Differentiation of human bone marrow mesenchymal stem cells on decellularized extracellular matrix materials.

Mesenchymal bone marrow stromal cells may be a source of cells to preseed decellularized biologic mesh materials for improved cellularization and promote a more physiologic tissue after remodeling. Spontaneous differentiation of mesenchymal stromal cells on the decellularized material would be undesirable. Conversely, induced differentiation of mesenchymal stem cells (MSC) on the material would suggest that these materials may have promise as scaffold materials for bone, cartilage, or adipocyte formation. Two sources of mesenchymal cells were evaluated for induced differentiation in control wells. These MSCs were also evaluated for spontaneous or induced differentiation on decellularized porcine dermis and mesothelium materials. Primarily harvested bone marrow MSCs and commercially obtained MSCs were induced into osteoblasts and adipocytes on decellularized dermis and mesothelium materials. The MSCs were able to be induced into chondrocytes in pellet form but not when grown as a monolayer on the materials. The MSCs did not undergo spontaneous differentiation when grown on the materials for up to four weeks. MSC grown on decellularized porcine dermis or mesothelium do not spontaneously differentiate and may serve as a source of autologous cells for preseeding these extracellular matrix materials prior to implantation.

Bioengineered dental tissues grown in the rat jaw.

Our long-term objective is to develop methods to form, in the jaw, bioengineered replacement teeth that exhibit physical properties and functions similar to those of natural teeth. Our results show that cultured rat tooth bud cells, seeded onto biodegradable scaffolds, implanted into the jaws of adult rat hosts and grown for 12 weeks, formed small, organized, bioengineered tooth crowns, containing dentin, enamel, pulp, and periodontal ligament tissues, similar to identical cell-seeded scaffolds implanted and grown in the omentum. Radiographic, histological, and immunohistochemical analyses showed that bioengineered teeth consisted of organized dentin, enamel, and pulp tissues. This study advances practical applications for dental tissue engineering by demonstrating that bioengineered tooth tissues can be regenerated at the site of previously lost teeth, and supports the use of tissue engineering strategies in humans, to regenerate previously lost and/or missing teeth. The results presented in this report support the feasibility of bioengineered replacement tooth formation in the jaw.

Engineering of implantable cartilaginous structures from bone marrow-derived mesenchymal stem cells.

Fabrication of implantable cartilaginous structures that could be secured in the joint defect could provide an alternative therapeutic approach to prosthetic joint replacement. Herein we explored the possibility of using biodegradable hydrogels in combination with a polyglycolic acid (PGA) scaffold to provide an environment propitious to mesenchymal stem cells (MSCs) chondrogenic differentiation. We examined the influence of type I collagen gel and alginate combined with PGA meshes on the extracellular matrix composition of tissue-engineered transplants. MSCs were isolated from young rabbits, expanded in monolayers, suspended in each hydrogel, and loaded on PGA scaffolds. All constructs (n=48) were cultured in serum-free medium containing transforming growth factor beta-1, under dynamic conditions in specially designed bioreactors for 3-6 weeks. All cell-polymer constructs had a white, shiny aspect, and retained their initial size and shape over the culture period. Their thickness increased substantially over time, and no shrinkage was observed. All specimens developed a hyalin-like extracellular matrix containing glycosaminoglycans (GAGs) and type II collagen, but significant differences were observed among the three different groups. In PGA/MSCs and collagen-PGA/MSCs constructs, the cell growth phase and the chondrogenic differentiation phase of MSCs occurred during the first 3 weeks. In alginate-PGA/MSCs constructs, cells remained round in the hydrogel and cartilage extracellular matrix deposition was delayed. However, at 6 weeks, alginate-PGA/MSCs constructs exhibited higher contents of GAGs and lower contents of type I collagen. These results suggest that the implied time for the transplantation of in vitro engineered constructs depends, among other factors, on the nature of the scaffold envisioned. In this study, we demonstrated that the use of a composite hydrogel-PGA scaffold supported the in vitro growth of implantable cartilaginous structures cultured in a bioreactor system.

Developmental analysis and computer modelling of bioengineered teeth.

Here we present the developmental progression of bioengineered pig teeth from 1 to 25 weeks of development. We demonstrate that 2-25 week implants contained embryonic tooth bud- and cap-stage tooth structures consisting of dental epithelium expressing the sonic hedgehog gene and condensed dental mesenchyme. Implants harvested at 18-25 weeks also contained tooth bud-like structures, as well as mature tooth structures containing enamel, dentin and pulp tissues. Immunohistochemical analyses confirmed the expression of dentin- and enamel-specific proteins in differentiated bioengineered tooth tissues. Three-dimensional computer modelling further demonstrated a spatial organization of enamel, dentin and pulp tissues resembling that of natural teeth. We conclude that bioengineered teeth commonly exhibit morphological stages characteristic of naturally forming teeth. Furthermore, the presence of immature tooth buds at all times assayed and increased numbers of bioengineered tooth structures over time suggests that porcine dental progenitor cells maintain the ability to form teeth for at least 25 weeks.

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.

Bioengineered teeth from cultured rat tooth bud cells.

The recent bioengineering of complex tooth structures from pig tooth bud tissues suggests the potential for the regeneration of mammalian dental tissues. We have improved tooth bioengineering methods by comparing the utility of cultured rat tooth bud cells obtained from three- to seven-day post-natal (dpn) rats for tooth-tissue-engineering applications. Cell-seeded biodegradable scaffolds were grown in the omenta of adult rat hosts for 12 wks, then harvested. Analyses of 12-week implant tissues demonstrated that dissociated 4-dpn rat tooth bud cells seeded for 1 hr onto PGA or PLGA scaffolds generated bioengineered tooth tissues most reliably. We conclude that tooth-tissue-engineering methods can be used to generate both pig and rat tooth tissues. Furthermore, our ability to bioengineer tooth structures from cultured tooth bud cells suggests that dental epithelial and mesenchymal stem cells can be maintained in vitro for at least 6 days.

Contractile cardiac grafts using a novel nanofibrous mesh.

Cardiomyoctes are terminally differentiated cells and therefore unable to regenerate after infarction. The use of autologous bioengineered cardiac grafts has been suggested to replace infarcted myocardium and enhance cardiac function. Here we report the development of an in vitro system for engineered myocardium. Cardiac nanofibrous meshes (CNM) were developed by culturing cardiomyocytes from neonatal Lewis rats on electrospun, nanofibrous polycaprolactone (PCL) meshes. The mesh had an ECM-like topography and was suspended across a wire ring that acted as a passive load to contracting cardiomyocytes. The cardiomyocytes started beating after 3 days and were cultured in vitro for 14 days. The cardiomyocytes attached well on the PCL meshes and expressed cardiac-specific proteins such as alpha-myosin heavy chain, connexin43 and cardiac troponin I. The results demonstrate the formation of contractile cardiac grafts in vitro. Using this technique, cardiac grafts can be matured in vitro to obtain sufficient function prior to implantation. It is conjectured that cardiac grafts with clinically relevant dimensions can be obtained by stacking CNMs and inducing vascularization with angiogenic factors.

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.

A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering.

Microporous, non-woven poly( epsilon -caprolactone) (PCL) scaffolds were made by electrostatic fiber spinning. In this process, polymer fibers with diameters down to the nanometer range, or nanofibers, are formed by subjecting a fluid jet to a high electric field. Mesenchymal stem cells (MSCs) derived from the bone marrow of neonatal rats were cultured, expanded and seeded on electrospun PCL scaffolds. The cell-polymer constructs were cultured with osteogenic supplements under dynamic culture conditions for up to 4 weeks. The cell-polymer constructs maintained the size and shape of the original scaffolds. Scanning electron microscopy (SEM), histological and immunohistochemical examinations were performed. Penetration of cells and abundant extracellular matrix were observed in the cell-polymer constructs after 1 week. SEM showed that the surfaces of the cell-polymer constructs were covered with cell multilayers at 4 weeks. In addition, mineralization and type I collagen were observed at 4 weeks. This suggests that electrospun PCL is a potential candidate scaffold for bone tissue engineering.

Tissue engineering of complex tooth structures on biodegradable polymer scaffolds.

Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continues to affect most adults adversely at some time in their lives. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments. To pursue this goal, we dissociated porcine third molar tooth buds into single-cell suspensions and seeded them onto biodegradable polymers. After growing in rat hosts for 20 to 30 weeks, recognizable tooth structures formed that contained dentin, odontoblasts, a well-defined pulp chamber, putative Hertwig's root sheath epithelia, putative cementoblasts, and a morphologically correct enamel organ containing fully formed enamel. Our results demonstrate the first successful generation of tooth crowns from dissociated tooth tissues that contain both dentin and enamel, and suggest the presence of epithelial and mesenchymal dental stem cells in porcine third molar tissues.

Tissue engineering of heart valves -- current aspects.

Tissue engineering of heart valves is an evolving research field. Driven by the shortcomings of the heart valve substitutes currently available, such as need for anticoagulation, susceptibility to infections, inability to grow and autorepair, the multidisciplinary approach for designing and growing viable heart valves identical to the native heart valves has begun. The following will give an update of the recent developments, current limitations and potential future applications of tissue-engineered heart valves.

Tissue engineering: a 21st century solution to surgical reconstruction.

Tissue engineering has emerged as a rapidly expanding approach to address the organ shortage problem. It is an "interdisciplinary field that applies the principles and methods of engineering and the life sciences toward the development of biological substitutes that can restore, maintain, or improve tissue function." Much progress has been made in the tissue engineering of structures relevant to cardiothoracic surgery, including heart valves, blood vessels, myocardium, esophagus, and trachea.

Hepatocyte transplantation using biodegradable matrices in ascorbic acid-deficient rats: comparison with heterotopically transplanted liver grafts.

BACKGROUND: Hepatocyte transplantation using polymeric matrices is under investigation as an alternative therapy for metabolic liver diseases. Long-term engraftment of hepatocytes in polymers has been demonstrated. However, the metabolic activity of hepatocytes in such devices has never been assessed in direct comparison with liver grafts. METHODS: Hepatocyte and partial liver transplantation were evaluated in the scurvy-prone osteogenic disorder Shionogi rat model. Biodegradable poly glycolic acid matrices seeded with hepatocytes equivalent to 20% of the recipient's liver mass, or 20% liver grafts were heterotopically transplanted into ascorbic acid- (AsA) deficient recipients. Recipients of cell-free matrices or AsA-deficient liver grafts served as controls. Recipients were set on AsA-free diet after transplantation. Plasma AsA levels, AsA concentrations in liver and adrenal gland tissue, and body weight ratios were assessed and H&E histology was performed. RESULTS: Recipients from the control groups showed symptoms of scurvy at 1 month after cessation of AsA supply. Hepatocyte transplantation and auxiliary liver transplantation prevented symptoms of scurvy and increased plasma and tissue AsA levels and body weight ratios. AsA levels in recipients of 20% liver grafts were comparable to normal control animals. CONCLUSIONS: Hepatocytes transplanted in polymeric matrices are able to compensate for liver-based metabolic deficiencies. Hepatocyte transplantation improves plasma AsA levels in AsA-deficient recipients. However, auxiliary liver grafts are superior to hepatocyte grafts in improving metabolic parameters. Further research work is needed to increase the efficiency of liver cell transplantation with regard to a clinical application.

A new artificial nerve graft containing rolled Schwann cell monolayers.

This study hypothesized that introducing high numbers of Schwann cells in monolayers via a novel rolled graft architecture would promote robust nerve regeneration. The objective was to place adherent Schwann cells in artificial nerve grafts and to assess regeneration through the Schwann cell-laden grafts compared with that through acellular grafts and autografts. Schwann cells were isolated from neonatal Fisher rats. Small intestinal submucosa (SIS) was harvested from adult Fisher rats, cut into 7 mm x 8 cm pieces, and pinned out. Schwann cells were plated onto the strips, allowed to reach confluence, and subsequently rolled into a laminar structure and implanted across a 7-mm gap in the rat sciatic nerve (n = 12). Control animals received SIS conduits without Schwann cells (n = 11) or autograft repair (n = 12). At 10.5 weeks, functional regeneration through the Schwann cell-laden grafts, measured by both sciatic function index and extensor postural thrust testing, exceeded that through the cell-free grafts and approached that achieved through autografts. These results highlight the role of Schwann cells in nerve regeneration. Regenerative results approaching autograft levels in the Schwann cell-laden group suggest that this methodology may ultimately be useful in clinical nerve repair.

Tissue-engineered cells producing complex recombinant proteins inhibit ovarian cancer in vivo.

Techniques of tissue engineering and cell and molecular biology were used to create a biodegradable scaffold for transfected cells to produce complex proteins. Mullerian Inhibiting Substance (MIS) causes regression of Mullerian ducts in the mammalian embryo. MIS also causes regression in vitro of ovarian tumor cell lines and primary cells from ovarian carcinomas, which derive from Mullerian structures. In a strategy to circumvent the complicated purification protocols for MIS, Chinese hamster ovary cells transfected with the human MIS gene were seeded onto biodegradable polymers of polyglycolic acid fibers and secretion of MIS confirmed. The polymer-cell graft was implanted into the right ovarian pedicle of severe combined immunodeficient mice. Serum MIS in the mice rose to supraphysiologic levels over time. One week after implantation of the polymer-cell graft, IGROV-1 human tumors were implanted under the renal capsule of the left kidney. Growth of the IGROV-1 tumors was significantly inhibited in the animals with a polymer-cell graft of MIS-producing cells, compared with controls. This novel MIS delivery system could have broader applications for other inhibitory agents not amenable to efficient purification and provides in vivo evidence for a role of MIS in the treatment of ovarian cancer.

Dynamics of extracellular matrix production and turnover in tissue engineered cardiovascular structures.

Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial requirements for their long-term patency and function. This complex process requires the deposition and accumulation of extracellular matrix molecules as well as the remodeling of this extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). In this study, we have investigated the dynamics of ECM production and the activity of MMPs and TIMPs in long-term tissue-engineered vascular conduits using quantitative ECM analysis, substrate gel electrophoresis, radiometric enzyme assays and Western blot analyses. Over a time period of 169 days in vivo, levels of elastin and proteoglycans/glycosaminoglycans in tissue-engineered constructs came to approximate those of their native tissue counter parts. The kinetics of collagen deposition and remodeling, however, apparently require a much longer time period. Through the use of substrate gel electrophoresis, proteolytic bands whose molecular weight was consistent with their identification as the active form of MMP-2 (approximately 64--66 kDa) were detected in all native and tissue-engineered samples. Additional proteolytic bands migrating at approximately 72 kDa representing the latent form of MMP-2 were detected in tissue-engineered samples at time points from 5 throughout 55 days. Radiometric assays of MMP-1 activity demonstrated no significant differences between the native and tissue-engineered samples. This study determines the dynamics of ECM production and turnover in a long-term tissue-engineered vascular tissue and highlights the importance of ECM remodeling in the development of successful tissue-engineered vascular structures.

Tissue engineering: current state and prospects.

Organ shortage and suboptimal prosthetic or biological materials for repair or replacement of diseased or destroyed human organs and tissues are the main motivation for increasing research in the emerging field of tissue engineering. No organ or tissue is excluded from this multidisciplinary research field, which aims to provide vital tissues with the abilities to function, grow, repair, and remodel. There are several approaches to tissue engineering, including the use of cells, scaffolds, and the combination of the two. The most common approach is biodegradable or resorbable scaffolds configured to the shape of the new tissue (e.g. a heart valve). This scaffold is seeded with cells, potentially derived from either biopsies or stem cells. The seeded cells proliferate, organize, and produce cellular and extracellular matrix. During this matrix formation, the starter matrix is degraded, resorbed, or metabolized. First clinical trials using skin or cartilage substitutes are currently under way. Both the current state of the field and future prospects are discussed.