Bony defect repair in rabbit using hybrid rapid prototyping polylactic-co-glycolic acid/ß-tricalciumphosphate collagen I/apatite scaffold and bone marrow mesenchymal stem cells.

BACKGROUND: In bone tissue engineering, extracellular matrix exerts critical influence on cellular interaction with porous biomaterial and the apatite playing an important role in the bonding process of biomaterial to bone tissue. The aim of this study was to observe the therapeutic effects of hybrid rapid prototyping (RP) scaffolds comprising polylactic-co-glycolic acid (PLGA), ß-tricalciumphosphate (ß-TCP), collagen I and apatite (PLGA/ß-TCP-collagen I/apatite) on segmental bone defects in conjunction with combination with bone marrow mesenchymal stem cells (BMSCs). MATERIALS AND METHODS: BMSCs were seeded into the hybrid RP scaffolds to repair 15 mm defect in the radius of rabbits. Radiograph, microcomputed tomography and histology were used to evaluate new bone formation. RESULTS: Radiographic analysis done from 12 to 36 weeks postoperative period demonstrated that new bone formed at the radial defect site and continues to increase until the medullary cavity is recanalized and remodelling is complete. The bone defect remained unconnected in the original RP scaffolds (PLGA/ß-TCP) during the whole study. Histological observations conformed to the radiographic images. In hybrid RP scaffold group, woven bone united the radial defect at 12 weeks and consecutively remodeled into lamellar bone 24 weeks postoperation and finally matured into cortical bone with normal marrow cavity after another 12 weeks. No bone formation but connective tissue has been detected in RP scaffold at the same time. CONCLUSION: Collagen I/apatite sponge composite coating could improve new bone formation in vivo. The hybrid RP scaffold of PLGA/ß-TCP skeleton with collagen I/apatite sponge composite coating is a promising candidate for bone tissue engineering.

Channelled scaffolds for engineering myocardium with mechanical stimulation.

The characteristics of the matrix (composition, structure, mechanical properties) and external culture environment (pulsatile perfusion, physical stimulation) of the heart are important characteristics in the engineering of functional myocardial tissue. This study reports on the development of chitosan-collagen scaffolds with micropores and an array of parallel channels (~ 200 µm in diameter) that were specifically designed for cardiac tissue engineering using mechanical stimulation. The scaffolds were designed to have similar structural and mechanical properties of those of native heart matrix. Scaffolds were seeded with neonatal rat heart cells and subjected to dynamic tensile stretch using a custom designed bioreactor. The channels enhanced oxygen transport and facilitated the establishment of cell connections within the construct. The myocardial patches (14 mm in diameter, 1-2 mm thick) consisted of metabolically active cells that began to contract synchronously after 3 days of culture. Mechanical stimulation with high tensile stress promoted cell alignment, elongation, and expression of connexin-43 (Cx-43). This study confirms the importance of scaffold design and mechanical stimulation for the formation of contractile cardiac constructs.

Fabricating a pearl/PLGA composite scaffold by the low-temperature deposition manufacturing technique for bone tissue engineering.

Here we developed a composite scaffold of pearl/poly(lactic-co-glycolic acid) (pearl/PLGA) utilizing the low-temperature deposition manufacturing (LDM). LDM makes it possible to fabricate scaffolds with designed microstructure and macrostructure, while keeping the bioactivity of biomaterials by working at a low temperature. Process optimization was carried out to fabricate a mixture of pearl powder, PLGA and 1,4-dioxane with the designed hierarchical structures, and freeze-dried at a temperature of -40 degrees C. Scaffolds with square and designated bone shape were fabricated by following the 3D model. Marrow stem cells (MSCs) were seeded on the pearl/PLGA scaffold and then cultured in a rotating cell culture system. The adhesion, proliferation and differentiation of MSCs into osteoblasts were determined using scanning electronic microscopy, WST-1 assay, alkaline phosphatase activity assay, immunofluorescence staining and real-time reverse transcription polymerase chain reaction. The results showed that the composite scaffold had high porosity (81.98 +/- 3.75%), proper pore size (micropores: <10 microm; macropore: 495 +/- 54 microm) and mechanical property (compressive strength: 0.81 +/- 0.04 MPa; elastic modulus: 23.14 +/- 0.75 MPa). The pearl/PLGA scaffolds exhibited better biocompatibility and osteoconductivity compared with the tricalcium phosphate/PLGA scaffold. All these results indicate that the pearl/PLGA scaffolds fulfill the basic requirements of bone tissue engineering scaffold.

TGF-ß3 immobilized PLGA-gelatin/chondroitin sulfate/hyaluronic acid hybrid scaffold for cartilage regeneration.

Although most in vitro studies indicate that transforming growth factor ß3 (TGF-ß3) immobilized scaffold is suitable for cartilage tissue engineering, in vivo studies of implanting immobilized scaffold for chondral defect repair are still lacking. This study is to evaluate the potentials of TGF-ß3 immobilized poly-(lactic-co-glycolic acid)-gelatin/chondroitin sulfate/hyaluronic acid (PLGA-GCH) hybrid scaffold for cartilage regeneration. The scaffold was fabricated by incorporating GCH micro-sponges into PLGA frameworks and then crosslinked with TGF-ß3 to mimic natural cartilaginous extra cellular matrix (ECM). In vitro study demonstrated that MSCs proliferated vigorously and produced abundant ECM on scaffold. The immunohistochemistry staining and alcian blue staining confirmed the cartilaginous ECM production. The chondrogenic differentiation of MSCs on scaffold was proved by the expression of collagen II gene in mRNA and protein level. Then MSCs/TGF-ß3 immobilized scaffolds were implanted in rabbits for chondral defects repair. After eight weeks, histological observation showed that differentiated MSCs were located in lacunae within the metachromatic staining matrix and exhibited typical chondrocyte morphology. Histological grading scores also indicated the congruent cartilage was regenerated. In conclusion, the TGF-ß3 immobilized PLGA-GCH hybrid scaffold has great potential in constructing the tissue-engineered cartilage.

[Medical applications of rapid prototyping--three-dimensional bodies for planning and implementation of treatment and for tissue replacement]. / Teollisen pikavalmistuksen lääketieteelliset sovellukset.

The possibilities of medical applications of rapid prototyping are continuously expanding and developing. In current applications, five main groups are distinguished: (1) preoperative planning, surgical training and teaching, (2) inert implants, (3) surgical instruments and special equipment associated with the operations, (4) postoperative guides, long-term supports and aids and (5) artificial tissue. The first four of these are already in general use, whereas the last one is still under investigation.

An cell-assembly derived physiological 3D model of the metabolic syndrome, based on adipose-derived stromal cells and a gelatin/alginate/fibrinogen matrix.

One of the major obstacles in drug discovery is the lack of in vitro three-dimensional (3D) models that can capture more complex features of a disease.Here we established a in vitro physiological model of the metabolic syndrome (MS) using cell-assembly technique (CAT), which can assemble cells into designated places to form complex 3D structures. Adipose-derived stromal (ADS) cells were assembled with gelatin/alginate/fibrinogen. Fibrin was employed as an effective material to regulate ADS cell differentiation and self-organization along with other methods. ADS cells differentiated into adipocytes and endothelial cells, meanwhile, the cells were induced to self-organize into an analogous tissue structure. Pancreatic islets were then deposited at designated locations and constituted the adipoinsular axis with adipocytes. Analysis of the factors involved in energy metabolism showed that this system could capture more pathological features of MS. Drugs known to have effects on MS showed accordant effects in this system, indicating that the model has potential in MS drug discovery. Overall, this study demonstrated that cell differentiation and self-organization can be regulated by techniques combined with CAT. The model presented could result in a better understanding of the pathogenesis of MS and the development of new technologies for drug discovery.

Recent trends and challenges in complex organ manufacturing.

Presently, there is a recognized and imperative need for bioartificial organs. The technological advances in transgenosis, tissue engineering, and rapid prototyping have led to the development of spatially complex tissues. An ideal artificial organ should provide nutrient transport system, mechanical stable architecture, and synergetic multicellular organization in one construct. The multinozzle rapid prototyping technique simultaneously assembles vascular systems including hierarchical multicellular structures in an automated and reproducible manner and offers an effective way for treating organ failures. In this article, a brief overview of the recent trends and outstanding challenges in organ manufacturing is provided. From the viewpoint of disciplinary crossing, integration, and development, future directions in the coming years were pointed out.

Rapid prototyping of a double-layer polyurethane-collagen conduit for peripheral nerve regeneration.

A new technique for preparing double-layer polyurethane (PU)-collagen nerve conduits for peripheral nerve repair via a double-nozzle, low-temperature, deposition manufacturing (DLDM) system has been developed. The DLDM system is based on a digital prototyping approach, and uses a combination of thermally induced phase separation and freeze-drying. With this system, two kinds of biomaterials with different properties can be combined to produce scaffold structures with good biocompatibility in the inner layer and with the desired mechanical strength protruded by the outer. The forming precision is high, the wall thickness can be controlled, and a tight connection between the two layers can be achieved. The effects of changing the processing parameters and the material temperature on the structure of the scaffolds have been investigated. Additionally, the effect of material concentration on the mechanical strength and hydrophilic properties of the scaffolds has also been studied. Ideal peripheral nerve repair conduits, comprising an outer microporous layer of PU and internal oriented filaments of collagen, have been manufactured through optimizing the processing parameters and the biomaterial concentrations.

Multinozzle low-temperature deposition system for construction of gradient tissue engineering scaffolds.

Tissue engineering is a technology that enables us to construct complicated hominine organs composed of many different types of cells. One of the key points to achieve this goal is to control the material composition and porous structure of the scaffold accurately. A disposable syringe based volume-driven injecting (VDI) nozzle was proposed and designed to extrude both natural derived and synthetic polymers. A multinozzle low-temperature deposition and manufacturing (M-LDM) system is proposed to fabricate scaffolds with heterogeneous materials and gradient hierarchical porous structures. PLGA, collagen, gelatin, chitosan can be extruded without leaking to form hierarchical porous scaffolds for primary study. Composite scaffolds with two kinds of materials were fabricated via two different nozzles to get both hydrophilic and mechanical properties. The results from scanning electron microscopy (SEM) demonstrated that the natural-derived biomaterials were strongly absorbed onto the synthetic biomaterials to form a stable network. Several gradient PLGA/TCP scaffolds were also fabricated to supply several samples.

Fabrication of a two-level tumor bone repair biomaterial based on a rapid prototyping technique.

After the removal of the giant cell tumor (GCT) of bone, it is necessary to fill the defects with adequate biomaterials. A new functional bone repair material with both stimulating osteoblast growth and inhibiting osteoclast activity has been developed with phosphorylated chitosan (P-chitosan) and disodium (1 --> 4)-2-deoxy-2-sulfoamino-beta-D-glucopyranuronan (S-chitosan) as the additives of poly(lactic acid-co-glycolic acid) (PLGA)/calcium phosphate (TCP) scaffolds based on a double-nozzle low-temperature deposition manufacturing technique. A computer-assisted design model was used and the optimal fabrication parameters were determined through the manipulation of a pure PLGA/TCP system. The microscopic structures, water absorbability and mechanical properties of the samples with different P-chitosan and S-chitosan concentrations were characterized correspondingly. The results suggested that this unique composite porous scaffold material is a potential candidate for the repair of large bone defects after a surgical removal of GCT.

[Preliminary clinical application of Chinese-made invisible orthodontic technique].

OBJECTIVE: To treat simple malocclusions preliminarily using Chinese-made invisible orthodontic aligners and discuss the indications, problems existed and future development. METHODS: Forty-one cases with different malocclusions were selected, including crowding, spaces and spaces due to periodontal problems. Invisible aligners were made and worn by patients and they were changed every 2 - 3 weeks. RESULTS: Acceptable treatment results were obtained in all cases, with nice alignments and good overbite and overjet. Treatment time ranged from 6 - 25 months. CONCLUSIONS: Indications of this technique were still limited and the technique needed to be further developed in the future.

[Surface modification of biodegradable polymer/TCP scaffolds and related research].

Under laboratory condition, the compound materials of Poly (DL-lactic-co-glycolic acid)/Tricalcium phosphate [PLGA/TCP(L), with component ratio of 7:3] were fabricated by combining the thermally induced phase separation (TIPS) with solvent-casting particulate-leaching (SCPL) approach. On the other hand, rapid prototyping (RP) technique manufactured PLGA/TCP scaffolds [PLGA/TCP(RP)] were obtained. These two kinds of carriers were coated with collagen type I (Col I). The extracted bovine bone morphogenetic protein (bBMP) was loaded into carriers to establish biomimetic synthetic bones. PLGA/TCP(L) scaffolds, demineralized bone matrices (DBM) of bovine cancellous bone, PLGA/TCP(L) scaffolds, biomimetic synthetic bones and OsteoSet bone graft substitutes were investigated. Scanning electron microscopy revealed that the microarchitecture of PLGA/TCP(RP) scaffolds was much better than that of PLGA/TCP(L) scaffolds. The diameter of macropore of PLGA/TCP(RP) scaffold was 350 microm. The porosities of PLGA/ TCP(L) scaffolds, DBM, PLGA/TCP(RP) scaffolds and OsteoSet bone graft substitutes were 21.5%, 70.4%, 58.6% and 0%, respectively (P<0.01). Modification of PLGA/TCP scaffolds with collagen type I [PLGA/TCP(L)-Col I and PLGA/TCP(RP)-Col I] essentially increased the affinity of the carriers to bBMP. Among these synthetic materials, PLGA/TCP(RP)-Col I-bBMP composite is promising as a novel bone graft substitute due to its advanced fabrication technique, good tri-dimensional microarchitecture and ideal components.

Liver tissue responses to gelatin and gelatin/chitosan gels.

Gelatin and gelatin/chitosan gels, crosslinked using glutaraldehyde, were previously developed as substrates for three-dimensional cell-assembly techniques. In this study, the biocompatibility and biodegradation of gelatin and gelatin/chitosan gels were evaluated following implantation in rat livers for periods up to 16 weeks. The two gels were characterized by different inflammatory responses and degradation rates. The gelatin/chitosan gel is more efficient in inducing fibrin formation and vascularization at the implant-host interface. The degrees of inflammatory reaction for the gelatin/chitosan gel were significantly stronger than the gelatin gel. Advanced biodegradation of the gelatin gels was observed. These data indicate that the gelatin gel has better liver tissue biocompatibility and a faster biodegraded rate than the gelatin/chitosan gel.

Comparison of chondral defects repair with in vitro and in vivo differentiated mesenchymal stem cells.

The purpose of this study was to compare chondral defects repair with in vitro and in vivo differentiated mesenchymal stem cells (MSCs). A novel PLGA-gelatin/chondroitin/hyaluronate (PLGA-GCH) hybrid scaffold with transforming growth factor-beta1 (TGF-beta1)-impregnated microspheres (MS-TGF) was fabricated to mimic the extracellular matrix. MS-TGF showed an initial burst release (22.5%) and a subsequent moderate one that achieved 85.1% on day 21. MSCs seeded on PLGA-GCH/MS-TGF or PLGA-GCH were incubated in vitro and showed that PLGA-GCH/MS-TGF significantly augmented proliferation of MSCs and glycosaminoglycan synthesis compared with PLGA-GCH. Then MSCs seeded on PLGA-GCH/MS-TGF were implanted and differentiated in vivo to repair chondral defect on the right knee of rabbit (in vivo differentiation repair group), while the contralateral defect was repaired with in vitro differentiated MSCs seeded on PLGA-GCH (in vitro differentiation repair group). The histology observation demonstrated that in vivo differentiation repair showed better chondrocyte morphology, integration, and subchondral bone formation compared with in vitro differentiation repair 12 and 24 weeks postoperatively, although there was no significant difference after 6 weeks. The histology grading score comparison also demonstrated the same results. The present study implies that in vivo differentiation induced by PLGA-GCH/MS-TGF and the host microenviroment could keep chondral phenotype and enhance repair. It might serve as another way to induce and expand seed cells in cartilage tissue engineering.

Rapid prototyping as a tool for manufacturing bioartificial livers.

Rapid prototyping (RP) technologies are a set of manufacturing processes that can produce very complex structures directly from computer-aided design models without structure-specific tools or knowledge. These technologies might eventually enable the manufacture of human livers to create functional substitutes for treating liver failure or dysfunctionality. However, the approaches used currently face many challenges, such as the complex branched vascular and bile ductular systems and the variety of cell types, matrices and regulatory factors involved in liver development. Here, we discuss the challenges and provide evidence for the usefulness of RP in overcoming them.

Porous morphology, porosity, mechanical properties of poly(alpha-hydroxy acid)-tricalcium phosphate composite scaffolds fabricated by low-temperature deposition.

Tissue engineering is expected to construct complicated hominine organs composed of many different types of cells. One of the key points is the accurate controlling of scaffold material and porous morphology point by point. A new direct rapid prototyping process called low-temperature deposition manufacturing (LDM) was proposed to fabricate scaffolds. The new process integrated extrusion/jetting and phase separation and therefore could fabricate scaffolds with hierarchical porous structures creating a wonderful environment for the growth of new tissue. The interconnected computer-designed macropores allow cells in the new tissue to grow throughout the scaffold. Also, the parameter-controlled micropores let nutrition in and metabolic wastes out. The macrocellular morphology, microcellular morphology, porosity, and mechanical properties of the poly(alpha-hydroxy acid)-TCP composite scaffolds prepared by the proposed method are investigated. These scaffolds with high controllability would potentially play an important role in tissue engineering. LDM could also be combined with multinozzle deposition or cell deposition to exactly control materials or cells point by point. This might bring a breakthrough to the engineered fabrication of complicated organs.

Biomanufacturing: a US-China National Science Foundation-sponsored workshop.

A recent US-China National Science Foundation-sponsored workshop on biomanufacturing reviewed the state-of-the-art of an array of new technologies for producing scaffolds for tissue engineering, providing precision multi-scale control of material, architecture, and cells. One broad category of such techniques has been termed solid freeform fabrication. The techniques in this category include: stereolithography, selected laser sintering, single- and multiple-nozzle deposition and fused deposition modeling, and three-dimensional printing. The precise and repetitive placement of material and cells in a three-dimensional construct at the micrometer length scale demands computer control. These novel computer-controlled scaffold production techniques, when coupled with computer-based imaging and structural modeling methods for the production of the templates for the scaffolds, define an emerging field of computer-aided tissue engineering. In formulating the questions that remain to be answered and discussing the knowledge required to further advance the field, the Workshop provided a basis for recommendations for future work.

Generation of three-dimensional hepatocyte/gelatin structures with rapid prototyping system.

Using rapid prototyping technology, three-dimensional (3D) structures composed of hepatocytes and gelatin hydrogel have been formed. This technique employs a highly accurate 3D micropositioning system with a pressure-controlled syringe to deposit cell/biomaterial structures with a lateral resolution of 10 microm. The pressure-activated micro-syringe is equipped with a fine-bore exit needle for which a wide variety of 3D patterns with different arrays of channels (through-holes) were created. More than 30 layers of a hepatocyte/gelatin mixture were laminated into a high spacial structure using this method. The laminated hepatocytes remained viable and performed biological functions in the construct for more than 2 months. The rapid prototyping technology offers potential for eventual high-throughout production of artificial human tissues or organs.

Preparation and characterization of a collagen/chitosan/heparin matrix for an implantable bioartificial liver.

A new type of collagen/chitosan/heparin matrix, fabricated by gelation of collagen/ chitosan with heparin sodium containing ammonia, was produced to construct livers by tissue engineering and regenerative engineering. The obtained collagen/chitosan/heparin matrix was found to be highly porous, swelled rapidly in PBS solution and was stable in vitro for at least 60 days in collagenase/lysozyme containing buffered aqueous solution (PBS, pH 7.4) at 37 degrees C. The collagen/chitosan/heparin matrix resulted in a superior blood compatibility compared to the ammonia-treated collagen and collagen/chitosan matrices. The morphology and behavior of the cells on the collagen/chitosan/heparin membrane were found to be similar to those on the collagen membrane but different from those on the collagen/chitosan membrane. Hepatocytes cultured on the collagen/chitosan/heparin matrices exhibited highest urea and triglyceride secretion functions 25 days post seeding. These results suggest that this collagen/chitosan/heparin matrix is a potential candidate for liver tissue engineering.

Preparation and evaluation of ammonia-treated collagen/chitosan matrices for liver tissue engineering.

To further enhance the properties of existing collagen/chitosan scaffolds for liver tissue engineering, a very simple method was developed to form noncovalently linked mimic of the liver extracellular matrices. Collagen/chitosan mixtures in various proportions (i.e., 1:0, 3:2, 1:1, 2:3, and 0:1 v/v) were lyophilized or evaporated to form sponges or flat films before they were gelled using an aqueous 25% ammonia solution. The porosities of the obtained sponges were above 90% with various pore sizes. The highest mechanical strength (1.9+/-0.7 MPa) and the lowest degradation time (65+/-1.7 days) were achieved by the collagen/chitosan (1:1) matrices. Hepatocytes cultured on the collagen/chitosan (1:1) matrices exhibited relatively high glutamate-oxaloacetate transaminase and glucose secretion functions 25 days post-seeding. Nuclues of the hepatocytes were more elongated and arranged in certain directions on the 1:1 matrices. The cytocompatibility and enhanced biostability of our new ammonia-treated collagen/chitosan matrices suggest that they could be used as scaffolds for liver tissue engineering.