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PubMed2006

Scaffolds for liver tissue engineering.

This review focuses on the expanding role for biomaterials and polymer scaffolds in liver tissue engineering. Studies are subdivided into in vitro and in vivo approaches. The in vitro section of the review discusses the challenges specific to liver tissue engineering, and how the choice of scaffold and its structure influences the success of the regenerative medicine strategy. The in vivo section evaluates early attempts to stimulate liver repair with cell and growth factor therapies, their failings and how current approaches aim to solve these problems.

Animals↗

PubMed1999

[Bone tissue engineering seeded with bone marrow stromal cells].

OBJECTIVE: To assess the ability of bone graft substitutes to support the growth of cells in vitro and new bone formation in vivo. METHODS: The methods of tissue engineering were employed, the bone marrow derived stromal cells were seeded onto blocks of hydroxypatite (HA), cultured for two weeks, and implanted intermuscularly into the rabbit back muscle, scanning electron microscopy and undecalcified bone tissue sections were used to evaluate the the growth of cells in vitro and new bone formation in vivo. RESULTS: The stromal cells were attached to and grew upon the surface of the periphery HA pores, especially in the pores adjacent to the bottom of culture flasks. No cell growth was found on the surface of the central pores. At four weeks after implantation, new bone formation was observed in most HA blocks, almost all of the new bones were lamellar bone and deposited directly on the surface of the HA pore. Active osteoblast and osteoid were found on the surface of the newly formed bone, invasion of bone marrow was observed in some newly formed bone. CONCLUSION: Bone marrow stromal cells are potentially the source of seeded cells for use in bone tissue engineering, and the HA may be considered as a suitable scaffold for these cells. The findings may be useful for detailed researches on seeded cells, scaffold and implanting fashion of bone tissue engineering.

Animals↗

PubMed2004

Biomimetic polymer/apatite composite scaffolds for mineralized tissue engineering.

The material surface must be considered in the design of scaffolds for bone tissue engineering so that it supports bone cells adhesion, proliferation and differentiation. A biomimetic approach has been developed as a 3D surface modification technique to grow partially carbonated hydroxyapatite (the bonelike mineral) in prefabricated, porous, polymer scaffolds using a simulated body fluid in our lab. For the rational design of scaffolding materials and optimization of the biomimetic process, this work focused on various materials and processing parameters in relation to apatite formation on 3D polymer scaffolds. The apatite nucleation and growth in the internal pores of poly(L-lactide) and poly(D,L-lactide) scaffolds were significantly faster than in those of poly(lactide-co-glycolide) scaffolds in simulated body fluids. The apatite distribution was significantly more uniform in the poly(L-lactide) scaffolds than in the poly(lactide-co-glycolide) scaffolds. After incubation in a simulated body fluid for 30 d, the mass of poly(L-lactide) scaffolds increased approximately 40%, whereas the mass of the poly(lactide-co-glycolide) scaffolds increased by about 15% (see Figure). A higher ionic concentration and higher pH value of the simulated body fluid enhanced apatite formation. The effects of surface functional groups on apatite nucleation and growth were found to be more complex in 3D scaffolds than on 2D films. Surprisingly enough, it was found that carboxyl groups significantly reduced the apatite formation, especially on the internal pore surfaces of 3D scaffolds. These findings are critically important in the rational selection of materials and surface design of 3D scaffolds for mineralized tissue engineering and may contribute to the understanding of biomineralization as well.SEM micrograph of a poly(L-lactide) scaffold.

Apatites↗

PubMed2003

[Mineralized collagen based composite for bone tissue engineering].

OBJECTIVE: To construct a mineralized collagen based composite by biomimetic synthesis for bone tissue engineering. METHODS: Using the molecular collagen as the template, the calcium phosphate is deposited on it to produce a mineralized collagen based composite, then is combined with minute amount of poly lactic acid (PLA), the three-dimensional scaffold composite is prepared by liquid phase separation. Using osteoblast culture technique, the biocompatibility of this biomaterial in vitro is detected by x-ray diffraction, SEM, TEM, fluoroscopy and CLSM. RESULTS: Both degree and the size of crystals in the composite are low, which are similar to that of nature bone. It possesses porous structure and the porosity of the composite is high. The typical fibrillar microstructure is self-assembled of the collagen and the nano-crystal hydroxyapatite (HA) in the composite, moreover, the x-ray diffraction graphic of HA crystal shows the [002]-oriented. CONCLUSIONS: The biomimetic three-dimensional composite can serve as one of the optimal scaffold material for bone tissue engineering both on structure and on property.

Animals↗

PubMed2005

Non-destructive studies of tissue-engineered phalanges by magnetic resonance microscopy and X-ray microtomography.

One of the intents of tissue engineering is to fabricate biological materials for the augmentation or replacement of impaired, damaged, or diseased human tissue. In this context, novel models of the human phalanges have been developed recently through suturing of polymer scaffolds supporting osteoblasts, chondrocytes, and tenocytes to mimic bone, cartilage, and tendon, respectively. Characterization of the model constructs has been accomplished previously through histological and biochemical means, both of which are necessarily destructive to the constructs. This report describes the application of two complementary, non-destructive, non-invasive techniques, magnetic resonance microscopy (MRM) and X-ray microtomography (XMT or quantitative computed tomography), to evaluate the spatial and temporal growth and developmental status of tissue elements within tissue-engineered constructs obtained after 10 and 38 weeks of implantation in athymic (nude) mice. These two times represent respective points at which model middle phalanges are comprised principally of organic components while being largely unmineralized and later become increasingly more mineralized. The spatial distribution of mineralized deposits within intact constructs was readily detected by XMT (qCT) and was comparable to low intensity zones observed on MRM hydration maps. Moreover, the MRM-derived hydration values for mineralized zones were inversely correlated with mineral densities measured by XMT. In addition, the MRM method successfully mapped fat deposits, collagenous tissues, and the hydration state of the soft tissue elements comprising the specimens. These results support the application of non-destructive, non-invasive, quantitative MRM and XMT for the evaluation of constituent tissue elements within complex constructs of engineered implants.

Animals↗

PubMed2005

Second generation of meniscus transplantation: in-vivo study with tissue engineered meniscus replacement.

INTRODUCTION: The options available after meniscus loss offer only limited chances for a long-term success. In the following experimental study, we investigated the effect of meniscus tissue engineering on properties of the collagen meniscus implant (CMI). METHODS: Autologous fibrochondrocytes, obtained per biopsy from adult Merino sheep (n=25), were released from the matrix, cultured in-vitro and seeded into CMI scaffolds (n=10, group 1). Following a 3-week in-vitro culture, the tissue engineered menisci were used for autologous transplantation. Macroscopical and histological evaluation were performed in comparison with non-seeded CMI controls (n=10, group 2) and with meniscus-resected controls (n=5, group 3) after 3 weeks (each 1 animal group 1 and 2) and 3 months. RESULTS: The lameness score did not show any difference between the groups. Meniscus tissue was found in seven knee joints (group 1), in five knee joints (group 2) and in two knee joints (group 3). The size of the transplants reduced from 25.9+/-4.5 to 20.1+/-10.8 mm (group 1) and from 25.9+/-1.5 to 14.4+/-12.5 mm (group 2). Histologically, enhanced vascularisation, accelerated scaffold re-modelling, higher content of extra-cellular matrix and lower cell number were noted in the pre-seeded menisci in comparison with non-seeded controls. Dense high-cellular fibrous scar tissue was found in two of five cases in the resection control group. CONCLUSION: Tissue engineering of meniscus with autologous fibrochondrocytes demonstrates a macroscopic and histological improvement of the transplants. However, further development of the methods, especially of the scaffold and of the cell-seeding procedure must prove the feasibility of this procedure for human applications.

Animals↗

PubMed2005

Alginate and chitosan polyion complex hybrid fibers for scaffolds in ligament and tendon tissue engineering.

Selecting the material for a scaffold is critically important for the success of tissue engineering. To simplify complicated biosynthetic matrices and achieve a novel class of potential materials, a model of polyion complex fibers was prepared from alginate and chitosan. In the current in vitro study, we thought that alginate-based chitosan hybrid biomaterials could provide excellent supports for fibroblast adhesion. In the current study, alginate polymer fiber (alginate group) and alginate-based chitosan hybrid polymer fibers (alginate with 0.05% chitosan, alginate-chitosan 0.05% group; alginate with 0.1% chitosan, alginate-chitosan 0.1% group) were originally prepared. We investigated the adhesion behavior of rabbit tendon fibroblast onto alginate polymer fibers versus the adhesion of the fibroblast onto alginate-based chitosan hybrid polymer fibers. Furthermore, mechanical properties and synthesis of the extracellular matrix were investigated. Mechanically, the novel fiber has considerable tensile strength of more than 200 MPa. We demonstrated that the alginate-based chitosan hybrid polymer fibers showed much improved adhesion capacity with fibroblast compared with alginate polymer fiber. Additionally, morphologic studies revealed the dense fiber of the type I collagen produced by the fibroblast in the hybrid polymer fibers. We concluded that an alginate-based chitosan hybrid polymer fiber has considerable potential as a desirable biomaterial scaffold for tendon and ligament tissue engineering.

Alginates↗

PubMed2001

Insulin in tissue engineering of cartilage: a potential model system for growth factor application.

Investigation of novel experimental application systems for growth factors or other bioactive substances in tissue engineering is often limited by high costs of substances and would benefit from a defined and easily controllable model tissue system. Herein, we demonstrate a potential three-dimensional in vitro system using engineered cartilage as a model tissue and readily available insulin as a model drug. Previously it has been shown that insulin-like growth factor-I (IGF-I) has profound effects on tissue-engineered cartilage in vitro. Insulin is known to bind to the IGF-I receptor and to elicit significant responses in cartilage. In this study, bovine articular chondrocytes were seeded onto biodegradable polyglycolic acid (PGA) scaffolds and cultured for up to 7 weeks. Exogenous insulin (0.05-50 microg/ml) increased the growth rate and the glycosaminoglycan fraction of tissue-engineered cartilage, decreased the cell number in the tissue constructs, and improved the morphological appearance, with 2.5 microg/ml being the most favorable concentration. The observed effects of insulin were similar to effects of IGF-I (0.05 microg/ml) and were in agreement with the reported binding constants of IGF-I and insulin at the IGF-I receptor. Besides the possibility to employ insulin as a potent substance to improve tissue-engineered cartilage, the presented easily controllable in vitro system may be used in the future to evaluate experimental growth factor application devices using economically favorable insulin as a model protein.

Animals↗

PubMed2006

Cell and tissue engineering and clinical applications: an overview.

Most human tissues do not regenerate spontaneously; this is why cell therapies and tissue engineering are promising alternatives. The principle is simple: cells are collected in a patient and introduced in the damaged tissue or in a tridimentional porous support and harvested in a bioreactor in which the physico-chemical and mechanical parameters are controlled. Once the tissues (or the cells) are mature they may be implanted. In parallel, the development of biotherapies with stem cells is a field of research in turmoil given the hopes for clinical applications that it brings up. Embryonic stem cells are potentially more interesting since they are totipotent, but they can only be obtained at the very early stages of the embryo. The potential of adult stem cells is limited but isolating them induces no ethical problem and it has been known for more than 40 years that bone marrow does possess the regenerating functions of blood cells. Finally, the properties of foetal stem cells (blood cells from the umbilical cord) are forerunners of the haematopoietic system but the ability of these cells to participate to the formation of other tissues is more problematic. Another field for therapeutic research is that of dendritic cells, antigen presenting cells. Their efficiency in cell therapy relies on the initiation of specific immune responses. They represent a promising tool in the development of a protective immune response against antigens which the host is usually unable to generate an efficient response (melanomas, breast against cancer, prostate cancer, ..). Finally, gene therapy, has been nourishing high hopes but few clinical applications can be envisaged in the short term, although potential applications are multiple (haemophilia, myopathies, ..). A large number of clinical areas stand as candidates for clinical applications: leukaemia and cancers, cardiac insufficiency and vascular diseases, cartilage and bone repair, ligaments and tendons, liver diseases, ophthalmology, diabetes, neurological diseases (Parkinson, Huntington disease, ..), .. Various aspects of this new regenerative therapeutic medicine are developed in this work.

Animals↗

PubMed2005

Molecular aspects of vascular tissue engineering.

Cardiovascular disease remains the number one cause of death in the United States. Most current surgical procedures to alleviate this disease rely on the availability of suitable small diameter vascular grafts, which are constrained by several limitations. Tissue engineering brings new hope to this field, but still faces many challenges. This review focuses on the molecular aspects of the different components of vascular tissue engineering. The topics addressed include the cell type, extracellular matrix, and physical and biochemical stimulation with respect to their role in the development of a tissue engineered vascular graft.

Animals↗

PubMedDate not supplied

Quantitative study of tissue-engineered cartilage with human bone marrow mesenchymal stem cells.

OBJECTIVES: To assess the possibility of cartilage tissue engineering using human mesenchymal stem cells (hMSCs) and to investigate the quantitative relationship between hMSCs and engineered cartilage. DESIGN: Human mesenchymal stem cells were cultured, cryopreserved, and expanded in vitro. Surface antigens were detected by flow cytometry. In vitro chondrogenesis of hMSCs and cryopreserved hMSCs was performed. The chondrogenesis-induced hMSCs were seeded onto polyglycolic acid scaffolds, cultured in vitro for 3 weeks in chondrogenic medium, and then implanted into nude mice. The implants were harvested after 10 weeks and examined with histologic and immunochemical staining. RESULTS: The construction of cartilages was identified grossly and histologically: 1.9 to 2.5 x 10(7) nucleated cells were obtained from 1 mL of bone marrow, and about 1 to 2 x 10(6) hMSCs were obtained from the primary culture. The number of hMSCs tripled at every passage and reached 1.4 to 2.8 x 10(12) at passage 15. The purity of hMSCs was 95% and 98% at the primary and the fourth passages, respectively. Twenty-one days was the optimal (induction rate, 95%) induction time, with no apparent differences in induction rates among different passages. Based on our findings, hMSCs from 0.07 to 0.14 mL of bone marrow, expanded during 4 passages and induced for 21 days, would be sufficient to engineer 1 cm(2) of cartilage, 3-mm thick. CONCLUSION: Quantitative standards of hMSCs as seed cells for cartilage tissue engineering were established and may have value for later clinical work.

Animals↗

PubMed2006

Towards injectable cell-based tissue-engineered bone: the effect of different calcium phosphate microparticles and pre-culturing.

Bone tissue engineering by combining bone marrow stromal cells (BMSCs) with a porous scaffold is a promising technology. Current major challenges are to upscale the technique for clinical application and to improve the handling characteristics. With respect to minimal invasive surgery, moldable and/or injectable formulations are highly preferable. Ceramic microparticles of different HA/TCP formulations (100/0, 70/30, 60/40, 40/60, and 0/100) with varying surface roughness were sieved to select 200 microg aliquots of the 212-300 microm fraction. Goat BMSCs were seeded on different aliquots one week prior to in vivo implantation. These constructs and remaining cells were cultured for one week. By then, the remaining cells were harvested and resuspended in a specific binder: hyaluronic acid, alginate, or blood plasma, combined with aliquots of 60/40 microparticles peroperatively. All constructs were implanted in nude rats (n = 10) and analyzed for their bone yield histomorphometrically after 6 weeks. All precultured constructs showed consistent bone formation of comparable quantity. No significant differences were observed between the different material compositions. Peroperatively prepared constructs hardly showed any bone formation. The present study demonstrated the osteogenic potential of a tissue- engineered bone substitute made of microparticles of various HA/TCP compositions. There was an obvious advantage when the constructs were pre-cultured.

Alginates↗

PubMed2006

Tissue engineering approach to the treatment of bone tumors: three cases of cultured bone grafts derived from patients' mesenchymal stem cells.

A novel approach to the treatment of bone tumors using tissue-engineered implants is reported in this study. The number of mesenchymal stem cells (MSCs) obtained from each patient's bone marrow cells was first increased, and the MSCs were forced to differentiate into osteoblasts followed by bone matrix formation on hydroxyapatite (HA) ceramics. The strong osteogenic ability of the implants, as evidenced by high osteoblastic activity, was confirmed. Consequently, the HA surface was covered with the patient's derived cultured osteoblast/bone matrix. The tissue-engineered HA was used to fill the patient's bone cavity after tumor curettage. Immediate healing potential was found by serial plain radiographs and computed tomograhy images, and no adverse reactions were noted in these patients. The results indicate that tissue-engineered osteogenic ceramics might be an alternative to autologous bone grafts.

Adolescent↗

PubMed2003

Efficient and stable retroviral transfection of ovine endothelial cells with green fluorescent protein for cardiovascular tissue engineering.

To determine whether cellular components of tissue-engineered cardiovascular structures are derived from cells harvested and seeded onto an acellular scaffold, or from cells originating from surrounding tissue (e.g., proximal and distal anastomosis), cellular retroviral transfection with green fluorescent protein (GFP) was used. Ovine endothelial cells (ECs) were transfected with a Moloney murine leukemia virus (Mo-MuLV)-based retroviral vector expressing GFP. Transfection was evaluated by fluorescence microscopy and fluorescence-activated cell sorting. The rate of transfection of the primary cells was 33.4% for ECs, 48 hours after transfection. Stable transfection could be observed for at least 25 subsequent passages. Retroviral transfection with GFP enables stable and reliable long-term labeling of ovine ECs. This approach might offer an attractive pathway to study tissue development, with emphasis on distinguishing between cellular components initially seeded onto a construct and those occurring as a result of cell ingrowth from surrounding tissue.

Animals↗

PubMed1994

Symbiosis of biotechnology and biomaterials: applications in tissue engineering of bone and cartilage.

The three ingredients for the successful tissue engineering of bone and cartilage are regulatory signals, cells, and extracellular matrix. Recent advances in cellular and molecular biology of the growth and differentiation factors have set the stage for a symbiosis of biotechnology and biomaterials. Recent advances permit one to enunciate the rules of architecture for tissue engineering of bone and cartilage. The purification and cloning of bone morphogenetic proteins (BMPs) and growth factors such as platelet derived growth factors (PDGF), transforming growth factor-beta (TGF-beta), and insulin-like growth factors (IGF-I) will allow the design of an optimal combination of signals to initiate and promote development of skeletal stem cells into cartilage and bone. Successful and optimal bone and cartilage formation is a synergy of inductive and conductive strategies governed by biomechanics, optimal load bearing, and motion. BMPs function as inductive signals. Biomaterials (both natural and synthetic) mimic the extracellular matrix and play a role in conduction of bone and cartilage. Examples of biomaterials include hydroxyapatite, polyanhydrides, polyphosphoesters, polylactic acid, and polyglycolic acid. The prospects for novel biomaterials are immense, and they likely will be a fertile growth industry. Cooperative ventures between academia and industry and technology transfer from the federal government augur well for an exciting future for clinical applications.

Animals↗

PubMedDate not supplied

A clinically relevant large-animal model for evaluation of tissue-engineered cardiac surgical patch materials.

Extracellular matrix (ECM) scaffolds may be useful as a tissue engineering approach toward myocardial regeneration in the infarcted heart. An appropriate large-animal model for testing the utility of biologically derived ECM in this application is needed. The purpose of this study was to develop such a model for optimal procedural success during and after patch implantation surgery. Myocardial infarction (MI) was created by embolization of the diagonal artery (DA) branch of the left anterior descending coronary artery with collagen suspension. After 4 to 6 weeks, 14 pigs received patch implant (ECM or expanded polytetrafluoroethylene). Six pigs were infarcted in the first DA and seven pigs in the second DA. Electrophysiology study was performed within 3 days before surgery. During surgery, the size and location of the infarct were measured. Infarcted myocardium (1.5-cm diameter) was transmurally excised under partial cardiopulmonary bypass. Patches (3-cm diameter) were sutured to the endomyocardial defect. Four pigs died postoperatively. After 1 month, 10 pigs were euthanized and the locations of patches were examined. Success rate of patch implant in the second DA (85.7%) was higher than the first DA (50%) group. Infarct size in the second DA was smaller than in the first DA (4.6+/-1.2 vs. 10.8+/-2.4 cm(2), P<.05). The second DA was more anteriorly positioned, which enabled easier access from the midsternal thoracotomy. However, the first DA was more laterally located requiring more manipulation of the heart during surgery. Electrophysiology revealed no ventricular tachyarrhythmia in the second DA but 33.3% in the first DA group (P<.05). At necropsy, the endocardial position of the first DA-infarct patches was anteroapical, whereas the second DA-infarct patches were more basolateral and often involved the anterior papillary muscle. The success rate of patch implant was associated with infarction size and location, and may be related to arrhythmic substrate. Experimental MI created by the second DA embolization is a feasible model for investigation of tissue-engineered cardiac patch implantation. This large-animal model is also suitable for study of cell therapy via endocardial catheter-based approaches or open surgical methods.

Animals↗

PubMed2003

Thoracic Surgery Directors Association Award. Bone marrow as a cell source for tissue engineering heart valves.

BACKGROUND: This study was designed to assess the feasibility of using ovine bone marrow-derived mesenchymal stem cells to develop a trileaflet heart valve using a tissue engineering approach. METHODS: Bone marrow was aspirated from the sternum of adult sheep. Cells were isolated using a Ficoll gradient, cultured, and characterized based on immunofluorescent staining and the ability to differentiate down a specific cell lineage. Two million cells per centimeter squared were delivered onto a polyglycolic acid (PGA), poly-4-hydroxybutyrate (P4HB) composite scaffold and cultured for 1 week before being transferred to a pulse duplicator for an additional 2 weeks. The tissue-engineered valves were assessed by histology, scanning electron microscopy, and biomechanical flexure testing. RESULTS: Cells expressed SH2, a marker for mesenchymal stem cells, as well as specific markers of smooth muscle cell lineage including alpha-smooth muscle actin, desmin, and calponin. These cells could be induced to differentiate down an adipocyte lineage confirming they had not fully committed to a specific cell lineage. Preliminary histologic examination showed patchy surface confluency confirmed by scanning electron microscopy, and deep cellular material. Biomechanical flexure testing of the leaflets showed an effective stiffness comparable to normal valve leaflets. CONCLUSIONS: Mesenchymal stem cells can be isolated noninvasively from the sternum of sheep and can adhere to and populate a PGA/P4HB composite scaffold to form "tissue" that has biomechanical properties similar to native heart valve leaflets. Thus, bone marrow may be a potential source of cells for tissue engineering trileaflet heart valves, particularly in children with congenital heart disease.

Animals↗

PubMed1999

Bioactive factors for bone tissue engineering.

Orthopaedic surgery is currently in the midst of a transformation from bone grafting and the use of bone graft substitutes to bone tissue engineering. Bioactive bone growth factors likely will play a particularly important role in this emerging field. This article will review the three leading strategies for using bioactive factors for bone tissue engineering: extraction and partial purification of growth factors, recombinant protein synthesis, and gene therapy. Preclinical and early clinical trial results with bone morphogenetic protein-2, bone morphogenetic protein-7 (osteogenic protein-1), and NeOsteo bovine bone protein extract will be reviewed. In addition, the current obstacles to clinical implementation of bone tissue engineering will be reviewed.

Animals↗