Journal of Bioactive and Compatible Polymers

A new double-layer conduit that combined an outer synthetic Polyurethane (PU) layer and inner natural collagen layer was fabricated via a double-nozzle low-temperature deposition manufacturing (DLDM) technology. The outer PU layer provided a mechanically stable tunnel against scar tissue invasion in vivo, while the inner collagen layer promoted Schwann cell adhesion, migration, and proliferation. Microporous structures were found in both the layers. A tight connection between the double layers was achieved by adjusting the distance between the two deposit nozzles and adjusting other processing parameters. Schwann cells from rat sciatic nerves were cultured in the layered PU-collagen conduits for one week; a significant enhancement in their retention and viability was seen compared to those made of pure PU. The poly(urethane-collagen) double layer conduit had better Schwann cell compatibility and so has a great potential use in clinical peripheral nerve repair.

A functional-region/separate-interface/single-cell-type of tissue engineering pathway was evaluated to regenerate osteochondral defects that are deep in the marrow cavity. A gradient osteochondral scaffold fabricated via a rapid prototyping technology, called multi-nozzle low-temperature deposition manufacturing, was composed of three parts with different materials and pore-structures, respectively, for bone, cartilage and a separate interface between them. The separate interface was composed of micro-pores, that were less than 5 urn and with low porosity, to reduce or to avoid the destruction of the micro-environment in vivo by preventing blood and cells and reducing the amount of oxygen and nutrients moving from the marrow cavity to the articulate marrow. The preliminary results after 6 weeks of implantation into 4 mm diameter osteochondral defects in the knee joints of rabbits showed that the defects with the scaffold/cells composition had bone-like or cartilage-like tissue filling the defects with smooth surface, while the defects with nothing (blank) showed only fibrous tissue.

One of the major obstacles in engineering thick and complex tissues while vascularizing tissues in vitro is to maintain cell viability during tissue growth and structural organization. Adipose-derived stromal (ADS) cells were used to establish a multicellular system through a cell-assembly technique. Attempts were made to control ADS cells differentiation into different targeted cell types according to their positions within an orderly 3D structure. Oil red 0 staining confirmed that the ADS cells in the structure differentiated into adipocytes with a spherical shape while immunostaining tests confirmed that the endothelial growth factor induced ADS cells on the walls of channels differentiated into mature endothelial cells and then organized into tubular structures throughout the engineered 3D structure. The endothelin-1 and nitric oxide release rules of the endothelial cells were coincidental with those in vivo.

This study provides a new approach to engineer orderly endothelial vessel networks in vitro and has potential applications in adipose-tissue engineering.

A cell encapsulation device based on the principle of high-voltage dispersion technology was designed and constructed. The parameters that influenced the formation of cell microcapsules, such as; voltage, push speed, electrode distance, and syringe size were analyzed and optimized. Unlike the traditional cell microencapsulating devices, both of the electrodes were separated from the CaCl2 solution in this device, which simplified the procedure of keeping the operation within a sanitary environment. Integrated adipose derived stem cell (ADSC) microcapsules with smooth surface and consistent diameter distribution were prepared. After 1-week in culture as a microcapsule and 3 days assembled in a construct, the ADSCs maintained a high viability and significant proliferation that established a favorable basis for the controlled assembly of cell microcapsules.

A β-TCP scaffold with a branched channel system was designed to create a novel micro-device that allowed culture perfusion and direct time observation of the cells attached. The scaffold was made by indirect solid free form fabrication (SFF) technology. The flow channel structure was exposed so that the perfusion of the mesenchymal stem cell (MSC) culture could be viewed directly. The cell-seeded scaffolds were continuously perfused for 7 days in the micro-device; during this time, it was possible to observe the dynamic culture processes with cells adhering to the scaffolds and real time cell growth directly. This concept has great potential for use in bone tissue engineering and for versatile fabrication of enhanced scaffolds.

A 3D poly(lactic acid-co-glycolic acid)/tricalcium phosphate (PLGA-TCP) composite scaffold, generated with the low-temperature deposition modeling rapid prototyping technique, was tested for its viability in a 3D cell cultivation in vitro. The aim was to find optimal cell culture conditions for the selected scaffold material and to monitor cell division, differentiation, and migration of selected cell types in this environment. In addition, the behavior and cell-matrix interactions of selected cell types were monitored as well as the biodegradation rate of the tested scaffold material. Chinese hamster ovary cells as well as a human cell line 293 epithelial cells were cultured on the scaffolds. A variety of different preconditioning protocols were deployed to prepare the scaffolds before seeding with the cells. Cell cultivations were conducted for 1–4 weeks and the coverage of the luminal surfaces was analyzed with light microscopy. Long cultivation periods were required to achieve partial coverage of the luminal surfaces of the scaffolds. Tissue engineering with 3D cell cultures and biomaterials represents a promising approach for organ manufacturing research. It may have potential for eventual on-demand high-throughput production of artificial tissues but the process has many challenges. The culture system in a well controlled bioreactor environment is discussed.

In this study, a two-step crosslinking method derived from commercial rapid prototyping equipment was used to fabricate a gradient hydrogel scaffold in vitro. This system contained two types of nozzles; one was double-nozzle unit, used for relatively simple gradient hydrogel scaffold composed of two nonmiscible hydrogel materials. The other was single nozzle that was used for mixing a gradient hydrogel scaffold composed of two types of hydrogel materials. Different types of scaffolds were formed by modifying the gradient in one of the relevant nozzles. To improve the extrusion controlling effect, accessorial parameters were introduced. A 3D gradient construct containing neuron cells and Schwann cells was fabricated and cultured for 7 days. This construct was helpful in designing a gradient mode to observe the relationship between different cells in vitro. This work has improved tissue-engineering techniques for later manufacturing of very complicated organ analogs.

The virtual human plan has been the hot point of recent research. The objective of this study is to explore the possibility of three-dimensional (3D) reconstruction of functional fascicular groups inside short segmental peripheral nerve. A 5 cm length of common peroneal nerve was horizontally sliced at 0.25 mm intervals, and each section was stained with acetycholinesterase histochemical staining. The 2D panorama images were acquired by high-resolution digital camera under 100 x microscope and mosaic software; different functional fascicular groups were distinguished and marked. The topographic database was then matched using image processing software, through the 3D reconstruction achieved using 3D reconstruction software (Amira 3.1). The reconstructed 3D images could be rotated or zoomed in any direction and the intercross and recombination processes of nerve bundles could be observed. Based on the serial histological sections and computer technology, the 3D microstructure of short segmental peripheral nerve functional fascicular groups was reconstructed. These results provide the possibility of 3D reconstruction of long segmental peripheral nerve functional fascicular groups.

A rapid prototyping and rapid tool technique-based method was developed to fabricate chitosan fiber calcium phosphate cement composites (CF/CPC) for bone tissue engineering scaffold applications. The products were characterized and the in vitro performance with canine bone marrow stem cells (BMCs) on CF/CPC scaffold with controlled fiber structures evaluated. The X-ray diffraction analysis showed that about 91% of the inorganic part of the CF/CPC scaffold was hydroxyapatite (HA) and the variation in CF had little effect on the percentage of HA content. The results from in vitro study demonstrated that the interconnected macropores rapidly formed inside the CF/CPC scaffolds and that the patterns were related to the fiber structures used. The differences in the fiber structures altered the morphology of the BMCs without affecting the proliferation of the BMCs.

The Rapid Prototyping (RP) manufacture technology, computer tomography (CT) image processing, and mechanical analyses were adopted to repair a skull defect injury. After the patient was inspected by spiral CT the data was exported to Digital Imaging and Communications in Medicine (DICOM) format. The data was processed with Mimics software to eliminate noise and gray-level registration. The processing filtered out the parenchyma data and preserved the bone data. The STL files were analyzed and processed by the Magics and put into the RP equipment. The RP equipment is one type of Stereo Lithography Apparatus (SLA) with laser light spot diameter that is <0. 2mm The 3D models were analyzed to comply with the patients’ requirements. The stereo lithography model emulated the mechanical properties and shape with the patient's bone. The titanium-alloy plate matched the anatomy well. The 3D reconstructed freeform model was accurate to within 0.2 mm.

The in vitro degradation and cytotoxicity of bacterial cellulose (BC) and its degradation products were studied for potential applications in bone tissue engineering. Emission scanning electron microscope was used to observe the morphology of original materials and their degradation products. The degradation was evaluated by measuring the concentration of reducing sugar by using ultraviolet spectrophotometer. Bone forming osteoblast (OB) cells and infinite culture cell line L929 fibroblasts were used to measure the cytotoxicity of materials using the MTT assay. Both types of cells proliferated normally with the BC and its degradation products with a cytotoxicity graded of 0–1. Nevertheless, the bone-forming target OB cells were more susceptible to cytotoxicity than the infinite culture fibroblast cells L929 fibroblasts. The results indicate that the BC is not very cytotoxic and that tissue functional cells are more suitable for evaluating the cytotoxicity of biomedical materials.

Three-dimensional highly porous poly(DL-lactic-co-glycolic acid)/tricalcium phosphate (PLGA/TCP) scaffolds were synthesized via a rapid prototyping (RP) technique. Bovine bone morphogenetic protein (bBMP) was loaded into the biopolymer scaffolds (PLGA/TCP/bBMP). Both the PLGA/TCP scaffolds and the PLGA/TCP/bBMP composites were evaluated by scanning electron microscopy. Lumbar intervertebral body fusion at L2~3 and L4~5 levels were performed on 15 goats using one of the following graft materials: RP synthesized PLGA/TCP scaffolds (group A), PLGA/TCP/bBMP composites (group B), and autogenous iliac bone graft (group C). All animals were sacrificed 24 weeks after surgery and the spine fusions evaluated by manual palpation tests, histological analyses, and radiography. In group A, the histological analyses showed that the PLGA/TCP scaffolds were biocompatible and biodegradable; however, no new bone was found. In group B, highly cellular bone marrow between the new trabecular bone was present in the fusion mass. In group C, there was a lesser amount of new bone. Twenty-four weeks after surgery, the fusion rate of lumbar intervertebral body fusion in group A, B, and C was 10% (1/10), 80% (8/10), and 50% (5/10), respectively. The fusion rate was significantly higher in group B compared with groups of A and C (p<0.01). Based on these results, extracted bBMP can be loaded in vitro into RP-based highly porous structural PLGA/TCP scaffolds to fabricate new graft composites that appear to be more effective for intervertebral spinal fusions. This biomimetic artificial grafting material holds promise as a tool for spine surgery.

A biomaterial patch of electrospun collagen type fibers was designed and produced by electrospinning seven different concentrations (8%-20% w/v) of collagen solutions. The tensile strength, yield strength, and elastic modulus of the electrospun collagen fibrous patches were found to be suitable for clinical transplantation. No significant differences versus fresh porcine pericardium as controls were observed. The SEM images of the groups showed that the patches were smooth with uniform interwoven and porous morphology. The fibrous patches were biocompatible and did not elicit local or systemic toxic effects when implanted in vivo. These electrospun collagen fibrous patches have significant potential as surgical biomaterial patches.

In this study, a hydroxyapatite (HA) was coated on a pure titanium surface by means of a complex oxidation and hydrothermal treatment. First an anodic oxidation was done on the titanium plates, followed by micro-arc oxidation. The HA-coated specimens and pure titanium specimens were immersed in SLB for 1, 5, and 10 days, respectively, to study their electrochemical behavior. The corrosion currents of HA-coated specimens were less than pure titanium specimens. This indicated that HA coating prevented surface metal ions of the implant from dissolving, thereby, reducing the tissue toxicity. The cytotoxic effect on fibroblasts L929 cells was measured by cell counting after being seeded for 2, 4, 8, 12, and 24 h. The number of surface cell attachments on the HA-coated specimens was much greater than on pure titanium specimens. The morphology of the cells on the HA coating had normal shapes and spread well with some cells climbing onto surface pores while cells on the pure titanium were oval shaped. The results confirm that the cell compatibility on HA-coated ion titanium surfaces is much better than pure titanium.

To provide an ideal test-base for the development of an intelligent bionic leg, a new pattern humanoid robot – biped robot with heterogeneous legs (BRHL) was designed. To simulate a normal human gait, a multi-axis knee mechanism was included into the artificial leg design that was different from the artificial leg of a common biped robot. The conception and research purposed for the BRHL was developed. Based on human bioscience, the bionic design of multi-axis knee artificial leg was analyzed and a virtual prototype was made. The kinematics model and dynamics model were deduced in detail. Based on human normal gait data, a simulation of dynamics model was carried. An optimized mechanical design of multi-axis knee artificial leg is discussed and a simulation was done. The optimal mechanism parameters for the multi-axis knee artificial leg and a BRHL prototype were given. This research indicated that a multi-axis knee artificial leg can simulate a human leg.