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Tissue-engineered lung: an in vivo and in vitro comparison of polyglycolic acid and pluronic F-127 hydrogel/somatic lung progenitor cell constructs to support tissue growth.

In this study, we describe the isolation and characterization of a population of adult-derived or somatic lung progenitor cells (SLPC) from adult mammalian lung tissue and the promotion of alveolar tissue growth by these cells (both in vitro and in vivo) after seeding onto synthetic polymer scaffolds. After extended in vitro culture, differentiating cells expressed Clara cell 10kDa protein, surfactant protein-C, and cytokeratin but did not form organized structures. When cells were combined with synthetic scaffolds, polyglycolic acid (PGA) or Pluronic F-127 (PF-127), and maintained in vitro or implanted in vivo, they expressed lung-specific markers for Clara cells, pneumocytes, and respiratory epithelium and organized into identifiable pulmonary structures (including those similar to alveoli and terminal bronchi), with evidence of smooth muscle development. Although PGA has been shown to be an excellent polymer for culture of specific cell types in vitro, in vivo culture in an immunocompetent host induced a foreign body response that altered the integrity of the developing lung tissue. Use of PF-127/cell constructs resulted in the development of tissue with less inflammatory reaction. These data suggest that the therapeutic use of engineered tissues requires both the use of specific cell phenotypes, as well as the careful selection of synthetic polymers, to facilitate the assembly of functional tissue.

Animals↗

Equibiaxial strain stimulates fibroblastic phenotype shift in smooth muscle cells in an engineered tissue model of the aortic wall.

Many cells in the body reside in a complex three-dimensional (3D) environment stimulated by mechanical force. In vitro bioreactor systems have greatly improved our understanding of the mechanisms behind cell mechanotransduction. Current systems to impose strain in vitro are limited either by the lack of uniform strain profile or inability to strain 3D engineered tissues. In this study, we present a system capable of generating cyclic equibiaxial strain to an engineered vascular wall model. Type I collagen hydrogels populated with rat aortic smooth muscle cells (RASMCs) were created either as a compacting disk or constrained hemisphere. Both models were adhered to silicone membranes precoated with collagen I, fibronectin, or Cell-Tak and assayed for adhesion characteristics. The best performing model was then exposed to 48 h of 10% strain at 1Hz to simulate wall strain profiles found in vascular aneurysms, with static cultures serving as controls. The finite strain profile at the level of the membrane and the free surface of the construct was quantified using microbeads. The results indicate that the hemisphere model adhered with Cell-Tak had the most stable adhesion, followed by fibronectin and collagen I. Disk models did not adhere well under any coating condition. Uniform strain propagation was possible up to a maximum area strain of 20% with this system. RASMC responded to 10% equibiaxial strain by becoming less elongated, and immunohistochemistry suggested that stretched RASMC shifted to a more synthetic phenotype in comparison to static controls. These results suggest that equibiaxial strain may induce smooth muscle cell differentiation. We conclude that this system is effective in stimulating cells with cyclic equibiaxial strain in 3D cultures, and can be applied to a variety of biomaterial and tissue engineering applications.

Animals↗

Prospective use of electrospun ultra-fine silicate fibers for bone tissue engineering.

Electrospinning involves the generation of a jet of viscous solution, and results in the formation of ultra-fine fibers. Silicate fibers prepared using this technology and via the sol-gel process were evaluated as scaffolds for bone tissue engineering. We found that human osteoblastic MG63 cells successfully adhered on individual silicate fibers, and proliferated on them. In an apatite-formation ability study, spherical particles covered the fibers after soaking in simulated body fluid for 7 days. Energy dispersive X-ray analysis revealed that Ca/P atomic ratio of the particles was similar to that of human bone. In addition, X-ray diffraction analysis revealed that crystalline structure of the particles agreed with that of apatite. These results suggest that electrospun silicate fibers are a potential candidate for scaffolding material in bone tissue engineering.

Apatites↗

Biodegradable elastomeric scaffolds for soft tissue engineering.

Elastomeric copolymers of 1,3-trimethylene carbonate (TMC) and epsilon-caprolactone (CL) and copolymers of TMC and D,L-lactide (DLLA) have been evaluated as candidate materials for the preparation of biodegradable scaffolds for soft tissue engineering. TMC-DLLA copolymers are amorphous and degrade more rapidly in phosphate-buffered saline (PBS) of pH 7.4 at 37 degrees C than (semi-crystalline) TMC-CL copolymers. TMC-DLLA with 20 or 50 mol% TMC loose their tensile strength in less than 5 months and are totally resorbed in 11 months. In PBS, TMC-CL copolymers retain suitable mechanical properties for more than a year. Cell seeding studies show that rat cardiomyocytes and human Schwann cells attach and proliferate well on the TMC-based copolymers. TMC-DLLA copolymers with either 20 or 50 mol% of TMC are totally amorphous and very flexible, making them excellent polymers for the preparation of porous scaffolds for heart tissue engineering. Porous structures of TMC-DLLA copolymers were prepared by compression molding and particulate leaching techniques. TMC-CL (co)polymers were processed into porous two-ply tubes by means of salt leaching (inner layer) and fiber winding (outer layer) techniques. These grafts, seeded with Schwann cells, will be used as nerve guides for the bridging of large peripheral nerve defects.

Animals↗

Flow-dependent re-endothelialization of tissue-engineered heart valves.

BACKGROUND AND AIM OF THE STUDY: The generation of a functional, non-immunogenic, non-thrombogenic construct based on autologous cells seeded onto an acellular extracellular matrix is the major goal in heart valve tissue engineering. The study aim was to identify culturing conditions required to achieve a stable endothelial cell (EC) layer under physiological flow conditions, a prerequisite for the requested characteristics. METHODS: Eleven detergent-decellularized ovine pulmonary valves (PVs) were statically reseeded in special bioreactors with ovine venous ECs (1.2x10(7) cells per valve). The dynamic culture was started with 0.1 l/min in eight bioreactors. In four bioreactors the initial flow rate was slow, and increased by 0.1 l/min twice each day until maximal flow was 0.5 l/min and pulsation rate (PR) was 20 beats/min; in four other bioreactors the flow was increased by 0.7 l/min/day and reached 2.0 l/min with a PR of 50 beats/min. The mean system pressure was maintained at 25 +/- 5 mmHg during the whole dynamic cultivation in both groups. Three statically reseeded valves served as baseline. After achieving maximal appointed flow, the valves were investigated morphologically (hematoxylin and eosin staining, electron microscopy, von Willebrand factor, endothelial nitric oxide synthase immunostaining) and for metabolic activity (MTS assay). RESULTS: After reseeding, the endothelium appeared on the luminal surface of the PV as a non-confluent monolayer. Moderate pulsatile circulation induced complete confluence of EC monolayers on both cusp sides and the pulmonary wall. A high flow rate led to a partial loss of cells on the wall surface with large defects, and to complete cell wash-off from cusps. Cusp and wall metabolic activity was significantly higher after culture under moderate flow (p < 0.001) than in other groups, and was absent from cusps in high-flow bioreactors. CONCLUSION: Moderate pulsatile flow with small increments stimulates EC proliferation on the ovine decellularized valve scaffold. A rapid increase in bioreactor flow to physiological levels leads to significant damage of the reseeded endothelium and complete loss of cusp cellularity. This effect may be responsible for the in-vivo failure of static reseeded tissue-engineered valves exposed to physiological hemodynamic forces.

Animals↗

Bone tissue engineering: hope vs hype.

The requirement for new bone to replace or restore the function of traumatised, damaged, or lost bone is a major clinical and socioeconomic need. Bone formation strategies, although attractive, have yet to yield functional and mechanically competent bone. Bone tissue engineering has been heralded as the alternative strategy to regenerate bone. In essence, the discipline aims to combine progenitor or mature cells with biocompatible materials or scaffolds, with or without appropriate growth factors, to initiate repair and regeneration. This brief review outlines the concepts, challenges, and limitations in bone tissue engineering and the potential that could improve the quality of life for many as a result of interdisciplinary collaboration.

Bone Regeneration↗

Bone tissue engineering for spine fusion: an experimental study on ectopic and orthotopic implants in rats.

Alternatives to the use of autologous bone as a bone graft in spine surgery are needed. The purpose of this study was to examine tissue-engineered bone constructs in comparison with control scaffolds without cells in a posterior spinal implantation model in rats. Syngeneic bone marrow cells were cultured in the presence of bone differentiation factors and seeded on porous hydroxyapatite particles. Seven rats underwent a posterior surgical approach, in which scaffolds with (five rats) or without cells (two rats) were placed on both sides of the lumbar spine. In addition, separate scaffolds were inserted intramuscularly and subcutaneously during the surgical procedure. After 4 weeks, all rats were killed and examined radiographically, by manual palpation of the excised spine and histologically for signs of bone formation or spine fusion. All rats that received cell-seeded scaffolds showed newly formed bone in all three locations, whereas none of the locations in the control rats showed bone formation. The results of this study support the concept of developing tissue-engineering techniques in posterior spine fusion as an alternative to autologous bone.

Animals↗

A device for long term, in vitro loading of three-dimensional natural and engineered tissues.

In vitro studies of mechanical loads applied to three-dimensional tissue constructs are important to the design and production of functional, engineered bone tissue. This study reports the development and characterization of a mechanical device capable of subjecting a three-dimensional section of natural or engineered tissue to precise, reproducible four-point bending deformations over a range of programmable magnitudes and frequencies. To test the biological and mechanical capabilities of the system, a low-cycle (360 cycles/day), medium-range strain (2500 microstrain), long-term (16 day) loading regime was applied to rat bone marrow stromal cells cultured in porous DL-polylactic acid scaffolds. Cells proliferated in culture throughout the experiment, and with time showed an increase in alkaline phosphatase expression per cell. Calcium and phosphorus mineral deposition by the unloaded group was significantly greater (p<0.05) than that deposited by the loaded group. The molar ratio of calcium to phosphorus in the unloaded group (0.94:1) was significantly greater (p<0.05) than that of the loaded group (0.41:1). The loading device presented here is a tool which can be used to help elucidate contributions of mechanical loading/fatigue on biodegradable materials, as well as study the effects of mechanical loading on natural or engineered tissues in vitro.

Animals↗

Comparative study on seeding methods of human bone marrow stromal cells in bone tissue engineering.

BACKGROUND: In general the traditional static seeding method has its limitation while the dynamic seeding method reveals its advantages over traditional static method. We compared static and dynamic seeding method for human bone marrow stromal cells (hBMSCs) in bone tissue engineering. METHODS: DNA assay was used for detecting the maximal initial seeding concentration for static seeding. Dynamic and static seeding methods were compared, when scaffolds were loaded with hBMSCs at this maximal initial cell seeding concentration. Histology and scanning electron microscope (SEM) were examined to evaluate the distribution of cells inside the constructs. Markers encoding osteogenic genes were measured by fluorescent RT-PCR. The protocol for dynamic seeding of hBMSCs was also investigated. RESULTS: DNA assay showed that the static maximal initial seeding concentration was lower than that in dynamic seeding. Histology and SEM showed even distribution and spread of cells in the dynamically seeded constructs, while their statically seeded counterparts showed cell aggregation. Fluorescent RT-PCR again showed stronger osteogenic potential of dynamically seeded constructs. CONCLUSION: dynamic seeding of hBMSCs is a promising technique in bone tissue engineering.

Bone Marrow Cells↗

[Biomechanical properties of tissue-engineered tendons after repairing digital flexor tendons in chickens].

Experiments have been performed to investigate why the biomechanical strength of repaired tendons is lower than that of the normal tendon when the engineered tendons are implanted in vivo to replace the tendon defects. We seeded the primary culture tendon cells derived from Roman chickens' digital flexor tendons on the degradable polyglycolic acid meshes to construct tissue-engineered tendons. The flexor tendon defects (0.5 cm-0.8 cm) excised in second digit bilaterally in 20 Roman chickens, had been repaired with the constructed tissue-engineered tendons. The samples of repaired tendons were collected at 2, 4, 6 and 8 weeks after operation. Tests for scaffold weight, hydroxyproline content, and mechanical strength of the samples were performed. We found that from 2 weeks to 8 weeks afteroperation, the weight of the scaffolds decreased significantly, almost disappearing at 8 weeks; the hydroxyproline content determining the total collagen content increased gradually without significance; mechanically, both energy at break and tensile strength showed a tendency of drastic decrease at first 4 weeks afteroperation and a gradual increase afterwards, but the tensile strength at 8 weeks afteroperation was only 23% of that of the normal tendon. We conclude that the lower biomechanical strength of repaired tendons is owing to the serious mismatch between scaffold degradation and collagen synthesis.

Achilles Tendon↗

Capillary vessel network integration by inserting a vascular pedicle enhances bone formation in tissue-engineered bone using interconnected porous hydroxyapatite ceramics.

The aim of the present study was to investigate the possibility of integrating porous hydroxyapatite (HA) ceramics with a capillary vessel network via insertion of a vascular pedicle, and to determine whether this procedure enhances new bone formation in tissue engineering of bone. First, synthetic interconnected porous HA (IP-CHA) was implanted subcutaneously into rat groin with or without insertion of superficial inferior epigastric vessels. At 6 weeks, IP-CHA with vascular insertion contained thick fibrous connective tissue with a number of large blood vessels that seemed to derive from the inserted vascular bundle. Next, IP-CHA loaded with recombinant human bone morphogenetic protein 2 (BMP, 2 or 10 microg/block) was implanted with or without vascular insertion. At 3 weeks, IP-CHA/BMP (10 microg) composite with vascular insertion exhibited abundant new bone formation in the pores of the deep portion close to the inserted vessels. In contrast, IP-CHA/BMP (10 microg) without vascular insertion showed poor bone formation. Histomorphometric analysis demonstrated that vascular insertion significantly increased new bone formation. In IP-CHAs with a lower dose of BMP (2 microg), no bone formation was found, with or without vascular insertion. These results suggest that the present system of integrating a vascular network with IP-CHA is a useful technique for bone tissue engineering.

Animals↗

Heart valve and arterial tissue engineering.

In the industrialized world, cardiovascular disease alone is responsible for almost half of all deaths. Many of the conditions can be treated successfully with surgery, often using transplantation techniques; however, autologous vessels or human-donated organs are in short supply. Tissue engineering aims to create specific, matching grafts by growing cells on appropriate matrices, but there are many steps between the research laboratory and the operating theatre. Neo-tissues must be effective, durable, non-thrombogenic and non-immunogenic. Scaffolds should be bio-compatible, porous (to allow cell/cell communication) and amenable to surgery. In the early days of cardiovascular tissue engineering, autologous or allogenic cells were grown on inert matrices, but patency and thrombogenicity of grafts were disappointing. The current ethos is toward appropriate cell types grown in (most often) a polymeric matrix that degrades at a rate compatible with the cells' production of their own extracellular matrical proteins, thus gradually replacing the graft with a living counterpart. The geometry is crucial. Computer models have been made of valves, and these are used as three-dimensional patterns for mass-production of implant scaffolds. Vessel walls have integral connective tissue architecture, and application of physiological level mechanical forces conditions bio-engineered components to align in precise orientation. This article reviews the concepts involved and successes achieved to date.

Arteries↗

Patellofemoral joint biomechanics and tissue engineering.

Recent advances in the study of patellofemoral joint biomechanics have provided promising diagnosis and treatment modalities for patellofemoral joint disorders, such as quantitative assessment of cartilage lesions from noninvasive imaging, computer simulations of surgical procedures for optimizing surgical parameters and potentially predicting outcomes, and cartilage tissue engineering for the treatment of advanced degenerative joint disease. These technologies are still in development and their clinical potentials remain an ongoing topic of investigation. We review some of our progress in addressing these issues, and the important role of cartilage mechanics and lubrication in understanding the challenges regarding patellofemoral surgery and cartilage tissue engineering.

Biomechanical Phenomena↗

Tissue engineering applications of therapeutic cloning.

Few treatment options are available for patients suffering from diseased and injured organs because of a severe shortage of donor organs available for transplantation. Therapeutic cloning, where the nucleus from a donor cell is transferred into an enucleated oocyte in order to extract pluripotent embryonic stem cells, offers a potentially limitless source of cells for replacement therapy. Scientists in the field of tissue engineering apply the principles of cell transplantation, material science, and engineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. The present chapter reviews recent advances that have occurred in therapeutic cloning and tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure.

Animals↗

A comparative histologic analysis of tissue-engineered bone using platelet-rich plasma and platelet-enriched fibrin glue.

OBJECTIVE: The aim of this study was to compare the effects of platelet-rich plasma (PRP) and platelet-enriched fibrin glue on bone formation in bone tissue engineering. STUDY DESIGN: PRP was mixed with bone marrow mesenchymal stem cells and bone morphogenetic protein-2 (BMP-2), and the composites were injected into the subcutaneous space on the dorsum of nude mice. On the contralateral side of the dorsum, platelet-enriched fibrin glue/bone marrow mesenchymal stem cells/BMP-2 composites were injected. Bone formation was evaluated after 12 weeks. RESULTS: The volumes of subcutaneous nodules formed in nude mice were 55 +/- 18 microL at the PRP/bone marrow mesenchymal stem cells/BMP-2 sites and 135 +/- 27 microL at the platelet-enriched fibrin glue/bone marrow mesenchymal stem cells/BMP-2 sites. Histomorphometric analysis demonstrated that the nodules contained 14.9 +/- 4.1% newly formed bone when using PRP and 19.8 +/- 3.6% newly formed bone when using platelet-enriched fibrin glue. CONCLUSION: The results indicated that the osteogenic characteristics of platelet-enriched fibrin glue are superior to PRP in bone tissue engineering.

Animals↗

[Tissue engineering for heart valves and vascular grafts].

Current prosthetic substitutes for heart valves and blood vessels have numerous limitations such as limited durability (biological valves), susceptibility to infection, the necessity of lifelong anticoagulation therapy (prosthetic valves), and reduced patency in small-caliber grafts, for example. Tissue engineering using either polymers or decellularized native allogeneic or xenogenic heart valve/vascular matrices may provide the techniques to develop the ideal heart valve or vascular graft. The matrix scaffold serves as a basis on which seeded cells can organise and develop into the valve or vascular tissue prior to or following implantation. The scaffold is either degraded or metabolised during the formation and organisation of the newly generated matrix, leading to vital living tissue. This paper summarises current research and first clinical developments in the tissue engineering of heart valves and vascular grafts.

Animals↗

Bone formation on tissue-engineered cartilage constructs in vivo: effects of chondrocyte viability and mechanical loading.

Interactions between bone and cartilage formation are critical during growth and fracture healing and may influence the functional integration of osteochondral repair constructs. In this study, the ability of tissue-engineered cartilage constructs to support bone formation under controlled mechanical loading conditions was evaluated using a lapine hydraulic bone chamber model. Articular chondrocytes were seeded onto polymer disks, cultured for 4 weeks in vitro, and then transferred to empty bone chambers previously implanted into rabbit femoral metaphyses. The effects of chondrocyte viability within the implanted constructs and in vivo mechanical loading on bone formation were tested in separate experiments. After 4 weeks in vivo, biopsies from the chambers consisted of a complex composite of bone, cartilage, and fibrous tissue, with bone forming in direct apposition to the cartilage constructs. Microcomputed tomography imaging of the chamber biopsies revealed that the implantation of viable constructs nearly doubled the bone volume fraction of the chamber tissue from 0.9 to 1.6% as compared with the implantation of devitalized constructs in contralateral control chambers. The application of an intermittent cyclic mechanical load was found to increase the bone volume fraction of the chamber tissue from 0.4 to 3.6% as compared with no-load control biopsies. The results of these experiments demonstrate that tissue-engineered cartilage constructs implanted into a well-vascularized bone defect will support direct appositional bone formation and that bone formation is significantly influenced by the viability of chondrocytes within the constructs and the local mechanical environment in vivo.

Animals↗

Hepatic tissue engineering for adjunct and temporary liver support: critical technologies.

The severe donor liver shortage, high cost, and complexity of orthotopic liver transplantation have prompted the search for alternative treatment strategies for end-stage liver disease, which would require less donor material, be cheaper, and less invasive. Hepatic tissue engineering encompasses several approaches to develop adjunct internal liver support methods, such as hepatocyte transplantation and implantable hepatocyte-based devices, as well as temporary extracorporeal liver support techniques, such as bioartificial liver assist devices. Many tissue engineered liver support systems have passed the "proof of principle" test in preclinical and clinical studies; however, they have not yet been found sufficiently reliably effective for routine clinical use. In this review we describe, from an engineering perspective, the progress and remaining challenges that must be resolved in order to develop the next generation of implantable and extracorporeal devices for adjunct or temporary liver assist.

Biomedical Technology↗