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PubMed2005

Assimilating cell sheets and hybrid scaffolds for dermal tissue engineering.

Cell sheets can be used to produce neo-tissue with mature extracellular matrix. However, extensive contraction of cell sheets remains a problem. We devised a technique to overcome this problem and applied it to tissue engineer a dermal construct. Human dermal fibroblasts were cultured with poly(lactic-co-glycolic acid)-collagen meshes and collagen-hyaluronic acid foams. Resulting cell sheets were folded over the scaffolds to form dermal constructs. Human keratinocytes were cultured on these dermal constructs to assess their ability to support bilayered skin regeneration. Dermal constructs produced with collagen-hyaluronic acid foams showed minimal contraction, while those with poly(lactic-co-glycolic acid)-collagen meshes curled up. Cell proliferation and metabolic activity profiles were characterized with PicoGreen and AlamarBlue assays, respectively. Fluorescent labeling showed high cell viability and F-actin expression within the constructs. Collagen deposition was detected by immunocytochemistry and electron microscopy. Transforming Growth Factor-alpha and beta1, Keratinocyte Growth Factor and Vascular Endothelial Growth Factor were produced at various stages of culture, measured by RT-PCR and ELISA. These results indicated that assimilating cell sheets with mechanically stable scaffolds could produce viable dermal-like constructs that do not contract. Repeated enzymatic treatment cycles for cell expansion is unnecessary, while the issue of poor cell seeding efficiency in scaffolds is eliminated.

Actins↗

PubMed2005

Tissue engineered cartilage on collagen and PHBV matrices.

Cartilage engineering is a very novel approach to tissue repair through use of implants. Matrices of collagen containing calcium phosphate (CaP-Gelfix), and matrices of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) were produced to create a cartilage via tissue engineering. The matrices were characterized by scanning electron microscopy (SEM) and electron diffraction spectroscopy (EDS). Porosity and void volume analysis were carried out to characterize the matrices. Chondrocytes were isolated from the proximal humerus of 22 week-old male, adult, local albino rabbits. For cell type characterization, Type II collagen was measured by Western Blot analysis. The foams were seeded with 1x10(6) chondrocytes and histological examinations were carried out to assess cell-matrix interaction. Macroscopic examination showed that PHBV (with or without chondrocytes) maintained its integrity for 21 days, while CaP-Gelfix was deformed and degraded within 15 days. Cell-containing and cell-free matrices were implanted into full thickness cartilage defects (4.5 mm in diameter and 4 mm in depth) at the patellar groove on the right and left knees of eight rabbits, respectively. In vivo results at 8 and 20 weeks with chondrocyte seeded PHBV matrices presented early cartilage formation resembling normal articular cartilage and revealed minimal foreign body reaction. In CaP-Gelfix matrices, fibrocartilage formation and bone invasion was noted in 20 weeks. Cells maintained their phenotype in both matrices. PHBV had better healing response than CaP-Gelfix. Both matrices were effective in cartilage regeneration. These matrices have great potential for use in the repair of joint cartilage defects.

Absorbable Implants↗

PubMed2005

Fibronectin-hepatocyte growth factor enhances reendothelialization in tissue-engineered heart valve.

BACKGROUND: To overcome the limitations of tissue-engineered heart valves, which require cell seeding before implantation, a growth factor for in situ recellularization may be an important strategy. We developed a new decellularized valve containing a fusion protein combined fibronectin and hepatocyte growth factor. Here, we tested the hypothesis that our valve might accelerate in situ recellularization by inducing the proliferation of endothelial cells. METHODS: Porcine aortic valves were decellularized using detergent. Fibronectin-hepatocyte growth factor was introduced into the decellularized valves. The decellularized valves with fibronectin-hepatocyte growth factor were implanted into the pulmonary arterial trunk of dogs (F group: n = 15). As controls, decellularized valves without the growth factor (C group: n = 12), and with hepatocyte growth factor (H group: n = 12) were implanted in the same manner. Histologic examinations were performed 1 week and 1 month after implantation. RESULTS: One week after implantation, endothelial cells partially covered the surface of the graft in the F group but not the C and H groups. Although the C and H groups had inadequate recellularization 1 month after implantation, the F group showed a monolayer of endothelial cells, underneath which were areas of additional cell layers, which were vimentin positive. Quantitative evaluation demonstrated the amount of vimentin in the F group was 71% of the native control, and it was much lower in the other groups (C, 2.8%; H, 16.8%) 1 month after implantation. CONCLUSIONS: This study demonstrated that fibronectin-hepatocyte growth factor enhanced early in situ recellularization in decellularized valves.

Animals↗

PubMed2003

In vivo forces used to develop design parameters for tissue engineered implants for rabbit patellar tendon repair.

Previous studies in tissue engineering have shown that suspending undifferentiated mesenchymal stem cells in collagen gels and wrapping them about a suture causes alignment of cells and contraction of constructs in culture in a form that is suitable for implantation for tendon repair. Little is known about the patterns of these in vivo signals that might improve tendon repair biomechanics. Three hypotheses were tested in this study using the rabbit patellar tendon (PT) model: (1) peak in vivo forces and the rates of rise and fall in these forces will increase significantly with increasing levels of activity; (2) the PTs safety factor for all activities will be in the range of values found for tendons (2.5-3); (3) rabbits will not "favor" the operated limb at the time of evaluation but maintain similar vertical ground reaction forces in both limbs during quiet standing (QS). In vivo rabbit PT forces were measured during QS and while the animal hopped on a treadmill whose speed (0.04 and 0.13 m/s) and inclination (0 degrees and 12 degrees) were controlled. Implantable force transducers were surgically placed in one PT and data collected three days post surgery in each of eight New Zealand White rabbits. Peak tensile forces increased significantly with inclination of the treadmill and the rates of rise and fall in tendon force increased significantly with both speed and inclination (p<0.001). Such design criteria should be useful in mechanically stimulating cell-gel constructs for tendon repair.

Animals↗

PubMed2006

Biomimetic bilayered gelatin-chondroitin 6 sulfate-hyaluronic acid biopolymer as a scaffold for skin equivalent tissue engineering.

In order to develop an adequate scaffold for skin tissue engineering, a bilayered gelatin-chondroitin 6 sulfate-hyaluronic acid membrane with a different pore size on either side was prepared. A rete ridges-like topographic microporous structure, which provided the paracrine crosstalk in the epithelial-mesenchymal interactions, was formed. Chondroitin-6-sulfate and hyaluronic acid were incorporated within the gelatin membrane to mimic skin composition and create an appropriate microenvironment for cell proliferation, differentiation, and migration. In the study, the lower layer of the membrane (pore size: 150 microm) was seeded with dermal fibroblasts and acted as the feeder layer for keratinocyte inoculation. Meanwhile, the upper layer (pore size: 20-50 microm) was seeded with keratinocytes for epidermalization. The dermal fibroblasts were dynamically seeded in a self-designed spinner flask for more even cell distribution. The keratinocytes were cultured in submerged conditions for 5 days and then in an air-liquid interface condition for further differentiation. After being cultured for 21 days, the upper layer, seeded with keratinocytes, developed into an epidermis-like structure while the lower part, which was seeded with dermal fibroblasts developed into a dermis-like structure. A histological examination and immunostain were used to prove that keratinocytes maintain their phenotype and stratified epidermis layers were formed within 21 days. In brief, the bilayered skin substitute with biological dermal analog and epidermal structure was successfully fabricated. From this study, we can suggest that the culture model is suitable for autologous skin equivalent preparation.

Adjuvants, Immunologic↗

PubMed2006

Porogen-induced surface modification of nano-fibrous poly(L-lactic acid) scaffolds for tissue engineering.

In addition to suitable pore architecture of a tissue-engineering scaffold, surface modification after scaffolding fabrication is often needed to enhance the interactions between cells and the synthetic material. In this study, a novel one-step process was developed to fabricate surface-modified nano-fibrous poly(L-lactic acid) (NF-PLLA) scaffolds. First, gelatin spheres with smooth surface were prepared by non-surfactant emulsification, solvent extraction, and freeze-drying. A three-dimensional NF-PLLA scaffold was then fabricated by using gelatin spheres as porogen. Gelatin molecules were entrapped onto the scaffold surface during the fabrication process. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy analysis demonstrated the existence of gelatin molecules on the surface of the polymer scaffold. The amount of gelatin on the scaffold surface was controlled by the composition of the solvent mixture of gelatin solution. The compressive modulus of scaffold prepared with gelatin spheres was more than three times higher than that prepared with irregular gelatin particles of the same size range. The surface modification significantly improved initial cell adhesion and proliferation over a 2-week culture. SEM images indicated that cells spread on the gelatin-entrapped scaffolds in contrast to spherical or spindle morphology on the control 1 day after cell seeding. Furthermore, more matrix secretion was observed on the surface-modified scaffolds than on the control after 2 weeks of in vitro cultivation. In conclusion, this approach provides a simple one-step process to fabricate surface-modified collagen-like NF-PLLA scaffolds, which have improved cell adhesion and proliferation.

3T3 Cells↗

PubMed1993

Tissue engineering.

The loss or failure of an organ or tissue is one of the most frequent, devastating, and costly problems in human health care. A new field, tissue engineering, applies the principles of biology and engineering to the development of functional substitutes for damaged tissue. This article discusses the foundations and challenges of this interdisciplinary field and its attempts to provide solutions to tissue creation and repair.

Animals↗

PubMed2006

[A potential use of collagen-hyaluronan-chondroitin sulfate tri-copolymer scaffold for cartilage tissue engineering].

OBJECTIVE: To evaluate collagen(Col)-hyaluronan (HA)-chondroitin sulfate (CS) tri-copolymer as a new biomimetic biodegradable polymer scaffold for application of the articular cartilage tissue engineering. METHODS: The Col-HA-CS tri-copolymer was prepared by freezing and lyophilization and was cross-linked by 1-ethyl-3-(3-dimethy inaminoproyl) carbodiimide (EDC). The morphological characteristics of the matrices were evaluated by the SME and HE stainings. The rabbit chondrocytes were isolated and seeded in the tri-copolymer scaffold. Morphology, proliferation and differentiation of glycosaminoglycan (GAG), and phenotypic expression of the rabbit articular chondrocytes cultured within the tri-copolymer scaffold were indicated by the histological examination, SEM, biochemical analysis, and reverse transcriptase PCR for collagen type II (Col II). RESULTS: The chondrocytes proliferated and differentiated well, and they preserved the phenotypic expression of Col II in the Col-HA-CS scaffold. After the 21-day cell culture within the Col-HA-CS scaffolds, the cartilage-specific morphology and the structural characteristics such as lacunae appeared, and DNA and GAG contents increased with the time. In addition, DNA and GAG contents were significantly higher in the Col-HA-CS matrix than in the collagen matrix alone (P < 0.05). CONCLUSION: These results show that the Col-HA-CS tri-copolymer matrices can provide an appropriate environment for the generation of cartilage-like tissues and have a potential application in the cartilage tissue engineering scaffold field.

Animals↗

PubMed2004

Contractile skeletal muscle tissue-engineered on an acellular scaffold.

For the reconstructive surgeon, tissue-engineered skeletal muscle may offer reduced donor-site morbidity and an unlimited supply of tissue. Using an acellularized mouse extensor digitorum longus muscle as a scaffold, the authors produced engineered skeletal muscle capable of generating longitudinal force. Eight extensor digitorum longus muscles from adult mice were made acellular using a protocol developed in the authors' laboratory. The acellular muscles were then placed in a bath of 20% fetal bovine serum in Dulbecco's modified Eagle's medium and 100 U/ml penicillin for 1 week at room temperature. C2C12 myoblasts were injected into the acellular muscle matrix using a 26-gauge needle and a 100-microl syringe. The resulting constructs were placed in growth medium for 1 week at 37 degrees C under 5% carbon dioxide, with media changes every 48 hours. The constructs were then placed in differentiation medium for 1 week, with media changes every 48 hours. Isometric contractile force testing of the constructs demonstrated production of longitudinal contractile force on electrical stimulation. A length-tension, or Starling, relationship was observed. Light and electron microscopy studies demonstrated recapitulation of some of the normal histologic features of developing skeletal muscle.

Animals↗

PubMed2003

Tissue engineering skin flaps: which vascular carrier, arteriovenous shunt loop or arteriovenous bundle, has more potential for angiogenesis and tissue generation?

This study was designed to clarify which vascular carrier, the arteriovenous shunt loop or the arteriovenous bundle, has more potential as a vascular carrier for an artificial skin flap in rats. An arteriovenous shunt loop was constructed between the femoral artery and vein using an interpositional artery (group I) or vein (group II) graft. For arteriovenous bundle groups, the femoral artery and vein were used and subdivided into two groups: distal ligation type (group III) and flow-through type (group IV). The vascular pedicle was wrapped with an artificial dermis and implanted beneath the inguinal skin for 4 weeks. For the control group, a folded sheet of artificial dermis without any vascular carrier was embedded. In experiment 1, the volumes of generated tissue within the artificial dermis were measured in the experimental and control groups (n = 5 in each group). In experiment 2, the origin of new blood vessels sprouting from the arteriovenous shunt loop and arteriovenous bundle were evaluated histologically. The volume of generated tissue in the shunt groups was significantly greater than that in the bundle groups (p < 0.01). However, the bundle groups also showed a great potential for producing new tissue. Serial histological studies showed that new capillaries were derived not only from the vasa vasorum of the femoral vessels but directly from the femoral vein in both the shunt and the bundle groups. This "sprouting" was extensively exhibited in the group III. Although the arteriovenous shunt loop showed a greater potential for producing new tissue and capillaries, the distal ligation type of bundle was thought to be an effective and practical vascular carrier for producing a tissue-engineered skin flap.

Anastomosis, Surgical↗

PubMed2005

New frontiers in calvarial reconstruction: integrating computer-assisted design and tissue engineering in cranioplasty.

Repair of large and complex calvarial defects remains a particular challenge for reconstruction. The paucity of techniques and materials emphasizes the need for alternative bone formation strategies. Recent integrative approaches suggest that successful reconstruction requires interdisciplinary teams, with surgeons interacting with imaging experts, materials scientists, and engineers. In this review, the authors present an overview of current materials used in calvarial reconstruction. Subsequently, progress in computer-designed prostheses, tissue engineering, and osteoinduction strategies is discussed. Finally, the authors discuss their experience with the integration of computer-aided fabrication of customized implants and tissue engineering for calvarial reconstruction.

Animals↗

PubMed2005

[Tissue engineered bone reconstruction with modified PLGA/Type-I collagen compound scaffold].

OBJECTIVE: To fabricate bone grafts by bone marrow stromal cell combined with modified PLGA/Type-I collagen compound scaffold using tissue engineering method. METHODS: The modified PLGA/Type-I collagen compound scaffold was fabricated. The rabbit primary cultured osteoblasts were identified and seeded onto the modified compound scaffold for one week in vitro. The adhesion and growth of cells were observed with scanning electron microscope. The complex of cells and scaffold was implanted into the subcutaneous region of rabbits and new bone formation was evaluated. RESULTS: The rabbit bone marrow stromal cells were induced and differentiated into osteoblasts. The adhesion and growth of osteoblasts in cluster were observed on the surface of scaffolds. New bone formation was observed at one month postoperatively and active osteoblasts were found on the surface of the newly formed bone in vivo. CONCLUSION: The complex of PLGA and type-I collagen is an appropriate biodegradable scaffold and can be applied in bone tissue engineering.

Absorbable Implants↗

PubMed2006

Adventitia contribution in vascular tone: insights from adventitia-derived cells in a tissue-engineered human blood vessel.

Whether the adventitia component of blood vessels directly participates in the regulation of vascular tone remains to be demonstrated. We have recently developed a human tissue-engineered blood vessel comprising the three tunicae of a native blood vessel using the self-assembly approach. To investigate the role of the adventitia in the modulation of vascular tone, this tissue-engineering method was used to produce three vascular constructs from cells explanted and proliferated from donor vessel tunicae 1) an adventitia + a media, or only 2) an adventitia, or 3) a media. The vasoconstriction responses of these 3 constructs to endothelin, the most potent vasopressor known up-to-date, as well as to nonselective and selective agonists and antagonists, were compared. The adventitia contracted to endothelin-1, -2, whereas the media and the media+adventitia contracted to all three endothelins. Endothelin-induced contraction of the adventitia was dependent on ET(A) receptors, whereas that of the media and the adventitia+media was ET(A) and ET(B) receptor-dependent. RT-PCR studies corroborated these results. SNP induced a dose-dependent relaxation of the three tissue constructs. We also demonstrated that the endothelin-converting enzyme, responsible for the formation of the active endothelin peptides, was present and functional in the adventitia. In conclusion, this is the first direct demonstration that the adventitia has the capacity to contract and relax in response to vasoactive factors. The present study suggests that the adventitia of a blood vessel could play a greater role than expected in the modulation of blood vessel tone.

Aspartic Acid Endopeptidases↗

PubMed1996

Tissue-engineered heart valves. Autologous valve leaflet replacement study in a lamb model.

BACKGROUND: We have previously reported the successful creation of tissue-engineered valve leaflets and the implantation of these autologous tissue leaflets in the pulmonary valve position. This study was designed to trace cultured cells that were seeded onto a biodegradable polymer with the use of a 1,1'-dioctadecyl-3,3,3' 3'-tetramethylindo-carbocyanine perchlorate (Di-1) cell-labeling method. We also examined the time-related biochemical, biomechanical, and histological characteristics and evolution of these tissue constructs. METHODS AND RESULTS: Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted ovine arteries. Endothelial cells were selectively labeled with an acetylated low density lipoprotein marker and separated from fibroblasts with the use of a fluorescence-activated cell sorter. A synthetic biodegradable scaffold consisting of polyglycolic acid fibers was seeded first with fibroblasts, then coated with endothelial cells. Using these methods, we implanted autologous cell/polymer constructs in six animals. In two additional control animals, a leaflet of polymer was implanted without prior cell seeding. In each animal, cardiopulmonary bypass was used to completely resect the right posterior leaflet of the pulmonary valve and replace it with an engineered valve leaflet with (n = 6) or without (n = 2) prior cultured cell seeding. The animals were killed either after 6 hours or after 1, 6, 7, 9, or 11 weeks, and the implanted valve leaflets were examined histologically, biochemically, and biomechanically. 4-Hydroxyproline assays were performed to determine collagen content. Leaflet strength was evaluated in vitro with a mechanical tester Factor VIII and elastin stains were done to verify histologically that endothelial cells and elastin, respectively, were present. Animals receiving leaflets made from polymers without cell seeding were killed and examined in a similar fashion after 8 weeks. In the control animals, the acellular polymer leaflets were completely degraded, with no residual leaflet tissue at 8 weeks. The tissue-engineered valve leaflet persisted in each animal in the experimental group. 4-Hydroxyproline analysis of the constructs showed a progressive increase in collagen content. Immunohistochemical staining demonstrated elastin fibers in the matrix and factor VIII on the surface of the leaflet. The cell-labeling experiments demonstrated that the cells on the leaflets had persisted from the in vitro seeding of the leaflets. CONCLUSIONS: In the tissue-engineered heart valve leaflet, transplanted autologous cells generated a proper matrix on the polymer scaffold in a physiological environment at a period of 8 weeks after implantation.

Animals↗

PubMed2001

Vascularized three-dimensional skeletal muscle tissue-engineering.

Medical science continues to battle against the loss or failure of organs or tissues. Since, skeletal muscle loss lead not only to the functional compromise of the affected site, but also a structural deformation; tissue engineering of skeletal muscle attempts to provide solutions to replace loss of tissue contour and function. In our study, myoblasts seeded onto polyglycolic acid (PGA) meshes were used to engineer skeletal muscle tissue in vivo. The cell-polymer constructs harvested after a period of 6-weeks were well vascularized three-dimensional structures with the ability to generate neo-muscle-like tissue. This is the first time that the ability of myoblasts to survive in vivo in the absence of mature skeletal muscle tissue was demonstrated. The successful ability to transplant myoblasts using biodegradable polymer strands without using the traditional transplantation buffer mediums as carriers was also employed for the first time.

Animals↗

PubMed2004

Developmental approaches to kidney tissue engineering.

Recent advances in our understanding of the developmental biology of the kidney, as well as the establishment of novel in vitro model systems, have potential implications for kidney tissue engineering. These advances include delineation of the roles of a number of growth factors in the developmental programs of branching morphogenesis and mesenchymal differentiation, a new understanding of the roles of the extracellular matrix, identification of potential "renal" stem cells, the ex vivo propagation and subsequent recombination of isolated components of the kidney, and successful transplantation of renal primordia into adult hosts. This review will examine these advances in the context of approaches to tissue engineering. Finally, novel approaches that synthesize advances in both cell-based and organ-based approaches are proposed.

Animals↗

PubMed2003

A tissue engineered heart valve implanted in a juvenile sheep model.

BACKGROUND: The tissue-engineered (TE) heart valve was developed to improve the durability of tissue heart valves. The aim of this study was to evaluate morphological and histological changes in a TE heart valve consisting of decellularized porcine matrices seeded with viable autologous vascular endothelial cells (AVEC). MATERIAL/METHODS: TE valves were implanted into the right ventricular outflow tract of eight juvenile sheep and explanted after 7 days, 3 and 6 months. The valves were evaluated visually, by invasive pressure measurements, X-ray, light microscopy, scanning, and transmission electron microscopy. The calcium content of the cusps was determined quantitatively by atomic absorption spectrometry. RESULTS: After valve implantation, all animals showed fast recovery with no complications during the observation period. Invasive pressure measurements presented a mean TE valve pressure gradient of 1.5+/-0.5 mm Hg at 3 and the same at 6 months. Light microscopy showed a monolayer of AVEC on all explanted heart valves, confirmed by scanning electron microscopy and immunohistochemical staining. X-ray examination of explanted TE heart valves showed no cusp calcification, confirmed by atomic absorption spectrometry. CONCLUSIONS: All explanted TE heart valves showed AVEC at the inner surface and ingrowth of fibroblast into the decellularized matrix, increasing during the observation period. The calcium contents were very low at explantation in these viable new heart valves.

Animals↗

PubMed2006

Modeling evaluation of the fluid-dynamic microenvironment in tissue-engineered constructs: a micro-CT based model.

Natural cartilage remodels both in vivo and in vitro in response to mechanical stresses, hence mechanical stimulation is believed to be a potential tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis in engineered cartilage constructs. The quantification of the hydrodynamic environment is a condition required to study the biochemical response to shear of 3D engineered cell systems. We developed a computational model of culture medium flow through the microstructure of a porous scaffold, during direct- perfused culture. The 3D solid model of the scaffold micro-geometry was reconstructed from 250 micro-computed tomography (micro-CT) images. The results of the fluid dynamic simulations were analyzed at the central portions of the fluid domain, to avoid boundary effects. The average, median and mode shear stress values calculated at the scaffold walls were 3.48, 2.90, and 2.45 mPa respectively, at a flow rate of 0.5 cm(3)/min, perfused through a 15 mm diameter scaffold, at an inlet fluid velocity of 53 microm/s. These results were compared to results estimated using a simplified micro-scale model and to results estimated using an analytical macro-scale porous model. The predictions given by the CT-based model are being used in conjunction with an experimental bioreactor model, in order to quantify the effects of fluid-dynamic shear on the growth modulation of tissue-engineered cartilage constructs, to potentially enhance tissue growth in vitro.

Bioreactors↗