E1) was measured using a methoxyphenyl tetrazolium salt (MTS) assay and more than doubled after the Ti coating. The differentiation level of cells was measured using the alkaline phosphatase (ALP) assay and also doubled. Furthermore, the in vivo animal tests showed that the Ti-coated PEEK implants had a much higher bone-in-contact (BIC) ratio than the pure PEEK implants. These in vitro and in vivo results suggested that the e-beam deposited Ti coating significantly improved the potential of PEEK for hard tissue applications.

Functionally gradient chitosan/hydroxyapatite composite scaffolds for controlled drug release.

This study explored the potential of chitosan/hydroxyapatite (HA) composites to act as a controlled drug delivery system by developing functional scaffolds with a gradient of structure and drug concentration. Firstly, a porous composite scaffold was prepared and tetracycline hydrochloride (TCH) was impregnated in the scaffold as a model drug. The pore size of the scaffold was negatively dependent on the HA content and ranged about 40-250 microm. Subsequently, a porous chitosan/HA composite layer without drug was coated on the scaffold to create a gradient drug concentration in the specimen. The in vitro drug-release test demonstrated that the porous layer without drug on the outer surface of the scaffold significantly reduced the initial burst of drug release and extended the release period. Finally, a successive and dense chitosan/HA composite layer endowed the scaffold with a sustained, drug-release pattern without any initial drug burst. These findings confirmed the high effectiveness of the hybrid scaffolds in regulating the release of drugs, and hence their capability to serve as a temporary drug carrier in tissue regeneration. These functional scaffolds also have potential application to the delivery of some bioactive molecules such as growth factors.

Enhanced biocompatibility of Co-Cr implant material by Ti coating and micro-arc oxidation.

The biocompatibility of Co-Cr alloy was significantly improved by forming a rough TiO(2) layer on its surface. The TiO(2) layer was formed by coating the Co-Cr alloy with titanium (Ti) through electron beam deposition followed by microarc oxidation (MAO). When Ti was coated on the surface, the biocompatibility of the Co-Cr alloy was enhanced and it was further improved by the MAO treatment. There were close relationships between the phase, morphology, and thickness of the TiO(2) layer and the applied voltage. The biocompatibility of the specimens coated with Ti and subjected to MAO treatment was evaluated by in vitro alkaline phosphatase activity tests.