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Extrusion container is the most important tooling in steel hot extrusion processing. Traditional designs such as the multilayered cylinder are difficult to execute for large extrusion containers. A novel design is proposed in this article, a salient feature of which is that the container employs steel wire winding technology. The primary difficulty of applying wire winding to the design of extrusion container is that the container should be heated to 350 °C while the temperature of steel wire cannot exceed 75 °C due to accelerated creep and stress relaxation at high temperature. In addition, the prestress generated by steel wire should fulfill the requirements necessary for the container. The proposed design should meet both goals. The functional requirements including preheating, protection of steel wire from high temperature, and stress distribution are discussed, and solutions are proposed. A new method to generate prestress called thermally induced prestress is introduced and detailed. To verify the design, a proof-of-concept prototype model was fabricated and tested. Results showed that the container using the proposed design could meet the expected prestressing goals, and the temperature of the wire would not exceed the limit and can offer an alternative to conventional design methods of extrusion containers.

Rapid prototyping (RP)-based highly porous poly(DL-lactic-co-glycolic acid)/tricalcium phosphate (PLGA/TCP(RP)) scaffolds were fabricated. PLGA/TCP constructs (PLGA/TCP(TS)) were also made via thermally induced phase separation with solvent casting and by particulate leaching approach. Both scaffolds were loaded with bovine bone protein extract (BBPE). Sixty-four New Zealand white rabbits were randomized into four groups (groups of A, B, C, and D) and unilaterally underwent posterior lumbar interlaminar fusion at L2—L4 level. Spinal fusions were systematically evaluated. In groups of A (PLGA/TCP (RP)/BBPE constructs) and C (autogenous iliac bone grafts), good bone fusions occurred in vivo. Histological analyses indicated that endochondral ossification played an essential role in initiation of bone fusions in group A, whereas in group B (PLGA/TCP(TS)/BBPE constructs), few bone fusions were observed. In group D (PLGA/TCP(RP) scaffolds alone), the scaffolds were biocompatible and biodegradable; however, no newly formed bone mass or bone fusion was found. Twelve weeks after surgery, the fusion was significantly higher in groups of A and C compared with groups B and D (p(RP)/BBPE biomaterials have potential as grafting substitutes for bone healing and spinal fusion.

A poly(DL-lactic-co-glycolic acid) (PLGA) sandwich fibrinogen/ adipose stem cell (ADSC) construct was fabricated to generate smooth muscle tissue. The mechanical properties and ADSC compatibility of PLGA, poly(ethylene glycol-1,6-hexamethyl diisocyanate-caprolactone) i.e. polyurethane (PU), gelatin, alginate, and fibrin composites were evaluated for vascular smooth muscle tissue generation. Synthetic PLGA and PU combined with natural gelatin, alginate, and fibrin for strength and cell compatibility were found to be effective. A trilayer construct was designed and built with a microporous inner PLGA layer to provide nutrient, oxygen, and metabolite transfer while the outer PLGA layer with no pores prevented leakage during in vitro culture and in vivo implantation. The fibrin layer suitably accommodated ADSC growth, migration, proliferation, and differentiation inside the construct. This design has the potential for wide use in tissue engineering and complex organ construction.

The bumpy-ridge structure applied in the field of heavy equipment is analysed based on fracture mechanics and contact theory. At the same time, the elastic—plastic contact of the ridges is simulated using the finite element method. Having been researched, the separated bumpy-ridge structure can be seen as a special integral structure that has a through crack. It was also found that, as long as there are adequate normal forces and treated contact surfaces, this kind of separated structure has the ability to resist detachment and parallel slide. The height of the notch and radius of the contact zone are the foremost indexes affecting the application of the bumpy-ridge technique, and these parameters are influenced by several factors, such as the shape of the ridges, the curvature of the contact point, the distance between neighbouring ridges, etc. The elastic contact of the ridge can be solved completely by the use of theoretical calculation and graphical method. Through comparison with the elastic contact, some principles of the elastic—plastic contact can be obtained with a finite element analysis method. A bumpy-ridge structure has been successfully applied to a 350 MN die forging press and a 360 MN extrusion press in China.

Three-dimensional highly porous poly(DL-lactic-co-glycolic acid)/ tricalcium phosphate (PLGA/TCP) scaffolds were fabricated using a rapid prototyping technique (RP). The 3D rhombic lamellar PLGA/TCP carriers (20 mm × 20 mm × 3 mm) subsequently were coated with collagen type I (Col) to produce PLGA/TCP/Col composites. Both the RP-based PLGA/TCP scaffolds and the PLGA/TCP/Col composites were observed by scanning electron microscopy. Forty New Zealand white rabbits were equally randomized into 2 groups (group A and group B) and bilaterally underwent posterolateral intertransverse process arthrodesis at the L4—L5 level using the following graft materials: In group A, PLGA/TCP/Col/BMSCs-OB composites (on the right side, group A1, n = 20) and autogenous iliac bone grafts (on the left side, group A2, n = 20) were used; In group B, PLGA/TCP scaffolds plus fresh autogenous bone marrow (on the right side, group B1, n = 20) and PLGA/TCP scaffolds alone (on the left side, group B2, n = 20) were utilized. In group A1, rabbit bone marrow stromal cells (BMSCs) were isolated and cultured under the osteogenic conditions (BMSCs-OB). Structural PLGA/TCP/Col composites then were efficiently loaded with BMSCs-OB and cultured 5 days to make PLGA/TCP/ Col/BMSCs-OB biomaterials. Rabbits were sacrificed after 12-week follow-up and the spinal fusion were evaluated by a general observation, a manual palpation test, histological analyses and radiography. As a result, RP established PLGA/TCP scaffolds with appropriate biomaterial properties including satisfactory microstructure, inter-connectivity and porosity. Modifications to the structural highly porous PLGA/TCP scaffolds with Col (PLGA/TCP/Col) essentially increased the affinity of the carriers to seeding cells. In group A1, radiological evaluation revealed strong ability of new bone formation and bony fusion in the implanted sites and histological analyses showed highly cellular bone marrow between the newly formed trabecular bone was present in the fusion mass. In group A2, there was a reduced amount of newly formed bone. In group B1, only a few bony fusions were obtained. In group B2, PLGA/TCP scaffolds were biocompatible and biodegradable; whereas, no newly formed bone or bony fusion was found. Twelve weeks after surgery, spinal fusion rates in groups of A1, A2, B1, and B2 were 70.0%(14/20), 45.0%(9/20), 15.8%(3/19), and 0%(0/19), respectively. The rates of fusion were significantly higher in groups of A1 and A2 compared with groups of B1 and B2 (p0.05). Therefore, RP-based 3D PLGA/TCP/Col/BMSCs-OB biomaterial holds promise as a bone grafting substitute for spinal fusion. Our attempts may provide a novel method for biofabrication of the bionic construct.

Cryopreservation technology plays an important role in conserving three dimensional (3D) constructs containing cells. Besides preserving the characteristics of the construct, it can also save a lot of resources in many aspects, such as cell culture space, culture vessel, culture medium etc. Otherwise, the biological properties of the cells and the 3D geometrical configuration will disappear with the death of cells and breakage of configuration. Consequently, a cryopreservation method for the 3D construct fabricated with a controlled cell assembling technology was studied. 10% dimethylsulfoxide (DMSO) was incorporated into the adipose-derived stem cell (ADSC)/gelatin/ alginate/fibrinogen mixture before assembling. Results indicate that the 3D construct containing cells can be preserved below —80°C for more than 1 week. After the construct underwent the thawing process, cell viability and proliferation ability were regained. This technique holds potential to be used widely in tissue engineering and organ manufacturing fields.

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 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.

Assembling multitype cells and biomimic extracellular matrix materials in a single construct is a promising method to regenerate complex tissues/organs in vitro. A new double-nozzle assembling technique was used to fabricate a hybrid cell/hydrogel construct with a vascular-like network to perform as a mass exchange conduit. Using a digital model to mimic an anatomical liver structure, adipose-derived stromal cells (ADSC) were combined within a gelatin/alginate/fibrinogen hydrogel to form a vascular-like network and hepatocytes combined gelatin/alginate/chitosan were placed around it. The construct was stabilized in a thrombin/CaCl2/Na5P3O10 solution after assembly. The ADSC were induced to differentiate into endothelial-like cells with endothelial growth factor. The albumin secretion level of the embedded hepatocytes increased during the 2 weeks culture, while the levels of urea and alanine transaminase were decreased after an increasing profile. These preliminary results indicate that this double-nozzle assembling technique could be a powerful tool for fabricating complex constructs with special intrinsic/ extrinsic structures. This technology has the potential to be widely used in medical regeneration and drug screening.

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.

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

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.

Unique double layer polyurethane (PU)-collagen nerve guide conduits with desirable biocompatibility and mechanical properties were fabricated using a newly developed double-nozzle low-temperature deposition manufacturing (DLDM) technique. The inner collagen layer of the conduit had oriented nano-size filaments with micro-pores (pore size 20—100μm) that permitted nutrient infiltration, while the outer PU layer had micro-pores (pore size 15—20 μm) preventing fibrous tissue invasion. In vivo animal tests (n = 6) on bridging a 10 mm long peroneal nerve defect was conducted using rats. Single layer pure PU conduit (n = 6) was used as the positive control and the same size defect with no implantation (n = 2) as the negative control. Through walk track analysis as well as electrophysiological and histological evaluations of the regenerated nerves, the double layer PU-collagen conduit demonstrated better nerve repair than the controls. This new PU-collagen has the potential for significant clinical applications in peripheral nerve repair.

Using a cell assembly device developed in this laboratory, we coextruded adipose derived stem cells (ADSC) and gelatin/alginate hydrogel to form cubic 3D constructions (10 × 10 × 10 mm3) with regular distributed go-through pores. The ADSC grew, proliferated, and differentiated within these constructions. With the addition of basic fibroblast growth factor (bFGF), cells located on the scaffold walls differentiated into endothelial like cells while cells embedded in the hydrogel differentiated into adipose like cells. The integrity of the constructions remained for more than 60 days. This new technology enables us to construct 3D adipose tissue with blood vessel-like structures in vitro which is a significant enhancement in adipose tissue engineering and provides a better biomimic 3D model for studying cell—cell interaction, stem cell differentiation conditions and cell organization mechanisms.

A novel 3D hybrid construct was developed for liver manufacturing by depositing biodegradable polyurethane (PU) and a naturally derived polymer gelatin simultaneously via a double nozzle rapid prototyping (RP) technique. A grid object was produced by precisely and simultaneously dispersing the PU and gelatin to form 3D constructs with interconnected macro-channels at a low temperature (-28°C). Micro-pores were formed by freeze-drying the constructs. The PU polymer provided mechanical support while gelatin provided accommodation for implant cells. The hydrophilicity of the hybrid constructs was between the pure PU and pure gelatin structures. The interconnected channels allow nutrients and oxygen to be supplied throughout the construct as well as provide space for the attachment of cells. The design and fabrication strategies, used to create complex physical objects directly from computer aided design (CAD) models, represent a promising technique for implantable bioartificial livers. It is anticipated that these PU-gelatin hybrid constructs will serve as a useful model for bioartificial liver manufacturing.

A precise control over the internal architecture was essential for manufacture of complex organs. To create vascular systems that mimic human livers, we designed and fabricated complex 3D objects with an intrinsic network of interconnected channels. A new elastomeric polyurethane, mainly based on polycaprolactone and poly(ethylene glycol) with excellent biocompatibility and tunable biodegradation properties, was used to fabricate these vascular systems using a low temperature deposition system based on the layer-by-layer manufacturing principle. A specific model was selected via computer aided design (CAD), solid free form fabrication processes are conducted under computer direction. Two example object patterns were produced by precision controlled dispensing of a biodegradable polyurethane into 3D multi-micro-tunnels with multi-micro-pores at a low temperature (-28°C). The design and fabrication strategies used to create physical objects directly from CAD models represent a promising route for the establishment of complex organ vascular systems.

There is a need for rapid fabrication of tissue or organs with well-defined structures and functions in regenerative medicine. Two patterns of cell/matrix constructs containing hepatic cells, gelatin and fibrinogen were successfully created by automated rapid prototyping techniques and stabilized with thrombin. No apparent cell damage was found during the process. Mechanical characterization demonstrated that a 1:1 ratio gelatin/fibrin mixture had the greatest elasticity modulus and compressive strength. Microscopic and histological observations showed that hepatic cells were embedded in the gelatin/fibrinogen matrix and were proliferating. Immunostaining and biochemical analysis indicated that the embedded hepatocytes secreted albumin. Fibrin appears to be a favorable component for a gelatin based cell assembly matrix in that it is bioresorbable, easily manipulated, and supports in vitro cell functions.

There is currently interest in the use of tube-shaped nerve guides to induce regeneration of severed peripheral nerve axons, to assist nerve growth in the correct directions and to provide protection for the regenerating nerves. In the present paper, preliminary studies on the degradation and biocompatibility of a new elastomeric polyurethane (PU), based on polycaprolactone, polyethylene glycol and 1,6-hexamethyl diisocyanate. The PU elastomer was evaluated in vivo for the repair of a 12-mm femoral nerve gap in rabbits. Histological and scanning electron microscope observations were carried out at three days and then 4, 8, 10 and 12 weeks postoperation. Electrophysiological recording was performed six months postoperation. Myelinated axon regeneration was observed from four weeks after implantation. Polymer degradation was observed over the duration of the study. The results suggest that PU channels can be used for the regeneration of peripheral nerves and are suitable for tissue engineering.

Brain tissue engineering has now emerged as one of the most promising treatments for the traumatic brain injury. In this article, two groups of three-dimensional (3D) hydrogel structures composed of gelatin and gelatin/hyaluronan have been formed using our 3D cell assembly technique for in vivo study in rats, in order to investigate their effects in reparation of injury in the central nervous system (CNS). The structures were implanted into cortical defects created in rat brains, and their abilities to improve the brain tissue reconstruction were then evaluated. After 4, 8, 10, and 13 weeks of implantation, sections of brains were processed with NISSL staining for observing the immigration of host neural cells into the implanted materials and the degradation property of the materials. The results showed that simplex gelatin and gelatin/hyaluronan (20:1) with 3D structures both have good biocompatibility with brain tissue while gelatin/hyaluronan has a better contiguity with the surrounding tissue. Through our primary study, it seems that 3D gelatin/hyaluronan structures may be useful in brain tissue repair.

The hemostatic capability, adhesion ability and biocompatibility of chitosan sponges were compared with commercial collagen sponges. The chitosan sponges were briefly immersed in an aqueous 20% ammonia solution before being applied to a rabbit cervical vein wound. The total amount of bleeding from the injured veins until hemostasis was similar for both chitosan and collagen sponges. The complete hemostasis success rates for both the chitosan and collagen sponges were also similar. Under hemostasis, the chitosan sponges strongly adhered to the surface of the rabbit muscles, whereas the collagen sponges were easily detached from the muscles. Under wet conditions, however, there was no significant difference in the adhesive ability between the two sponges. During implantation, the chitosan sponges were much more flexible and resistant to breakage than the collagen sponges. The biocompatibilities and biodegradation rates of the two sponges were totally different after subcutaneous implantation in rabbits. The chitosan sponge was degraded much slower than the collagen sponge, while tissue responses for the chitosan sponges were much greater than for the collagen sponges.

An organ manufacturing technique was developed that enables the formation of cell/extracellular matrix (ECM) complexes for in vitro or in vivo growth. In this study, a three-dimensional (3D) structure composed of hepatocytes and gelatin/alginate hydrogel was made using a cell assembler-I apparatus to thoroughly control cell assembling. Hepatocytes and ECM were constructed into 10 X 10 X 3mm3 structures according to a designed pattern. The embedded hepatocytes remained viable and performed biological functions in the construct for more than 12 days. This 3D structure has the potential to be used as a precursor for tissue or organ regeneration. This technology offers the potential for high-throughput production of artificial human tissues and organs.

To make an implantable bioartificial liver (IBL), a new biocompatible collagen/chitosan/heparin complex was prepared using a crosslinking agent. The X-ray photoelectron spectroscopy (XPS), mechanical strength and biocompatibility with whole blood and hepatocytes were measured. The collagen/chitosan/heparin complex resulted in a superior blood compatibility compared to 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) crosslinked collagen matrix. The morphology and behavior of the cells on the collagen/chitosan/heparin membrane were found to be different from those on the collagen and collagen/chitosan membranes. Cells on the collagen membrane formed smaller three-dimensional aggregates than those on the collagen/chitosan membrane, while on the collagen/chitosan/heparin membrane, a round shape with no junctions were manifested. No adverse effects were found on the viability and function of the hepatocytes on the collagen/chitosan/heparin membrane compared to the collagen and collagen/chitosan membranes. These results suggest that this collagen/chitosan/heparin matrix is a potential candidate for hepatic tissue engineering.

In the race to fabricate a product to market with increases in speed, cost and quality, the drive to economically decrease tooling lead times becomes more important. Rapid tooling (RT) fabricated at Tsinghua University uses a metal plasma spraying process and rapid prototyping (RP) to form a metal tool. The process uses plasma spraying as the heat pool to melt metal powder and then deposit molten metal on to the substrate made by RP. It provides a quick, accurate, simple and relatively cost effective route for producing metal parts or tools, especially for large tools. The process and key technologies are analysed and described in detail. Applications illustrate that the total costs and lead times for new products can be reduced.

The technique of precision rapid mould by transferred coating forming is an innovative rapid tooling (RT) process. It combines many techniques such as transferred coating and rapid prototyping. The main advantages of this process compared with other RT processes are low investment and production cost, shorter production cycles and many kinds of scale tool. The manufacturing process and the coating materials have been researched. The results of application show that net shape production may be fabricated in 2–7 days. The surface roughness of tools, Ra, can reach 1.6–6.3 äm, and dimensional accuracy may attain CT4-CT6.