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PubMed2004

[Regeneration of autologous tissue-engineered cartilage by using basic-fibroblast growth factor in vitro culture].

OBJECTIVE: To investigate the effect of the basic fibroblast growth factor (b-FGF) to regenerate an autologous tissue-engineered cartilage in vitro. METHODS: The Cells were harvested from the elastic auricular cartilage of swine,and were plated at the concentration of 1 x 10(4) cells/cm2 , studied in vitro at two different media enviroments: Group I contained Ham's F-12 with supplements and b-FGF, Group II contained Ham's F-12 only with supplements. The passage 2 cells (after 12.75 +/- 1.26 days) were harvested and mixed with 30% pluronic F-127/Ham's F-12 at the concentration of 50 x 10(6) cells/ml. It was injected subcutaneously at 0.5 ml per implant. The implants were harvested 8 weeks after the vivo culture and examined with the histological stains. RESULTS: The chondrocytes displayed morphologically similar to the fibroblasts in the media containing basic-FGF. The number of cell doublings (after 12.75 +/- 1.26 days) in vitro culture was as the following: Group I, 70; Group II, 5.4. Eight 8 weeks after the vivo autologous implantation, the average weight (g) and volume (cm3) in each group was as the following: Group I, 0.371 g/0.370 cm3 Group II, 0.179 g/0.173 cm3 (P < 0.01). With the b-FGF in vitro culture, the cells were expanded by 70 times after 2 weeks. Histologically, all of the engineered cartilage in the two groups were similar to the native elastic cartilage. CONCLUSION: These results indicate that the basic-FGF could be used positively to enhance the quality and quantity of the seeding cells for the generation of the well-engineered cartilage.

Animals↗
PubMed2004

Interaction between insulin-like growth factor-1 with other growth factors in serum depleted culture medium for human cartilage engineering.

The regulation roles of insulin-like growth factor-1 (IGF-1) with basic fibroblast growth factor (bFGF) and transforming growth factor beta 2 (TGFbeta2) in human nasal septum chondrocytes monolayer culture and cartilage engineering was investigated in this study. The role of IGF-1 with bFGF and TGFbeta2 was investigated by measuring chondrocyte growth kinetic and collagen genes expression. IGF-1 together with bFGF and TGFbeta2 promote cartilage tissue engineering, increase type II collagen expression and enhance the histological features of engineered cartilage.

Cartilage↗
PubMed2004

Gene expression characteristic in human auricular cartilage tissue engineering.

This study was to assess collagen type II and collagen type I gene expression in tissue-engineered human auricular: cartilage formed via tissue engineering technique. Large-scale culture expansions were transformed into 3D in vitro construct and were implanted subcutaneously on the dorsal of athymic mice. After 8 weeks, explanted construct was processed in the same manner of native cartilage to facilitate cells for gene expression analysis. Isolated cells from in vivo construct demonstrated expression of type II collagen gene comparable to native cartilage. This study verified that tissue-engineered auricular cartilage expressed cartilage specific gene, collagen type II after in vivo maturation.

Actins↗
PubMed2004

[Construct tissue-engineered bone by co-seeding marrow stromal cells and endothelial cells].

To identify the role of co-seeding marrow stromal cells (MSCs) and endothelial cells (ECs) onto biocomposite in constructing tissue-engineered bone for promoting osteogenesis and angiogenesis, we seeded rat marrow stromal cells (rMSCs) and human umbilical vein endothelial cells (hUVECs) onto poly (L-lactic acid)/beta-tricalcium phosphate (PLLA/beta-TCP) macroporous composite (the test group), and seeded rMSCs onto the same kind of composite (the control group). We implanted these biocomposites into the thighs of nude mice intramuscularly and sacrificed these mice at 1, 4, 8, 12, 16 weeks after implantation, respectively. Biocomposites were taken out and histomorphometry was performed. Image manipulation technology was used to calculate the percentages of new bone area and material area in the test group and control group. We found that the percentage of new bone area of test group increased faster than that of control group, the percentage of material of test group decreased faster than that of control group, and the growth of capillary network of test group was much better than that of control group. These results suggest that co-seeding MSCs and ECs onto scaffold for constructing tissue-engineered bone is beneficial to osteogenesis, to angiogenesis in the prosthesis, and to the degradation of scaffold, and it is of significance in the research of bone tissue engineering.

Absorbable Implants↗
PubMed2004

[Tissue engineered cartilage for biological repair of cartilage defects].

Recent developments in tissue engineering techniques in cartilage repair were discussed. Recently, the novel two-step method, the alginate-recovered-chondrocyte method (ARC method), which does not require the aid of an exogenous synthetic matrix, was developed. The first step of this method consists of culturing phenotypically stable chondrocytes under conditions optimal for the formation of a proteoglycan-rich cell-associated matrix (CM) in alginate beads. Then, the cells with their CM are recovered from the alginate and allowed to rapidly integrate into a solid mass of tissue on a culture insert with a porous membrane. The use of a growth factor, recombinant human osteogenic protein-1 (OP-1) maximized the formation of tissue engineered cartilaginous tissue from adult human articular cartilage. Using the ARC method, the enhancement of matrix formation by OP-1 will produce a larger volume of tissue-engineered cartilage to cover large defects.

Activin Receptors, Type I↗
PubMed2005

Selective laser sintering of biocompatible polymers for applications in tissue engineering.

The ability to use biological substitutes to repair or replace damaged tissues lead to the development of Tissue Engineering (TE), a field that is growing in scope and importance within biomedical engineering. Anchorage dependent cell types often rely on the use of temporary three-dimensional scaffolds to guide cell proliferation. Computer-controlled fabrication techniques such as Rapid Prototyping (RP) processes have been recognised to have an edge over conventional manual-based scaffold fabrication techniques due to their ability to create structures with complex macro- and micro-architectures. Despite the immense capabilities of RP fabrication for scaffold production, commercial available RP modelling materials are not biocompatible and are not suitable for direct use in the fabrication of scaffolds. Work is carried out with several biocompatible polymers such as Polyetheretherketone (PEEK), Poly(vinyl alcohol) (PVA), Polycaprolactone (PCL) and Poly(L-lactic acid) (PLLA) and a bioceramic namely, Hydroxyapatite (HA). The parameters of the selective laser sintering (SLS) process are optimised to cater to the processing of these materials. SLS-fabricated scaffold specimens are examined using a Scanning Electron Microscope (SEM). Results observed from the micrographs indicate the viability of them being used for building TE scaffolds and ascertain the capabilities of the SLS process for creating highly porous scaffolds for Tissue Engineering applications.

Biocompatible Materials↗
PubMedDate not supplied

Applying industrial engineering practices to radiology.

Seven hospitals in Oregon and Washington have successfully adopted the Toyota Production System (TPS). Developed by Taiichi Ohno, TPS focuses on finding efficiencies and cost savings in manufacturing processes. A similar effort has occurred in Canada, where Toronto's Hospital for Sick Children has developed a database for its diagnostic imaging department built on the principles of TPS applied to patient encounters. Developed over the last 5 years, the database currently manages all interventional patient procedures for quality assurance, inventory, equipment, and labor. By applying industrial engineering methodology to manufacturing processes, it is possible to manage these constraints, eliminate the obstacles to achieving streamlined processes, and keep the cost of delivering products and services under control. Industrial engineering methodology has encouraged all stakeholders in manufacturing plants to become participants in dealing with constraints. It has empowered those on the shop floor as well as management to become partners in the change process. Using a manufacturing process model to organize patient procedures enables imaging department and imaging centers to generate reports that can help them understand utilization of labor, materials, equipment, and rooms. Administrators can determine the cost of individual procedures as well as the total and average cost of specific procedure types. When Toronto's Hospital for Sick Children first implemented industrial engineering methodology to medical imaging interventional radiology patient encounters, it focused on materials management. Early in the process, the return on investment became apparent as the department improved its management of more than 500,000 dollars of inventory. The calculated accumulated savings over 4 years for 10,000 interventional procedures alone amounted to more than 140,000 dollars. The medical imaging department in this hospital is only now beginning to apply what it has learned to other factors contributing to case cost. It has started to analyze its service contracts with equipment vendors. The department also is accumulating data to measure room, equipment, and labor utilization. The hospital now has a true picture of the real cost associated with each patient encounter in medical imaging. It can now begin to manage case costs, perform better capacity planning, create more effective relationships with its material suppliers, and optimize scheduling of patients and staff.

Cost Control↗
PubMed2005

Sequential immunogene therapy with interleukin-12- and interleukin-15-engineered neuroblastoma cells cures metastatic disease in syngeneic mice.

PURPOSE: To investigate the potential synergistic effects of Neuro2a neuroblastoma cells engineered with IL-12 and/or IL-15 genes in improving survival of syngeneic mice bearing neuroblastoma metastatic disease. EXPERIMENTAL DESIGN: Neuro2a cells engineered with interleukin (IL)-12 (Neuro2a/IL-12), IL-15 (Neuro2a/IL-15), or both cytokines (Neuro2a/IL-12/IL-15) were injected s.c. in syngeneic A/J mice challenged i.v. with Neuro2a parental cells (Neuro2apc) using different schedules of administration in either preventive or therapeutic settings. RESULTS: A single injection of Neuro2a/IL-12 or Neuro2a/IL-15 cells induced resistance to a subsequent i.v. Neuro2apc challenge in 45% and 28% of mice, respectively. Neuro2a/IL-12/IL-15 cells protected 28% of mice, showing no synergistic effect. However, sequential vaccination with Neuro2a/IL-12 (day -30) followed by Neuro2a/IL-15 (day -15) protected 71% of mice from subsequent challenge with Neuro2apc. A single dose of Neuro2a/IL-12 prolonged the mean survival time of mice bearing established metastatic neuroblastoma from 21 +/- 3 to 46 +/- 27 days but failed to cure mice, whereas Neuro2a/IL-15 or Neuro2a/IL-12/IL-15 were ineffective. However, sequential vaccination with Neuro2a/IL-12 (day +3) followed by Neuro2a/IL-15 (day +13) cured 43% of mice as assessed by histologic analysis of different organs from long-term surviving mice. CTL activity against Neuro2apc cells was observed in splenocytes from treated mice, and CD8(+) T-cell depletion abrogated the therapeutic effect of vaccination. CONCLUSIONS: Sequential vaccination with IL-12- and IL-15-engineered neuroblastoma cells induced optimal preventive and therapeutic effects, which may be related to the Th1 priming effect of IL-12 followed by the enhancement of CD8(+) T-cell responses and their maintenance mediated by IL-15.

Animals↗
PubMed2005

[Study of antithrombotic function of endothelium in vascular tissue engineering].

OBJECTIVE: To investigate the current situation and developing trend of antithrombotic function study of endothelium in vascular tissue engineering. METHODS: The effect of several elements on the antithrombotic ability of endothelium, including the source of endothelium, the characteristic of the matrix materials, the cell culture methods, and the endothelium's gene modification were analyzed. RESULTS: The normal antithrombotic function of tissue engineered vascular relied on the source of endothelium, gene modification of seeding cells, the cell culture methods in vitro, and the characteristic of the scaffolds. CONCLUSION: The establishment of an ideal antithrombotic functional tissue engineering vascular still requires further studies in various aspects including seeding cells, matrix materials, and cell culture methods. Gene modification of vascular endothelium, which improves the antithrombotic ability, deserves more attention.

Animals↗
PubMed2005

The ectopic study of tissue-engineered bone with hBMP-4 gene modified bone marrow stromal cells in rabbits.

BACKGROUND: Tissue-engineering techniques combined with gene therapy have been recently reported to improve osteogenesis. In this study, tissue-engineered bone constructed by human Bone Morphogenetic Protein 4 (hBMP-4) gene-modified bone marrow stromal cells (bMSCs) was explored in an ectopic bone formation model in rabbits. METHODS: A pEGFP-hBMP-4 mammalian plasmid (EGFP: Enhanced Green Fluorescent Protein) was constructed by subcloning techniques. bMSCs obtained from rabbits were cultured and transfected with either pEGFP-hBMP-4, pEGFP or left uninfected in vitro. Transfer efficiency was detected through the expression of EGFP. Transcription of the target gene was detected by RT-PCR. Alkaline phosphatase (ALP) and Von Kossa tests were also conducted to explore the phenotypes of osteoblasts. The autologous bMSCs of the 3 groups were then combined with Natural Non-organic Bone (NNB), a porous hydroxyapatite implant with a dimension of 6 mm x 6 mm x 3 mm, at a concentration of 5 x 10(7) cells/ml. They were subsequently implanted into 6 rabbits subcutaneously using NNB alone as a blank control (6 implants per group). Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation. RESULTS: pEGFP-hBMP-4 expression plasmid was constructed. Under optimal conditions, gene transfer efficiency reached more than 30%. Target gene transfer could strengthen the transcription of BMP-4, and increase the expression of ALP as well as the number of calcium nodules. In the ectopic animal model, NNB alone could not induce new bone formation. The new bone area formed in the bMSCs group was (17.2 +/- 7.1)%, and pEGFP group was (14.7 +/- 6.1)%, while pEGFP-hBMP-4 group was (29.5 +/- 8.2)%, which was the highest among the groups (F = 7.295, P < 0.01). CONCLUSIONS: The mammalian hBMP-4 expression plasmid was successfully constructed and a comparatively high transfer efficiency was achieved. The gene transfer technique enhanced the expression of BMP-4 and promoted differentiation from bMSCs to osteoblasts. These in vivo results suggested that transfection of bMSCs with hBMP-4 might be a suitable method to enhance their inherent osteogenic capacity for bone tissue engineering applications.

Animals↗
PubMed2004

[Effect of expression of platelet-derived growth factor B gene on blood vessel reconstruction after tissue engineering skin grafting].

OBJECTIVE: To study the effect of PDGF on dermal blood vessel reconstruction by transplanted tissue-engineering skin containing PDGF-B gene to rats. METHODS: The recombined eukaryotic expression vector, pcDNA3.1-hPDGF-B, was constructed and transfected into fibroblasts mediated by LipofectAMINE. Keratinocytes + acellular dermal matrix (group A), keratinocytes + acellular dermal matrix + fibroblasts (group B), keratinocytes + acellular dermal matrix + fibroblasts with PDGF gene (group C) were recombined respectively, then transplanted them to rat dorsum and evaluated the reconstruction of blood vessels in the dermis after 2, 4, 6 week postoperation. RESULTS: In 2-4 weeks after skin grafting the vascularization rate in group C was higher than that of group B and group A. The vascularization rates in all groups had no significant differences in six weeks (P > 0.05). CONCLUSION: PDGF-B gene plays an important role in reconstruction of blood vessels in the dermis at early tissue-engineering skin grafting, which ensures the take of grafted tissue-engineering skin.

Acellular Dermis↗
PubMed2005

[Fabrication and properties of a composite chitosan/type II collagen scaffold for tissue engineering cartilage].

OBJECTIVE: To develop a novel porous three-dimensional scaffold and to investigate its physico-chemical properties for tissue engineering cartilage. METHODS: Refined 88% deacetylation degree chitosan was prepared and dissolved in 0.2 mol/L acetate acid and fully mixed with highly purified porcine type II collagen in 0.5 mol/L acetate acid solution in a ratio of 4 to 1 (wt/wt). Freeze-drying process was employed to fabricate the composite scaffold. The construct was cross-linked by use of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). A mechanical tester was utilized to determine the tensile strength change before and after cross-linking. The microstructure was observed via scanning electron microscopy (SEM). The lysozyme degradation was performed to evaluate the degradability of the scaffold in vitro. RESULTS: A bulk scaffold with desired configuration was obtained. The mechanical test showed that the cross-linking treatment could enhance the mechanical strength of the scaffold. The SEM results revealed that the two constituents evenly distributed in the scaffold and that the matrix was porous, sponge-like with interconnected pore sizing 100-250 microm. In vitro lysozyme degradation indicated that cross-linked or uncross-linked composite scaffolds had faster degradation rate than the chitosan matrix. CONCLUSION: Chitosan and type II collagen can be developed into a porous three-dimensional scaffold. The related physico-chemical tests suggest that the composite scaffold meets requirements for tissue engineered scaffold and may serve as an alternative cell-carrier for tissue engineering cartilage.

Cartilage↗
PubMed2001

New experimental and theoretical tools for metabolic engineering of micro-organisms.

Presently an increasing gap is developing between our experimental capabilities in metabolic engineering of microbial metabolism and our quantitative theoretical understanding of the kinetic interaction between primary metabolism and product pathways. Such theoretical understanding is absolutely needed for a rational design of said metabolic engineering experiments and targets. To obtain such understanding in-vivo kinetic experiments and in-vivo kinetic models are needed. To this end the following new methods for in-vivo kinetic studies of Saccharomyces cerevisiae have recently been developed. The Bioscope device allows the reliable and repeated perturbation (e.g. glucose pulse, or inhibitor etc.) of steady state biomass outside the fermentor and subsequent sampling and quenching to measure glycolytical intermediates and nucleotides in a time frame of 0-70 seconds. Dynamic modelling of fermentor off-gas O2/CO2-measurements allows to calculate O2 uptake and CO2 production rates in such a perturbation experiment (0-70 seconds time windows). A new LC-MSMS based method has been developed to measure large sets of intracellular metabolites in said in-vivo kinetic experiments. It has been shown for the first time that in long chemostat cultivation (up to 800 hrs.) intracellular metabolites levels drop, showing absence of a real steady state. A new kinetic format, lin log kinetics, has been developed for describing the intracellular kinetic behavior of metabolic networks. This format allows general analytical solutions of networks flux, metabolic levels. From simulation studies it appears that this approach is remarkable accurate in describing intracellular metabolite dynamics and in metabolic design questions of where to change enzyme levels and how much to achieve a desired change in fluxes and metabolite levels. At this moment these tools are being applied in metabolic engineering studies of Saccharomyces cerevisiae and Penicillin chrysogenum.

Adenosine Triphosphate↗
PubMed2005

[Advances in research on calcium polyphosphate bioceramic for bone tissue engineering scaffold].

Bone tissue engineering is a novel, developing and challenging science which provides a new way to repair bone lost from injury and disease. Porous calcium polyphosphate bioceramic is one kind of absorptable bioceramic. Owing to its fine biocompatibility and degradability, more and more pieces of research wark have been carried out in bone tissue engineering, and because of its special characteristics, calcium polyphosphate bioceramic is regarded as a promising material for solving the problem of how to match the degradation velocity of scaffold with the velocity of cell growth. The recent research of using calcium polyphosphate bioceramic as the scaffold in bone tissue engineering is summarized, including the property, synthesis and advances.

Bone Substitutes↗
PubMed2005

[A new loading bioreactor for bone tissue-engineering applications].

Bone cells live in an environment heavily influenced by mechanical force. The development of bone tissue is dependent on the environment that surrounds it, both in vivo and in vitro. A loading stimulator on research of bone tissue-engineering was developed based on the mechanism of mechanosensation, scaffolding composites with mechanical strains with more physiologic magnitude, frequency components, and waveform. It also achieves the mechanical environment particularly in hard scaffold enough strong like cancellous bone. The device was tested using a reference scaffold made of better elastic plastic material. The experiment results showed that the device could be used in precision strain controls. Since the drive of the stimulator comes from the usage of smart material, piezoceramics, the strain at physiological level is controlled precisely. The stimulator provides a mechanical condition under which the effects of loading applied on bone tissue-engineering culture are conveniently investigated. Furthermore, after the stimulator is improved, it will be an appropriate bioreactor for bone tissue-engineering culture.

Bioreactors↗
PubMed2005

[Construction and clinical application of tissue engineered epidermal membrane].

OBJECTIVE: To Construct tissue engineered epidermal membrane for promoting healing of clinical skin graft donor site wound. METHODS: Epidermal cells /Chitosan-Gelatin (CG) membrane was constructed with cultured human epidermal cells(EC) and CG membrane, at the donor site of split skin graft, the wound was divided into three groups: the control group was covered with CG membrane without KC, KC/CG membrane was grafted on the treatment part of the wound area, and blank group was covered with traditional vaseline gauze. The engineered epidermal membrane and its effect on wound were evaluated with gross observation, HE, immunohistochemistry, collagen type I/III ratio by picrosirius polarization method and RT - PCR test at various time points before and after operation. RESULTS: The result showed that human tissue engineered epidermis could be constructed with cultured human EC and CG membrane, and this artificial epidermal membrane could be used for promoting the healing of skin graft donor site wound successfully (16 cases with 3 months' oberservation). The average healing time is (16.2 +/- 3.8) days for control group, (8.1 +/- 1.3) days for experimental group and (23.0 +/- 5.7) days for blank group. The artificial epidermis was well survived with normal structure. In addition, less hypertrophic scar development was observed in treated wound at 90 days (3 in 16 cases, 20.0%) than in the blank sites (11 in 16 cases, 74.4%). The difference is statistically significant (chi2 = 8.127, P < 0.01). CONCLUSIONS: The constructed EC-CG membrane can be constructed in vitro, survived in vivo and has good clinical application in promoting healing of skin graft donor site wound and inhibiting hypertrophic scar formation.

Adult↗
PubMed2005

[Application of chitosan in cartilage tissue engineering].

OBJECTIVE: To introduce the application of polymer material, chitosan, in the cartilage tissue engineering. METHODS: The recent original articles on the application of chitosan in cartilage tissue engineering were extensively reviewed. The biocompatibility and biodegradation characters of chitosan and its application were analysed. RESULTS: Chitosan has a high degree of biocompatibility and a favorable chondrogenic characteristic. It can support the maintenance of the phenotypic morphology of chondrocytes besides being used as a scaffold for cell growth. CONCLUSION: The perspective of the application of chitosan in cartilage tissue engineering is hopeful.

Absorbable Implants↗
PubMed2005

[Bone defect repair with a new tissue-engineered bone carrying bone morphogenetic protein in rabbits].

OBJECTIVE: To construct a new tissue-engineered bone with poly (D, L-lactide-co-glycolide) (PLGA), bone morphogenetic protein (BMP) and bone marrow-derived stem cells (BMSCs) and observe its effect in repairing segmental bone defects. METHODS: A 15-mm bone defect in the right radius was induced in New Zealand white rabbits, and the models were randomized into three groups to receive implantation of the tissue-engineered bone grafts constructed with PLGA carrying 5 mg BMP and about 1 x 10(6) BMSCs (experimental group), grafts of PLGA with about 1 x 10(6) BMSCs (control group), or grafts of exclusive PLGA (blank control group), respectively. The osteogenesis in the bone defect after the implantation on was evaluated X-ray films, and the histological changes of the tissues sampled from the bone defect 4, 8, and 12 weeks after operation were observed and new bone formation was measured by image analysis. RESULTS: The bone defect was completely repaired in the experimental group 12 weeks after the implantation, showing the best results among the 3 groups. The bone defects in the blank control group was filled with only fibrous and connective tissues at 12 weeks. CONCLUSION: This tissue-engineered bone constructed with PLGA, BMP and BMSCs possesses good ability in repairing segmental bone defect.

Animals↗