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PubMed2006

Tissue engineering using human embryonic stem cells.

The possibility of using stem cells for tissue engineering has greatly encouraged scientists to design new platforms in the field of regenerative and reconstructive medicine. Stem cells have the ability to rejuvenate and repair damaged tissues and can be derived from both embryonic and adult sources. Among cell types suggested as a cell source for tissue engineering (TE), human embryonic stem cells (hESCs) are one of the most promising candidates. Isolated from the inner cell mass of preimplantation stage blastocysts, they possess the ability to differentiate into practically all adult cell types. In addition, their unlimited self-renewal capacity enables the generation of sufficient amount of cells for cell-based TE applications. Yet, several important challenges are to be addressed, such as the isolation of the desired cell type and gaining control over its differentiation and proliferation. Ultimately, combing scaffolding and bioactive stimuli, newly designed bioengineered constructs, could be assembled and applied to various clinical applications. Here we define the culture conditions for the derivation of connective tissue lineage progenitors, design strategies, and highlight the special considerations when using hESCs for TE applications.

Cell Culture Techniques↗

PubMed2001

Tissue engineering and its potential impact on surgery.

The loss or failure of an organ or tissue is one of the most frequent, devastating, and costly problems in healthcare. Current treatment modalities include transplantation of organs, surgical reconstruction, use of mechanical devices, or supplementation of metabolic products. A new field, tissue engineering, applies the principles and methods of engineering, material science, and cell and molecular biology toward the development of viable substitutes which restore, maintain, or improve the function of human tissues. In this review, we outline the opportunities and challenges of this emerging interdisciplinary field and its attempts to provide solutions to tissue creation and repair. Within this context, we present our experience using the basic tools of tissue engineering to guide regeneration of diverse tissues that include the liver, small intestine, cardiovascular structures, nerve, and cartilage. And in addition, we discuss the necessity of finding new strategies to achieve vascularization of complex tissues for transplant and present our approaches utilizing MicroElectroMechanical Systems (MEMS) technology and three-dimensional printing.

Artificial Organs↗

PubMed2004

Does low-intensity pulsed ultrasound stimulate maturation of tissue-engineered cartilage?

Traumatic events are a primary cause of local lesions of articular cartilage. Tissue engineered, cartilage-like structures represent an alternative to current treatment methods. The time necessary for tissue maturation and the mechanical quality of the regenerate at implantation are both critical factors for clinical success. Low-intensity pulsed ultrasound has proven to accelerate chondrogenesis in vitro. The goal of this study was to evaluate whether low-intensity pulsed ultrasound is capable of accelerating the process of cartilage maturation and increasing regenerate stability. Hyaline-like cartilage specimens were generated in vitro and subcutaneously implanted in the backs of nude mice. Twenty-eight animals received 20 min of low-intensity pulsed ultrasound treatment daily, and 28 animals received a sham treatment. Specimens were explanted after 1, 3, 6, and 12 weeks, mechanically tested with the use of an indentation test, histologically examined, and processed for RT-PCR. The Young's moduli significantly increased from 3 to 12 weeks, and at 6 weeks were comparable to those of native articular cartilage. In histological examination, specimens showed neocartilage formation. There was no significant difference between ultrasound-treated and sham-treated groups. The mechanical stability of the neocartilage specimens increased with treatment time and reached values of native cartilage after 6 weeks in vivo. Low-intensity pulsed-ultrasound stimulation showed no stimulatory effect on tissue maturation. In contrast, ultrasound-treated specimens showed a reduced Col 2 expression at 1 week and were significantly less stiff compared to native cartilage at 6 and 12 weeks. An acceleration of the maturation of tissue-engineered neocartilage in a clinical setting by means of low-intensity pulsed ultrasound therefore appears rather unrealistic.

Cartilage↗

PubMed2005

Tissue engineering of bone: search for a better scaffold.

BACKGROUND: Large bone defects still represent a major problem in orthopedics. Traditional bone-repair treatments can be divided into two groups: the bone transport (Ilizarov technology) and the graft transplant (autologous or allogeneic bone grafts). Thus far, none of these strategies have proven to be always resolving. As an alternative, a tissue engineering approach has been proposed where osteogenic cells, bioceramic scaffolds, growth factors and physical forces concur to the bone defect repair. Different sources of osteoprogenitor cells have been suggested, bone marrow stromal cells (BMSC) being in most cases the first choice. METHODS AND RESULTS: In association with mineral tridimensional scaffolds, BMSC form a primary bone tissue which is highly vascularized and colonized by host hemopoietic marrow. The chemical composition of the scaffold is crucial for the osteoconductive properties and the resorbability of the material. In addition, scaffolds should have an internal structure permissive for vascular invasion. Porous bioceramics [hydroxyapatite (HA) and tricalcium phosphate] are osteoconductive and are particularly advantageous for bone tissue engineering application as they induce neither an immune nor an inflammatory response in the implanted host. Earlier, we first reported a cell-based tissue engineering procedure to treat three patients with long bone segmental defects. Cells were loaded on a 100% HA porous ceramic. These scaffolds proved to have good osteoconductive properties resulting in a good functional recovery, but they have not been resorbed after more than 5 years from the implant. In addition, due to the high density of the mineral and the relatively low porosity (50-60%), it was very difficult to monitor the patient recovery during the post-surgery time using X-rays. CONCLUSIONS: We report here some pre-clinical testing of new scaffolds. To compare these second generation ceramic scaffolds more suitable for a tissue engineering approach we had to first establish animal models and analysis procedures including the use of X-ray-computed microtomography associated with X-rays synchroton radiation.

Animals↗

PubMed2006

Experimental study on low intensity ultrasound and tissue engineering to repair segmental bone defects.

In order to evaluate the efficacy of low intensity ultrasound and tissue engineering technique to repair segmental bone defects, the rabbit models of 1.5-cm long rabbit radial segmental osteoperiosteum defects were established and randomly divided into 2 groups. All defects were implanted with the composite of calcium phosphate cement and bone mesenchymal stem cells, and additionally those in experimental group were subjected to low intensity ultrasound exposure, while those in control group to sham exposure. The animals were killed on the postoperative week 4, 8 and 12 respectively, and specimens were harvested. By using radiography and the methods of biomechanics, histomorphology and bone density detection, new bone formation and material degradation were observed. The results showed that with the prolongation of time after operation, serum alkaline phosphatase (AKP) levels in both groups were gradually increased, especially in experimental group, reached the peak at 6th week (experimental group: 1.26 mmol/L; control group: 0.58 mmol/L), suggesting the new bone formation in both two group, but the amount of new bone formation was greater and bone repairing capacity stronger in experimental group than in control group. On the 4th week in experimental group, chondrocytes differentiated into woven bone, and on the 12th week, remodeling of new lamellar bone and absorption of the composite material were observed. The mechanical strength of composite material and new born density in experimental group were significantly higher than in control group, indicating that low intensity ultrasound could not only effectively increase the formation of new bone, but also accelerate the calcification of new bone. It was concluded that low intensity ultrasound could evidently accelerate the healing of bone defects repaired by bone tissue engineering.

Animals↗

PubMed2006

Depth-dependent biomechanical and biochemical properties of fetal, newborn, and tissue-engineered articular cartilage.

Adult articular cartilage has depth-dependent mechanical and biochemical properties which contribute to zone-specific functions. The compressive moduli of immature cartilage and tissue-engineered cartilage are known to be lower than those of adult cartilage. The objective of this study was to determine if such tissues exhibit depth-dependent compressive properties, and how these depth-varying properties were correlated with cell and matrix composition of the tissue. The compressive moduli of fetal and newborn bovine articular cartilage increased with depth (p<0.05) by a factor of 4-5 from the top 0.1 mm (28+/-13 kPa, 141+/-10 kPa, respectively) to 1 mm deep into the tissue. Likewise, the glycosaminoglycan and collagen content increased with depth (both p<0.001), and correlated with the modulus (both p<0.01). In contrast, tissue-engineered cartilage formed by either layering or mixing cells from the superficial and middle zone of articular cartilage exhibited similarly soft regions at both construct surfaces, as exemplified by large equilibrium strains. The properties of immature cartilage may provide a template for developing tissue-engineered cartilage which aims to repair cartilage defects by recapitulating the natural development and growth processes. These results suggest that while depth-dependent properties may be important to engineer into cartilage constructs, issues other than cell heterogeneity must be addressed to generate such tissues.

Animals↗

PubMed2006

Alveolar cleft osteoplasty using tissue-engineered osteogenic material.

The use of tissue-engineered osteogenic material comprising platelet-rich plasma and autologous mesenchymal stem cells isolated, expanded and induced to osteogenic potential in bone augmentation procedures as a replacement for autologous bone grafts, offers predictable results with minimal donor-site morbidity. This material was applied for an alveolar cleft osteoplasty of a 9-year-old female patient. Serial computed tomograms showed the regenerated bone extending from the cleft walls after 3 months and bridging the cleft after 6 months, with 79.1% of the grafted region after 9 months at the time when the canine and lateral incisor in the affected side erupted in the reconstructed alveolar ridge.

Alveolar Process↗

PubMed2004

Assessment of tissue-engineered stomach derived from isolated epithelium organoid units.

OBJECTIVE: Isolated stomach epithelial organoid units developed on biodegradable polymers were transplanted to assess the feasibility of a tissue-engineered stomach. BACKGROUND: Despite recent advances in reconstruction techniques, total gastrectomy is still accompanied by various complications. An alternative treatment would be a tissue-engineered stomach, which replaces the mechanical and metabolic functions of a normal stomach. METHODS: Stomach epithelial organoid units isolated from neonatal rats were seeded onto biodegradable polymers. The constructs implanted into the omenta of adult rats were harvested for examination at designated times. Nine rats underwent a second operation for anastomosis. RESULTS: The constructs resulted in cyst-like formations showing vascularized tissue with neomucosa lining the lumen. The surface morphology as assessed using scanning electron microscopy was similar to that of a native stomach. Immunohistochemical staining for alpha-actin smooth muscle and gastric mucin indicated the presence of a smooth muscle layer and a well-developed gastric epithelium, respectively. The luminal surface of the anastomosed tissue-engineered stomach was well-covered with epithelium. CONCLUSIONS: Epithelium-derived stomach organoid units seeded on biodegradable polymers and transplanted into donor rats were shown to vascularize, survive, and regenerate into complex tissue resembling native stomach. Anastomosis between the units and native small intestine may have the potential to stimulate epithelial growth. This research may provide insight into new approaches to alleviate complications following total gastrectomy.

Animals↗

PubMed2004

Characterization of statically loaded tissue-engineered mitral valve chordae tendineae.

Chordae tendineae are essential to the proper function of the mitral valve. Native chordae contain a dense collagenous core and an outer elastin sheath. We have been using the principle of directed collagen gel shrinkage to fabricate tissue-engineered mitral valve chordae. Because the microstructure of biologic tissues determines their mechanical behavior, the morphology of collagen and elastin in tissue-engineered chordae should mimic that of native chordae. The objective of this study, therefore, was to examine the morphology of our tissue-engineered constructs in comparison to native chordae. A collagen-cell suspension was cast into silicon rubber wells with microporous anchors at the ends and cultured in an incubator. The anchors allowed shrinkage to occur only transverse to the long axis of the wells, thus creating highly aligned collagen fibril constructs. The collagen constructs were cultured for 8 weeks and characterized mechanically, histologically, and biochemically at different culture time points. Histologic sections showed that in all mature constructs collagen fibers were oriented parallel to the long axis of the constructs. At the edge of the tissue collagen fibers were in general straight, whereas in the middle of the tissue they were wavy. Transmission electron microscopy showed a progressive increase in the density and longitudinal orientation of collagen fibrils with culture time. Light and scanning electron microscopy showed the presence of an elastin sheath around the collagen core. Immunostaining demonstrated that smooth muscle cells differentiate during tissue development and TUNEL assay showed that cells in the interior of the constructs undergo apoptosis. This study has demonstrated that collagen-cell constructs, with material properties and microstructure similar to native mitral valve chordae, can be developed using static culture.

Animals↗

PubMed1997

A novel gene delivery system using urothelial tissue engineered neo-organs.

PURPOSE: Presently gene delivery is most effectively achieved by ex vivo gene transfer, which includes removal of the target tissue, in vitro gene delivery to the target cells, possible selection to enhance the proportion of transfected cells and reintroduction of the gene modified cells. Reintroduction of transformed cells in vivo has been a challenging task. Based on the feasibility of tissue engineering techniques in which cells seeded on biodegradable polymer scaffolds form tissue when implanted in vivo, we explored the possibility of developing a neo-organ system for in vivo gene therapy. MATERIALS AND METHODS: Normal human urothelial cells were harvested, expanded in vitro and seeded on biodegradable polymer scaffolds. The cell-polymer complex was then transfected with PGL3-luc, pCMV-luc and pCMV beta-gal promoter reporter gene constructs. The transfected cell-polymer scaffolds were then implanted in athymic mice and the engineered tissue was retrieved 0, 1, 3, 5 and 7 days after implantation. RESULTS: The reporter gene assay demonstrated an expression of luciferase activity at days 1, 3, 5 and 7 with the peak at day 5. X-gal and beta-galactosidase antibody assays stained positive on the deoxyribonucleic acid treated transfection. CONCLUSIONS: Successful gene transfer can be achieved using biodegradable polymer scaffolds as a urothelial cell delivery vehicle. The transfected cell-polymer scaffold forms an organ-like structure with functional expression of the transfected genes. This study demonstrates that urothelial tissue engineered gene transfer is safe and effective.

Animals↗

PubMed2004

Tissue engineering trachea: Malaysian experience.

Management of severe tracheal anomalies remains a clinical challenge. Tissue engineering offers new hope in trachea reconstruction surgery. However to date no optimal technique achieved in the formation of human or animal trachea. The main problem lies on the biomaterial used and the complex city of forming trachea in vivo. This study was aimed at creating tissue-engineered trachea cartilage from easily accessible human and animal nasal septum cartilage using internal scaffold and biodegradable human and animal fibrin.

Absorbable Implants↗

PubMed2004

Tissue-engineered human nasal septal cartilage using the alginate-recovered-chondrocyte method.

OBJECTIVES: Tissue engineering of nasal septal cartilage has numerous potential applications in craniofacial reconstruction. Chondrocytes suspended in alginate gel have been shown to produce a substantial cell-associated matrix. The objective of this study was to determine whether cartilage tissue could be generated using the alginate-recovered-chondrocyte (ARC) method, in which chondrocytes are cultured in alginate as an intermediate step in tissue fabrication. METHODS: Nasal septal chondrocytes from five patient donors were isolated by enzymatic digestion, then expanded in monolayer culture. At confluency, a portion of those cells were seeded at high density onto a semipermeable membrane and cultured for 14, 21, or 28 days (monolayer group). The remaining cells were suspended in alginate and cultured until a cell-associated matrix was observed (10-17 days). Cells and their associated matrix were released from alginate (ARC group), seeded onto a semipermeable membrane, and cultured as already described. DNA (Hoechst 33258 Assay), glycosaminoglycan (GAG; dimethylmethylene blue assay), and collagen (hydroxyproline assay) were analyzed biochemically. Immunohistochemistry was performed to assess expression of collagens type I and type II. Histochemistry was performed to localize cells accumulating sulfated GAG (Alcian blue stain). RESULTS: The ARC constructs, in contrast to the monolayer constructs, had substantial structural stability and the histologic and gross appearance of cartilaginous tissue. ARC constructs demonstrated significantly greater GAG and collagen accumulation than monolayer constructs (P <.05). Histologic analysis revealed substantial GAG and collagen type II production and only moderate collagen type I production. The composition of the matrix was thus similar to that of native human septal cartilage. CONCLUSIONS: Tissue-engineered human nasal septal cartilage using the ARC method has the histologic and gross appearance of native cartilage and has biochemical composition more like that of native cartilage than monolayer constructs. This is the first report of human nasal septal neocartilage formation without the use of biodegradable scaffolds.

Alginates↗

PubMedDate not supplied

Rapid prototyping of tissue-engineering constructs, using photopolymerizable hydrogels and stereolithography.

One of the most important aspects of tissue engineering is the design of the scaffold providing the mechanical strength and access to nutrients for the new tissue. For customized tissue engineering, it is essential to be able to fabricate three-dimensional scaffolds of various geometric shapes, in order to repair defects caused by accidents, surgery, or birth. Rapid prototyping or solid free-form fabrication (SFF) techniques hold great promise for designing three-dimensional customized scaffolds, yet traditional cell-seeding techniques may not provide enough cell mass for larger constructs. This article presents a novel attempt to fabricate three-dimensional scaffolds, using hydrogels combined with cell encapsulation to fabricate high-density tissue constructs. A commercially available stereolithography technique was applied to fabricate scaffolds using poly(ethylene oxide) and poly(ethylene glycol)dimethacrylate photopolymerizable hydrogels. Mechanical characterization shows the constructs to be comparable with soft tissues in terms of elasticity. High cell viability was achieved and high-density constructs fabricated.

Animals↗

PubMed2005

Tissue-engineered cartilage using fibrin/hyaluronan composite gel and its in vivo implantation.

The importance of scaffold biomaterials has been emphasized for in vitro culture of tissue-engineered cartilage in a three-dimensional (3D) environment. In this study, we examined the feasibility of fibrin glue, mixed with hyaluronic acid (HA) as a composite scaffold. Fibrin glue has been a useful cell delivery matrix for cartilage tissue engineering and HA is a key component of normal articular cartilage. Our hypothesis is that compared to fibrin itself, a fibrin/HA composite can have significantly enhanced properties, due mainly to the added benefits of HA in the matrix. Pieces of cartilage were isolated from rabbit knees and the chondrocytes were harvested through enzymatic digestion. Both fibrin and fibrin/HA composite were prepared and subsequently implanted in nude mice (n = 9, each group) for 1, 2, and 4 weeks, respectively. The retrieved specimens were then analyzed and the results were compared. Cartilage-like tissue formation was detected earlier with fibrin/HA specimens. They produced significantly higher amounts of the extracellular matrix (ECM) molecules, GAG, and collagen at each time point than those in fibrin. Interestingly, the fibrin/HA composite was also competent in maintaining its initial size. Histology--Safranin O/fast green and Alcian blue--of the retrieved specimens found more intense, uniform staining in the fibrin/HA composites. Analysis of the gene expression of the ECM molecules also confirmed the benefits of the composite with added HA in the maintenance of phenotypic stability. The present study suggests that fibrin/HA composite may serve as a dependable cell delivery vehicle as well as a structural basis for tissue-engineered cartilage.

Adjuvants, Immunologic↗

PubMed2000

Smooth muscle cell adhesion to tissue engineering scaffolds.

Synthetic polyesters of lactic and glycolic acid, and the extracellular matrix molecule collagen are among the most widely-utilized scaffolding materials in tissue engineering. However, the mechanism of cell adhesion to these tissue engineering scaffolds has not been extensively studied. In this paper, the mechanism of adhesion of smooth muscle cells to these materials was investigated. Vitronectin was found to be the predominant matrix protein adsorbed from serum-containing medium onto polyglycolic acid, poly(lactic co-glycolic) acid, and collagen two-dimensional films and three-dimensional scaffolds. Fibronectin adsorbed to both materials as well, although to a much lower density. Smooth muscle cell adhesion was mediated through specific integrin receptors interacting with these adsorbed proteins, as evidenced by both immunostaining and blocking studies. The receptors involved in adhesion included the alpha(v)beta5 to vitronectin, the alpha5beta1 to fibronectin and the alpha2beta1 to collagen I. Identification of the specific receptors used to adhere to these polymers clarifies why smooth muscle tissue development differs on these scaffolds, and may allow one to design tissue formation by controlling the surface chemistry of tissue engineering scaffolds.

Adsorption↗

PubMed2006

Prevention of device-related tissue damage during percutaneous deployment of tissue-engineered heart valves.

BACKGROUND: Endovascular application of pulmonary heart valves has been recently introduced clinically. A tissue-engineering approach was pursued to overcome the current limitations of bovine jugular vein valves (degeneration and limited longevity). However, deployment of the delicate tissue-engineered valves resulted in severe tissue damage. Therefore the objective of this study was to prevent tissue damage during the folding and deployment maneuver. MATERIAL AND METHODS: Porcine pulmonary heart valves, small intestinal submucosa, and ovine carotid arteries were obtained from a slaughterhouse. After dissection and antimicrobial incubation, the valves were trimmed (removal of sinus and most of the muscular ring) to fit into the deployment catheter. The inside (in-stent group, n = 6) or outside (out-stent group, n = 6) of a nitinol stent was covered by an acellular small intestinal submucosa, and the valves were sutured into the stent. The valves were folded, tested for placement in the deployment catheter, and decellularized enzymatically. Myofibroblasts were obtained from carotid artery segments and seeded onto the scaffolds. The seeded constructs were placed in a dynamic bioreactor system and cultured for 16 consecutive days. After endothelial cell seeding, the constructs were folded, deployed, and processed for histology and surface electron microscopy. RESULTS: The valves opened and closed competently throughout the entire dynamic culture. Surface electron microscopy revealed an almost completely preserved tissue in the in-stent group. Stents covered with small intestinal submucosa on the outside, however, showed severe damage. CONCLUSION: This study demonstrates that small intestinal submucosa covering of the inside of a pulmonary valved stent can prevent stent strut-related tissue damage.

Animals↗

PubMed2003

The notochordal cell in the nucleus pulposus: a review in the context of tissue engineering.

An understanding of developmental biology can provide useful insights into how different tissue-engineered repairs might be designed. During embryogenesis of the intervertebral disk, the cells of the notochord play a critical role in initiating tissue formation, and may be responsible for development of the nucleus pulposus. In some species, including humans, these notochordal cells may eventually be lost, either through apoptosis or terminal differentiation, and are replaced by chondrocyte-like cells. However, there is some evidence that the notochordal cells may persist in at least some humans. This review discusses some of the potential applications of notochordal cells in tissue engineering of the nucleus pulposus.

Animals↗

PubMed2004

Fetal bone cells for tissue engineering.

We envision the use of human fetal bone cells for engineered regeneration of adult skeletal tissue. A description of their cellular function is then necessary. To our knowledge, there is no description of human primary fetal bone cells treated with differentiation factors. The characterization of fetal bone cells is particularly important as the pattern of secreted proteins from osteoblasts has been shown to change during aging. In the first part of this work, human primary fetal bone cells were compared to adult bone cells and mesenchymal stem cells for their ability to proliferate and to differentiate into osteoblasts in vitro. Cell proliferation, gene expression of bone markers, alkaline phosphatase (ALP) activity, and mineralization were analyzed during a time-course study. In the second part of this paper, bone fetal cells behavior exposed to osteogenic factors is further detailed. The doubling time of fetal bone cells was comparable to mesenchymal stem cells but significantly shorter than for adult bone cells. Gene expression of cbfa-1, ALP, alpha1 chain of type I collagen, and osteocalcin were upregulated in fetal bone cells after 12 days of treatment, with higher inductions than for adult and mesenchymal stem cells. The increase of ALP enzymatic activity was stronger for fetal than for adult bone cells reaching a maximum at day 10, but lower than for mesenchymal stem cells. Importantly, the mineralization process of bone fetal cells started earlier than adult bone and mesenchymal stem cells. Proliferation of fetal and adult bone cells was increased by dexamethasone, whereas 1alpha,25-dihydroxyvitamin D3 did not show any proliferative effect. Mineralization studies clearly demonstrated the presence of calcium deposits in the extracellular matrix of fetal bone cells. Nodule formation and calcification were strongly increased by the differentiation treatment, especially by dexamethasone. This study shows for the first time that human primary fetal bone cells could be of great interest for bone research, due to their fast growth rate and their ability to differentiate into mature osteoblasts. They represent an interesting and promising potential for therapeutic use in bone tissue engineering.

Adult↗