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PubMedDate not supplied

Fabrication and endothelialization of collagen-blended biodegradable polymer nanofibers: potential vascular graft for blood vessel tissue engineering.

Electrospun collagen-blended poly(L-lactic acid)-co-poly(epsilon-caprolactone) [P(LLA-CL), 70:30] nanofiber may have great potential application in tissue engineering because it mimicks the extracellular matrix (ECM) both morphologically and chemically. Blended nanofibers with various weight ratios of polymer to collagen were fabricated by electrospinning. The appearance of the blended nanofibers was investigated by scanning electron microscopy and transmission electron microscopy. The nanofibers exhibited a smooth surface and a narrow diameter distribution, with 60% of the nanofibers having diameters between 100 and 200 nm. Attenuated total reflectance-Fourier transform infrared spectra and X-ray photoelectron spectroscopy verified the existence of collagen molecules on the surface of nanofibers. Human coronary artery endothelial cells (HCAECs) were seeded onto the blended nanofibers for viability, morphogenesis, attachment, and phenotypic studies. Five characteristic endothelial cell (EC) markers, including four types of cell adhesion molecule and one EC-preferential gene (von Willebrand factor), were studied by reverse transcription-polymerase chain reaction. Results showed that the collagen-blended polymer nanofibers could enhance the viability, spreading, and attachment of HCAECs and, moreover, preserve the EC phenotype. The blending electrospinning technique shows potential in refining the composition of polymer nanofibers by adding various ingredients (e.g., growth factors) according to cell types to fabricate tissue-engineering scaffold, particularly blood vessel-engineering scaffold.

Biocompatible Materials↗

PubMed2004

Evaluation of chitosan-alginate-hyaluronate complexes modified by an RGD-containing protein as tissue-engineering scaffolds for cartilage regeneration.

In this study, a series of natural biodegradable materials in the form of chitosan (C)-alginate (A)-hyaluronate (H) complexes are evaluated as tissue-engineering scaffolds. The weight ratio of C/A is 1 : 1 or 1 : 2. Sodium hyaluronate is mixed in 2%. The complexes can be cast into films or fabricated as scaffolds. Their surface can be further modified by an Arg-Gly-Asp (RGD)-containing protein, a cellulose-binding domain-RGD (R). Cytocompatibility tests of the films are conducted using immortalized rat chondrocyte (IRC) as well as primary articular chondrocytes harvested from rabbits. The neocartilage formation in cell-seeded scaffolds is examined in vitro as well as in rabbits, where the scaffolds are implanted into the defect-containing joints. The results from cytocompatibility tests demonstrate that R enhances cell attachment and proliferation on C-A and C-A-H complex films. Complex C1A1HR (C : A = 1 : 1 with H and R) has better performance than the other formulation. Cells retain their spherical morphology on all C-A and C-A-H complexes. The in vitro evaluation of the seeded scaffolds indicates that the C1A1HR complex is the most appropriate for 3-D culture, manifested by the better cell growth as well as higher glycosaminoglycan and collagen contents. When the chondrocyte scaffolds are implanted into rabbit knee cartilage defects, partial repair is observed after 1 month in C1A1HR as well as in C1A1 (C : A = 1 : 1 without H and R) scaffolds. The defects are completely repaired in 6 months when C1A1HR constructs are implanted. It is concluded that C1A1HR is a potential tissue-engineering scaffold for cartilage regeneration.

Alginates↗

PubMed2005

The application of tissue engineering to regeneration of pulp and dentin in endodontics.

Caries, pulpitis, and apical periodontitis increase health care costs and attendant loss of economic productivity. They ultimately result in premature tooth loss and therefore diminishing the quality of life. Advances in vital pulp therapy with pulp stem/progenitor cells might give impetus to regenerate dentin-pulp complex without the removal of the whole pulp. Tissue engineering is the science of design and manufacture of new tissues to replace lost parts because of diseases including cancer and trauma. The three key ingredients for tissue engineering are signals for morphogenesis, stem cells for responding to morphogens and the scaffold of extracellular matrix. In preclinical studies cell therapy and gene therapy have been developed for many tissues and organs such as bone, heart, liver, and kidney as a means of delivering growth factors, cytokines, or morphogens with stem/progenitor cells in a scaffold to the sites of tissue injury to accelerate and/or induce a natural biological regeneration. The pulp tissue contains stem/progenitor cells that potentially differentiate into odontoblasts in response to bone morphogenetic proteins (BMPs). There are two strategies to regenerate dentin. First, is in vivo therapy, where BMP proteins or BMP genes are directly applied to the exposed or amputated pulp. Second is ex vivo therapy and consists of isolation of stem/progenitor cells from pulp tissue, differentiation into odontoblasts with recombinant BMPs or BMP genes and finally transplanted autogenously to regenerate dentin. This review is focused on the recent progress in this area and discusses the barriers and challenges for clinical utility in endodontics.

Animals↗

PubMed2003

Fetal tissue engineering from amniotic fluid.

BACKGROUND: We have recently shown, in an animal model, that amniotic fluid can be a source of cells for fetal tissue engineering. This study was aimed at determining whether fetal tissue constructs could also be engineered from cells normally found in human amniotic fluid. STUDY DESIGN: Cells obtained from the amniotic fluid of pregnant women at 15 to 19 weeks of gestation (n=6) were cultured in Dulbecco's Modified Eagle's medium (Sigma Chemical, St Louis, MO) containing 20% fetal bovine serum and 5 ng/mL basic fibroblast growth factor in a 95% humidified, 5% CO(2) chamber at 37 degrees C. A subpopulation of morphologically distinct cells was then mechanically isolated from the rest and selectively expanded. The lineage of this subpopulation of amniocytes was determined by immunofluorescent staining with antibodies against standard intermediate filaments and surface antigens. Cell proliferation rates were determined by oxidation assay. After cell expansion, colonies of amniocytes were statically and dynamically seeded onto both unwoven, 1-mm-thick polyglycolic acid polymer scaffold and acellular human dermis for 72 hours. The resulting constructs were analyzed by scanning electron microscopy. RESULTS: Amniocytes stained positively for smooth muscle actin, vimentin, cytokeratin 18, and fibroblast surface protein, and negatively for desmin, cluster of differentiation 31, and von Willebrand's factor (Dako, Carpenteria, CA). These findings are consistent with a mesenchymal, fibroblast-myofibroblast cell lineage. Mesenchymal amniocytes could be rapidly expanded in culture, based on results of the proliferation assay. Scanning electron microscopy of amniocyte constructs revealed dense, confluent layers of cells surrounding the polymer matrices and firm cell adhesion to both PGA and Alloderm (Lifecell Corp, Branchburg, NJ) scaffolds. No evidence of cell death was observed. CONCLUSIONS: Subpopulations of fetal mesenchymal cells can be consistently isolated from human amniotic fluid and rapidly expanded in vitro. Human mesenchymal amniocytes attach firmly to both polyglycolic acid polymer and acellular human dermis. The amniotic fluid can be a valuable and practical cell source for fetal tissue engineering.

Amniotic Fluid↗

PubMed2004

Tissue engineering of cartilage with the use of chitosan-gelatin complex scaffolds.

Chitosan has been shown to be a promising scaffold for various applications in tissue engineering. In this study, a chitosan-gelatin complex was fabricated as a scaffold by a freezing and lyophilizing technique. Chitosan's structure and characteristics are similar to those of glycosaminoglycan (GAG) and its analogs, and possesses various biological activities, whereas gelatin can serve as a substrate for cell adhesion, differentiation, and proliferation. With the use of autologous chondrocytes isolated from pig's auricular cartilage and seeded onto the chitosan-gelatin scaffold, elastic cartilages have been successfully engineered at the porcine abdomen subcutaneous tissue. After 16 weeks of implantation, the engineered elastic cartilages have acquired not only normal histological and biochemical, but also mechanical properties. The tissue sections of the engineered elastic cartilages showed that the chondrocytes were enclosed in the lacuna, similar to that of native cartilage. The presence of elastic fibers in the engineered cartilages was also demonstrated by Vehoeff's staining, and immunohistochemical staining confirmed the presence of type II collagen in the engineered cartilages. Quantitatively, the GAG in the engineered cartilages reached 90% of the concentration in native auricular cartilage. Furthermore, biomechanical analysis demonstrated that the extrinsic stiffness of the engineered cartilages reached 85% of the level in native auricular cartilage when it was harvested at 16 weeks. Thus, this study demonstrated that the chitosan-gelatin complex may serve as a suitable scaffold for cartilage tissue engineering.

Animals↗

PubMed2002

Of layers and spheres: the reaggregate approach in tissue engineering.

The reaggregate approach involves the regeneration of histotypical three-dimensional spheres from dispersed cells of a given tissue in suspension culture. Reaggregated spheres are used as tumour, genetic, toxicological, biohybrid and neurosphere models, and often replace animal experimentation. A particularly instructive example is the use of reaggregation to regenerate complete laminar tissue from avian embryonic retina. By revealing constraints of layered tissue formation, such retinal spheres could be instrumental for regenerative medicine, including stem cell-based tissue engineering.

Animals↗

PubMed2006

Tissue engineering of a small hand phalanx with a porously casted polylactic acid-polyglycolic acid copolymer.

In this study, we evaluate a porous polylactic acid-polyglycolic acid (PLGA) polymer in tissue engineering a small hand phalanx. The PLGA polymer was processed with a unique solvent cast/salt-leaching method in the shape of a little finger distal phalanx. An 85% polylactic acid (PLA)/15% polyglycolic acid (PGA) copolymer was determined to be the most favorable mixture for creating a small digital phalanx in precise shape and size. The pore sizes averaged 250-425 microm. Bovine periosteum was wrapped around each phalangeal polymer. Chondrocytes were injected into the articular end. Six constructs were surgically placed into a dorsal subcutaneous pocket of an ethylic mouse. After 8 (n=2) or 16 (n = 4) weeks, all implants demonstrated growth of bone and cartilage in the shape and size of a small hand phalanx, which was maintained throughout the study period. Histologic evaluation showed a homogeneous presence of bone and cartilage, with progressive tissue differentiation and matrix production from 8 to 16 weeks. The tissue-engineered bone formed through an endochondral ossification process. Articular cartilage was maintained where the chondrocytes were placed. These findings suggest that the porous PLGA polymer is an effective synthetic biodegradable device to support and guide tissue growth of bone and articular cartilage in the shape of a human phalanx.

Absorbable Implants↗

PubMed2006

[Reconstruction of bone using tissue engineering and nanoscale technology].

OBJECTIVE: To explore the method of fabricating freeze-dried demineralized bone matrix with nanoscale topography (nFDBM) and to investigate the feasibility of reconstruction of tissue-engineered bone with the novel scaffold. METHODS: Allogenic dogs' phalangeal cortical bone was fabricated into freeze-dried demineralized bone (FDBM) with modified Urist's method. FDBM was subjected to Nd : YAG laser irradiation under special conditions. The surface topography was identified by atomic force microscope (AFM) and scanning electron microscope (SEM). The osteoblasts were induced from autologous mesenchymal stem cells (MSCs) and mixed with nFDBM and FDBM in vitro. The effects of the different topography on cell-behavior was identified by SEM. The complex of nFDBM and osteoblasts were implanted into fascial bags on dogs' back (experimental group A) and dogs' phalangeal defects on right (experimental group C), while FDBM-osteoblast complex (control group B) and unique FDBM (control group D) were implanted into the corresponding sites on left as control groups. The osteogenic status was assessed by X-ray, HE and SEM at 4, 8 and 12 weeks after surgery. RESULTS: The surface of FDBM subjected to Nd : YAG laser irradiation resulted in well-defined three-dimensional nanoscale grooves (150 nm in depth and 600 to 800 nm in width). When the osteoblasts were implanted on the scaffold, the cells adhering to nFDBM were more than those to FDBM and secreted more extracellular matrix. Either new bone-like thin layer on the nanoscale surface or a lot of new bone-formation inner the experimental complex was observed by HE after 12 weeks of surgery and the experimental complexes were partially calcified at the same time, while the control groups almost had no osteogenic phenomena. CONCLUSION: Nd : YAG laser could produce nanoscale grooves on the FDBM surface. The nanoscale grooves are conductive to adherence, proliferation and matrix secretion of osteoblasts. Complexes by tissue engineering and nanoscale technology have some osteogenic abilities in vivo after implanted the animal model.

Animals↗

PubMed2003

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells.

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 106 smooth muscle cells (SMCs) obtained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biodegradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6~8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6~8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.

Animals↗

PubMed2005

The application of polyhydroxyalkanoates as tissue engineering materials.

Polyhydoxyalkanoates (PHA) are polyesters produced by microorganisms under unbalanced growth conditions. They are generally biodegradable and thermoprocessable, making them attractive as biomaterials for applications in both conventional medical devices and tissue engineering. Over the past years, PHA, particularly poly 3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly 4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) and poly 3-hydroxyoctanoate (PHO) and its composites have been used to develop devices including sutures, repair devices, repair patches, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings. The changing PHA compositions also allow favorable mechanical properties, biocompatibility, and degradation times within desirable time frames under specific physiological conditions. This paper reviews what have been achieved in the PHA tissue engineering area and concluded that the PHA prospective will look very bright in the near future.

3-Hydroxybutyric Acid↗

PubMed2007

Biopreservation of cells and engineered tissues.

The development of effective preservation and long-term storage techniques is a critical requirement for the successful clinical and commercial application of emerging cell-based technologies. Biopreservation is the process of preserving the integrity and functionality of cells, tissues and organs held outside the native environment for extended storage times. Biopreservation can be categorized into four different areas on the basis of the techniques used to achieve biological stability and to ensure a viable state following long-term storage. These include in vitro culture, hypothermic storage, cryopreservation and desiccation. In this chapter, an overview of these four techniques is presented with an emphasis on the recent developments that have been made using these technologies for the biopreservation of cells and engineered tissues.

Cell Culture Techniques↗

PubMed2004

Tissue engineering of ovine aortic blood vessel substitutes using applied shear stress and enzymatically derived vascular smooth muscle cells.

Compared to native blood vessels, all clinically available blood vessel substitutes perform suboptimally. Numerous approaches to tissue engineer (TE) blood vessels have been pursued using different scaffold materials, cell types, and culture conditions. Several limitations however remain to be overcome prior to the potential application in the arterial system. This study aimed at tissue engineering viable ovine blood vessels suitable for implantation into the systemic circulation of sheep. In recent studies vascular smooth muscle cells (vSMC) were derived by an explant technique. However, in this study we show that homogenous populations of differentiated vSMC were only obtained by enzymatic dispersion as characterized by immunostaining for specific vSMC marker proteins. In contrast the explant method yielded predominantly less differentiated myofibroblast-like cells. Enzymatically derived vSMC were seeded onto P-4-HB scaffolds and incubated either in a pulsatile flow bioreactor or under static conditions. Dynamically cultured TE blood vessel substitutes showed confluent layered tissue formation and were completely water resistant. They displayed significantly increased ECM synthesis, DNA, and protein content as well as vSMC marker expression. Mechanical properties of bioreactor cultured TE blood vessels approached those of native aorta. In conclusion ovine, aortic blood vessel substitutes were successfully created using enzymatically derived vSMC, bioabsorbable scaffolds, and applied shear stress.

Animals↗

PubMedDate not supplied

Fibrin-polyurethane composites for articular cartilage tissue engineering: a preliminary analysis.

In this study we investigated the use of a fibrin hydrogel to improve the potential of a polyurethane (PU) scaffold-based system for articular cartilage tissue engineering. PU-only ("no-fibrin") and PU-fibrin ("fibrin") composites were cultured for up to 28 days and analyzed for DNA content, glycosaminoglycan (GAG) content, type II collagen content, GAG release, and gene expression of aggrecan, collagen I, and collagen II. The use of fibrin allowed for higher viable cell-seeding efficiency (10% higher DNA content on day 2 in fibrin versus no-fibrin composites) and more even cell distribution on seeding, a more than 3-fold increase in the percentage of newly synthesized GAG retained in the constructs, and 2- to 6-fold higher levels of type II collagen and aggrecan gene expression through day 14. Addition of aprotinin to the medium inhibited fibrin degradation, most noticeably in the center of the constructs, but had little effect on biochemical composition or gene expression. Short-term mechanical compression (0-10% sinusoidal strain at 0.1 Hz for 1 h, applied twice daily for 3 days) doubled the rate of GAG release from the constructs, but had little effect on gene expression, regardless of the presence of fibrin. Although further work is needed to optimize this system, the addition of fibrin hydrogel to encapsulate cells in the stiff, macroporous PU scaffold is a step forward in our approach to articular cartilage tissue engineering.

Aggrecans↗

PubMed2000

[Advancement in the study of embryonic stem cells and the emerging potential promising future of ocular tissue engineering].

The advancement of the study in embryonic stem cells and tissue engineering makes it promising to effectively solve the intractable problems in the present ophthalmic practice. The problems include: no effective measures to regain the visual function of the patients with late stage glaucoma or retinal diseases, shortage of appropriate biocompatible donor tissues in treating ocular surface diseases.

Animals↗

PubMed2005

Evaluation of the safety and efficacy of periodontal applications of a living tissue-engineered human fibroblast-derived dermal substitute. I. Comparison to the gingival autograft: a randomized controlled pilot study.

BACKGROUND: Periodontists have found the gingival autograft to be an effective and predictable technique to increase the amount of attached gingiva around teeth, but this technique requires the surgeon to harvest donor tissue from a remote surgical site. The present study seeks to evaluate the safety and effectiveness of a tissue-engineered skin equivalent, a living human fibroblast-derived dermal substitute (HF-DDS), compared to a gingival autograft (GA) consisting of donor tissue harvested from the patient's palate in a procedure designed to increase the amount of keratinized tissue around teeth that do not require root coverage. METHODS: Twenty-five patients with insufficient attached gingiva associated with at least two teeth in contralateral quadrants of the same jaw were treated. One tooth in each patient was randomized to receive either a GA (control) or a HF-DDS graft (test). Clinical parameters measured at baseline and 3, 5, 7, 9, and 12 months included recession, clinical attachment level, keratinized tissue height, and plaque index. Probing depth was measured at 7, 9, and 12 months. Inflammation of each site was scored and texture and color of the grafted tissue were compared to the surrounding tissue. Resistance to muscle pull was evaluated and a questionnaire was used to determine patient preference. Surgical position of the graft and alveolar bone level were recorded at the surgical visit and patients were evaluated weekly for the first 4 weeks at which time recession and level of oral hygiene were measured. Biopsies and persistence studies were performed on a subset of the patients. RESULTS: Results for both test and control groups were similar for all measured clinical parameters with the exception of amount of keratinized tissue and percent shrinkage of keratinized tissue. The control group exhibited an average of 1.0 to 1.2 mm more keratinized tissue over time than the test group (P <0.001) and the control group had about half as much shrinkage as the test group over time (P <0.001). Test sites demonstrated significantly better color match over time compared to control sites. Similarly, tissue texture for test sites was significantly better than control sites over time. CONCLUSIONS: Based on the results of this investigation, the tissue engineered HF-DDS graft was safe and capable of generating keratinized tissue without the morbidity and potential clinical difficulties associated with donor site surgery. The GA generated more keratinized tissue and shrank less than the HF-DDS graft, but the test graft generated tissue that appeared more natural.

Adult↗

PubMed2001

Tissue engineering as a platform for controlled release of therapeutic agents: implantation of microencapsulated dopamine producing cells in the brains of rats.

Tissue engineering can lead to novel controlled release devices and controlled release strategies (e.g., of growth factors) can enhance the performance of tissue engineered constructs. There are however a number of technical challenges that must be overcome before these goals can be realized. The apparently 'simple' challenge of implanting the device (e.g., capsules) in the optimal site must be met. In addition, adequate nutrient supply to the capsules is required to maintain cell viability. To illustrate this problem we describe a guide and delivery cannula technique to provide reliable and reproducible delivery of up to 120 PC12 cell containing capsules into the caudate putamen (CPu). Microencapsulation of mammalian cells is potentially a powerful means of delivering therapeutically important molecules such as insulin. It can also have numerous applications as a platform for gene therapy. However, realizing this potential has been more difficult than first anticipated.

Animals↗

PubMed2006

Tissue engineering of injectable muscle: three-dimensional myoblast-fibrin injection in the syngeneic rat animal model.

BACKGROUND: Surgical treatment of skeletal muscle loss resulting from trauma, tumor ablation, or inborn tissue defects is hampered by the scarcity of functional substitute tissue. By using techniques of tissue engineering, reconstitution of skeletal muscle defects might become a more viable option. However, it is necessary to develop an adequate, practical method for delivering myoblasts within a three-dimensional scaffold in vivo. The aim of this study was to create and evaluate a novel method for the transfer of myoblasts with clinically approved components within a three-dimensional matrix. METHODS: The authors injected expanded primary male myoblasts into muscle defects in female syngeneic rats using a two-way syringe (Duploject) within a three-dimensional fibrin matrix. Detection and evaluation were performed using Y chromosome in situ hybridization, antidesmin immunostaining, and hematoxylin and eosin staining. To identify possible differences by means of integration, the injected myoblasts were compared with 7 days of precultivated constructs. RESULTS: Injected myoblasts showed increasing integration into host muscle fibers in a time-dependent manner, exclusively at the injection site. Antidesmin staining revealed a conserved myogenic phenotype of transplanted cells. The fibrin matrix resolved over a period of 12 weeks, with no indication of an inflammatory reaction. No significant difference in the number of detected Y chromosome-positive nuclei was found between the two transplantation groups. CONCLUSIONS: The presented technique of myoblast-fibrin injection indicates a clinical potential for reconstruction of skeletal muscle defects in vivo using a ready-to-use device in combination with tissue-engineering methods.

Animals↗

PubMedDate not supplied

Tissue engineered nerve constructs: where do we stand?

Driven by enormous clinical need, interest in peripheral nerve regeneration has become a prime focus of research and area of growth within the field of tissue engineering. While using autologous donor nerves for bridging peripheral defects remains today's gold standard, it remains associated with high donor site morbidity and lack of full recovery. This dictates research towards the development of biomimetic constructs as alternatives. Based on current concepts, this review summarizes various approaches including different extracellular matrices, scaffolds, and growth factors that have been shown to promote migration and proliferation of Schwann cells. Since neither of these concepts in isolation is enough, although each is gaining increased interest to promote nerve regeneration, various combinations will need to be identified to strike a harmonious balance. Additional factors that must be incorporated into tissue engineered nerve constructs are also unknown and warrant further research efforts. It seems that future directions may allow us to determine the "missing link".

Animals↗