Combining a Vascular Bundle and 3D Printed Scaffold with BMP-2 Improves Bone Repair and Angiogenesis.

Vascularization is currently considered the biggest challenge in bone tissue engineering due to necrosis in the center of large scaffolds. We established a new expendable vascular bundle model to vascularize a three-dimensional printed channeled scaffold with and without bone morphogenetic protein-2 (BMP-2) for improved healing of large segmental bone defects. Bone formation and angiogenesis in an 8 mm critical-sized bone defect in the rat femur were significantly promoted by inserting a bundle consisting of the superficial epigastric artery and vein into the central channel of a large porous polycaprolactone scaffold. Vessels were observed sprouting from the vascular bundle inserted in the central tunnel. Although the regenerated bone volume in the group receiving the scaffold and vascular bundle was similar to that of the healthy femur, the rate of union of the group was not satisfactory (25% at 8 weeks). BMP-2 delivery was found to promote not only bone formation but also angiogenesis in the critical-sized bone defects. Both insertion of the vascular bundle alone and BMP-2 loading alone induced comparable levels of angiogenesis and when used in combination, significantly greater vascular volume was observed. These findings suggest a promising new modality of treatment in large bone defects. Level of Evidence: Therapeutic level I. Impact statement Vascularization is currently the main challenge in bone tissue engineering. The combination of a vascular bundle and an osteoinductive three-dimensional printed graft significantly improved and accelerated bone regeneration and angiogenesis in critical-sized large bone defects, suggesting a promising new modality of treatment in large bone defects.

Effect of Zinc Oxide Nanoparticle Addition to Polycaprolactone Periodontal Membranes on Antibacterial Activity and Cell Viability.

During the design of membranes for guided tissue regeneration (GTR) to treat periodontal diseases, infection of the exposed membranes and postoperative complications can be prevented by increasing bacterial resistance. This study evaluated the antibacterial activity of PCL/ZnO membranes and their effect on cell viability via addition of antibacterial zinc oxide (ZnO) nanoparticles to a biocompatible and biodegradable material such as polycaprolactone (PCL). Neat PCL membranes and PCL/ZnO membranes containing 0.5 wt.% and 5 wt.% ZnO were produced, and divided into PCL (0% ZnO), LZ (0.5 wt.% ZnO), and HZ (5 wt.% ZnO) groups, respectively. The surface characteristics of the membranes including morphological features and changes in composition were analyzed. Adhesion of bacteria, including Streptococcus mutans and Porphyromonas gingi-valis, was analyzed using a crystal violet assay. The proliferation of MC3T3-E1 osteoblasts was evaluated using a WST-8 assay. Significant differences were analyzed using the Kruskal-Wallis test (P < 0.05). The results of groups were compared using the Mann-Whitney test (P < 0.017). ZnO nanoparticles were dispersed in the PCL matrix of PCL/ZnO membranes. Compared with neat PCL membranes, their ability to form crystals decreased and their amorphous structure increased. The adhesion of S. mutans and P. gingivalis in the LZ and HZ groups containing ZnO was significantly decreased compared with that of the neat PCL membranes (P < 0.05). No significant differences were observed in the proliferation of MC3T3-E1 cells between the PCL/ZnO membranes and the neat PCL membranes both on days 2 and 5 of culture (P > 0.05). This study has demonstrated that the PCL membranes carrying the ZnO nanoparticles inhibited bacterial adhesion without affecting the viability of osteoblasts, suggesting the potential application of ZnO in GTR to increase antibacterial activity of membranes.

Vascularized Bone-Mimetic Hydrogel Constructs by 3D Bioprinting to Promote Osteogenesis and Angiogenesis.

Bone is a highly vascularized tissue with a unique and complex structure. Long bone consists of a peripheral cortical shell containing a network of channels for vascular penetration and an inner highly vascularized bone marrow space. Bioprinting is a powerful tool to enable rapid and precise spatial patterning of cells and biomaterials. Here we developed a two-step digital light processing technique to fabricate a bone-mimetic 3D hydrogel construct based on octacalcium phosphate (OCP), spheroids of human umbilical vein endothelial cells (HUVEC), and gelatin methacrylate (GelMA) hydrogels. The bone-mimetic 3D hydrogel construct was designed to consist of a peripheral OCP-containing GelMA ring to mimic the cortical shell, and a central GelMA ring containing HUVEC spheroids to mimic the bone marrow space. We further demonstrate that OCP, which is evenly embedded in the GelMA, stimulates the osteoblastic differentiation of mesenchymal stem cells. We refined the design of a spheroid culture device to facilitate the rapid formation of a large number of HUVEC spheroids, which were embedded into different concentrations of GelMA hydrogels. It is shown that the concentration of GelMA modulates the extent of formation of the capillary-like structures originating from the HUVEC spheroids. This cell-loaded hydrogel-based bone construct with a biomimetic dual ring structure can be potentially used for bone tissue engineering.

Preclinical induced membrane model to evaluate synthetic implants for healing critical bone defects without autograft.

Critical bone defects pose a formidable orthopaedic problem in patients with bone loss. We developed a preclinical model based on the induced membrane technique using a synthetic graft to replace autograft for healing critical bone defects. Additionally, we used a novel osteoconductive scaffold coupled with a synthetic membrane to evaluate the potential for single-stage bone regeneration. Three experimental conditions were investigated in critical femoral defects in rats. Group A underwent a two-stage procedure with insertion of a polymethylmethacrylate (PMMA) spacer followed by replacement with a 3D printed polycaprolactone(PCL)/ß-tricalcium phosphate (ß-TCP) osteoconductive scaffold after 4 weeks. Group B received a single-stage PCL/ß-TCP scaffold wrapped in a PCL-based microporous polymer film creating a synthetic membrane. Group C received a single-stage bare PCL/ß-TCP scaffold. All groups were examined by serial radiographs for callus formation. After 12 weeks, the femurs were explanted and analyzed with micro-CT and histology. Mean callus scores tended to be higher in Group A. Group A showed statistically significant greater bone formation on micro-CT compared with other groups, although bone volume fraction was similar between groups. Histology results suggested extensive bone ingrowth and new bone formation within the macroporous scaffolds in all groups and cell infiltration into the microporous synthetic membrane. This study supports the use of a critical size femoral defect in rats as a suitable model for investigating modifications to the induced membrane technique without autograft harvest. Future investigations should focus on bioactive synthetic membranes coupled with growth factors for single-stage bone healing. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Systematic characterization of 3D-printed PCL/ß-TCP scaffolds for biomedical devices and bone tissue engineering: influence of composition and porosity.

This work aims at providing guidance through systematic experimental characterization, for the design of 3D printed scaffolds for potential orthopaedic applications, focusing on fused deposition modeling (FDM) with a composite of clinically available polycaprolactone (PCL) and ß-tricalcium phosphate (ß-TCP). First, we studied the effect of the chemical composition (0% to 60% ß-TCP/PCL) on the scaffold's properties. We showed that surface roughness and contact angle were respectively proportional and inversely proportional to the amount of ß-TCP, and that degradation rate increased with the amount of ceramic. Biologically, the addition of ß-TCP enhanced proliferation and osteogenic differentiation of C3H10. Secondly, we systematically investigated the effect of the composition and the porosity on the 3D printed scaffold mechanical properties. Both an increasing amount of ß-TCP and a decreasing porosity augmented the apparent Young's modulus of the 3D printed scaffolds. Third, as a proof-of-concept, a novel multi-material biomimetic implant was designed and fabricated for potential disk replacement.

Tunable Elastomers with an Antithrombotic Component for Cardiovascular Applications.

This study reports the development of a novel family of biodegradable polyurethanes for use as tissue engineered cardiovascular scaffolds or blood-contacting medical devices. Covalent incorporation of the antiplatelet agent dipyridamole into biodegradable polycaprolactone-based polyurethanes yields biocompatible materials with improved thromboresistance and tunable mechanical strength and elasticity. Altering the ratio of the dipyridamole to the diisocyanate linking unit and the polycaprolactone macromer enables control over both the drug content and the polymer cross-link density. Covalent cross-linking in the materials achieves significant elasticity and a tunable range of elastic moduli similar to that of native cardiovascular tissues. Interestingly, the cross-link density of the polyurethanes is inversely related to the elastic modulus, an effect attributed to decreasing crystallinity in the more cross-linked polymers. In vitro characterization shows that the antiplatelet agent is homogeneously distributed in the materials and is released slowly throughout the polymer degradation process. The drug-containing polyurethanes support endothelial cell and vascular smooth muscle cell proliferation, while demonstrating reduced levels of platelet adhesion and activation, supporting their candidacy as promising substrates for cardiovascular tissue engineering.

Synthesis and characterization of polycaprolactone urethane hollow fiber membranes as small diameter vascular grafts.

The design of bioresorbable synthetic small diameter (<6mm) vascular grafts (SDVGs) capable of sustaining long-term patency and endothelialization is a daunting challenge in vascular tissue engineering. Here, we synthesized a family of biocompatible and biodegradable polycaprolactone (PCL) urethane macromers to fabricate hollow fiber membranes (HFMs) as SDVG candidates, and characterized their mechanical properties, degradability, hemocompatibility, and endothelial development. The HFMs had smooth surfaces and porous internal structures. Their tensile stiffness ranged from 0.09 to 0.11N/mm and their maximum tensile force from 0.86 to 1.03N, with minimum failure strains of approximately 130%. Permeability varied from 1 to 14×10(-6)cm/s, burst pressures from 1158 to 1468mmHg, and compliance from 0.52 to 1.48%/100mmHg. The suture retention forces ranged from 0.55 to 0.81N. HFMs had slow degradation profiles, with 15 to 30% degradation after 8weeks. Human endothelial cells proliferated well on the HFMs, creating stable cell layer coverage. Hemocompatibility studies demonstrated low hemolysis (<2%), platelet activation, and protein adsorption. There were no significant differences in the hemocompatibility of HFMs in the absence and presence of endothelial layers. These encouraging results suggest great promise of our newly developed materials and biodegradable elastomeric HFMs as SDVG candidates.

Vascularization in bone tissue engineering constructs.

Vascularization of large bone grafts is one of the main challenges of bone tissue engineering (BTE), and has held back the clinical translation of engineered bone constructs for two decades so far. The ultimate goal of vascularized BTE constructs is to provide a bone environment rich in functional vascular networks to achieve efficient osseointegration and accelerate restoration of function after implantation. To attain both structural and vascular integration of the grafts, a large number of biomaterials, cells, and biological cues have been evaluated. This review will present biological considerations for bone function restoration, contemporary approaches for clinical salvage of large bone defects and their limitations, state-of-the-art research on the development of vascularized bone constructs, and perspectives on evaluating and implementing novel BTE grafts in clinical practice. Success will depend on achieving full graft integration at multiple hierarchical levels, both between the individual graft components as well as between the implanted constructs and their surrounding host tissues. The paradigm of vascularized tissue constructs could not only revolutionize the progress of BTE, but could also be readily applied to other fields in regenerative medicine for the development of new innovative vascularized tissue designs.

Annulated isoxazoles via [3 + 2] cycloaddition of alkenyl bromides and oximoyl chlorides and Ag(I) promoted elimination.

Substituted salicylaldehydes are converted to fused tetracyclic isoxazoles through a synthetic sequence incorporating substitution of 2-bromo-2-cyclohexen-1-ol, formation of an oxime function, conversion to an oximoyl chloride, intramolecular [3 + 2] cycloaddition, and elimination of an equivalent of hydrogen bromide using silver(I) carbonate. Six examples of this sequence are presented.