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Vitrification as a prospect for cryopreservation of tissue-engineered constructs.

Cryopreservation plays a significant function in tissue banking and will presume yet larger value when more and more tissue-engineered products will routinely enter the clinical arena. The most common concept underlying tissue engineering is to combine a scaffold (cellular solids) or matrix (hydrogels) with living cells to form a tissue-engineered construct (TEC) to promote the repair and regeneration of tissues. The scaffold and matrix are expected to support cell colonization, migration, growth and differentiation, and to guide the development of the required tissue. The promises of tissue engineering, however, depend on the ability to physically distribute the products to patients in need. For this reason, the ability to cryogenically preserve not only cells, but also TECs, and one day even whole laboratory-produced organs, may be indispensable. Cryopreservation can be achieved by conventional freezing and vitrification (ice-free cryopreservation). In this publication we try to define the needs versus the desires of vitrifying TECs, with particular emphasis on the cryoprotectant properties, suitable materials and morphology. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most promising modality of cryopreservation.

Animals↗

Elastin biosynthesis: The missing link in tissue-engineered blood vessels.

Nearly 20 years have passed since Weinberg and Bell attempted to make the first tissue-engineered blood vessels. Following this early attempt, vascular tissue engineering has emerged as one of the most promising approaches to fabricate orderly and mechanically competent vascular substitutes. In elastic and muscular arteries, elastin is a critical structural and regulatory matrix protein and plays an important and dominant role by conferring elasticity to the vessel wall. Elastin also regulates vascular smooth muscle cells activity and phenotype. Despite the great promise that tissue-engineered blood vessels have to offer, little research in the last two decades has addressed the importance of elastin incorporation into these vessels. Although cardiovascular tissue engineering has been reviewed in the past, very little attention has been given to elastin. Thus, this review focuses on the recent advances made towards elastogenesis and the challenges we face in the quest for appropriate functional vascular substitutes.

Arteries↗

Heart muscle engineering: an update on cardiac muscle replacement therapy.

Cardiac muscle engineering aims at providing functional myocardium to repair diseased hearts and model cardiac development, physiology, and disease in vitro. Several enabling technologies have been established over the past 10 years to create functional myocardium. Although none of the presently employed technologies yields a perfect match of natural heart muscle, it can be anticipated that human heart muscle equivalents will become available after fine tuning of currently established tissue engineering concepts. This review provides an update on the state of cardiac muscle engineering and its utilization in cardiac regeneration. We discuss the application of stem cells including the allocation of autologous cell material, transgenic technologies that may improve tissue structure as well as in vivo engraftment, and vascularization concepts. We also touch on legal and economic aspects that have to be considered before engineered myocardium may eventually be applied in patients and discuss who may be a potential recipient.

Animals↗

The outlook for implants and endodontics: a review of the tissue engineering strategies to create replacement teeth for patients.

Ideally, root canal therapy involves the removal of diseased pulp tissues and permanent replacement with healthy pulp to revitalize teeth. Rather than placing implants, the ideal solution is to grow new replacement teeth. Success rates of implants and endodontic treatments can exceed 90%, which presents a formidable challenge to tissue engineering researchers to ensure that future dental treatments are even more successful. The purpose of this article is to explain how tissue engineering can be used to create replacement teeth. The science of tissue engineering has evolved from growing simple tissues in cell culture incubators to a multistep process. Although the problems of introducing tissue engineering therapies as part of routine dental treatments are substantial, the potential benefits are equally ground breaking.

Biomimetic Materials↗

Advancing cartilage tissue engineering: the application of stem cell technology.

The treatment of cartilage pathology and trauma face the challenges of poor regenerative potential and inferior repair. Nevertheless, recent advances in tissue engineering indicate that adult stem cells could provide a source of chondrocytes for tissue engineering that the isolation of mature chondrocytes has failed to achieve. Various adjuncts to their propagation and differentiation have been explored, such as biomaterials, bioreactors and growth hormones. To date, all tissue engineered cartilage has been significantly mechanically inferior to its natural counterparts and further problems in vivo relate to poor integration and deterioration of tissue quality over time. However, adult stem cells--with their high rate of proliferation and ease of isolation--are expected to greatly further the development and usefulness of tissue engineered cartilage.

Adipose Tissue↗

Using engineering control principles to inform the design of adaptive interventions: a conceptual introduction.

The goal of this paper is to describe the role that control engineering principles can play in developing and improving the efficacy of adaptive, time-varying interventions. It is demonstrated that adaptive interventions constitute a form of feedback control system in the context of behavioral health. Consequently, drawing from ideas in control engineering has the potential to significantly inform the analysis, design, and implementation of adaptive interventions, leading to improved adherence, better management of limited resources, a reduction of negative effects, and overall more effective interventions. This article illustrates how to express an adaptive intervention in control engineering terms, and how to use this framework in a computer simulation to investigate the anticipated impact of intervention design choices on efficacy. The potential benefits of operationalizing decision rules based on control engineering principles are particularly significant for adaptive interventions that involve multiple components or address co-morbidities, situations that pose significant challenges to conventional clinical practice.

Drug Administration Schedule↗

Living patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells.

OBJECTIVE: A major shortcoming in contemporary congenital heart surgery is the lack of viable replacement materials with the capacity of growth and regeneration. Here we focused on living autologous patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells (EPCs) as a ready-to-use cell source for paediatric cardiovascular tissue engineering. METHODS: EPCs were isolated from 20 ml fresh umbilical cord blood by density gradient centrifugation and myofibroblasts were harvested from umbilical cord tissue. Cells were differentiated and expanded in vitro using nutrient media containing growth factors. Before seeding, cell-phenotypes were assessed by immuno-histochemistry. Biodegradable patches fabricated from synthetic polymers (PGA/P4HB) were seeded with myofibroblasts followed by endothelialization with EPCs. All patches were cultured in a perfusion bioreactor. A subgroup of patches was additionally stimulated by cyclic strain. Analysis of the neo-tissues comprised histology, immuno-histochemistry, extracellular matrix (ECM) analysis and biomechanical testing. RESULTS: Endothelial phenotypes of EPCs before seeding were confirmed by Ac-Dil-LDL, CD 31, von-Willebrand-Factor and eNOS staining. Histology of the seeded patches demonstrated layered viable tissue formation in all samples. The cells in the newly formed tissues expressed myofibroblast markers, such as desmin and alpha-SMA. The EPCs derived neo-endothelia showed constant endothelial phenotypes (CD 31, vWF). major constituents of ECM such as collagen and proteoglycans were biochemically detected. Stress-strain properties of the patches showed features of native-analogous tissues. CONCLUSIONS: Living tissue engineered patches can be successfully generated from human umbilical cord derived myofibroblasts and EPCs. This new cell source may enable the tissue engineering of versatile, living, autologous replacement materials for congenital cardiac interventions.

Biocompatible Materials↗

Formation of in vivo tissue engineered human hyaline cartilage in the shape of a trachea with internal support.

OBJECTIVE: Treatment and management of congenital as well as post-traumatic trachea stenosis remains a challenge in pediatric surgery. The aim of this study was to reconstruct a trachea with human nasal septum chondrocytes by using the combination of biodegradable hydrogel and non-biodegradable high-density polyethylene (HDP) as the internal predetermined shape scaffold. METHODS: Human nasal septum cartilage was harvested as excessive tissue after elective septoplasty and digested in 0.6% collagenase II. Chondrocytes were cultured in an equal volume mix of Ham's F12 medium and Dulbecco's modified eagle medium added with 10% fetal bovine serum and basic fibroblast growth factor. After two passages, the cultured chondrocytes were trypsinized and mixed with biodegradable hydrogel Pluronic F127. The chondrocytes-hydrogel admixture was then painted over the HDP as the internal support in a predetermined trachea shape. The composite was then implanted subcutaneously in athymic mice. RESULTS: After 8 weeks of in vivo implantation, the tissue engineered trachea constructs were harvested. Macroscopic appearance of the tissue engineered trachea constructs demonstrated that the HDP were 80-90% covered with yellowish glistering cartilage like tissue without any sign of inflammation. The tissue engineered trachea cartilage consisted of evenly spaced lacunae embedded in basophilic matrix and stained red with Safranin-O staining denoting abundant proteoglycans production. Type II collagen gene which was expressed in native cartilage was highly expressed in this tissue engineered trachea cartilage. CONCLUSION: We have successfully reconstructed a trachea in vivo with human nasal septum chondrocytes using HDP as the internal support. This construct has the advantage of bio-inert and strength in which both are important properties in tracheal reconstruction.

Animals↗

Depth-dependent biomechanical and biochemical properties of fetal, newborn, and tissue-engineered articular cartilage.

Adult articular cartilage has depth-dependent mechanical and biochemical properties which contribute to zone-specific functions. The compressive moduli of immature cartilage and tissue-engineered cartilage are known to be lower than those of adult cartilage. The objective of this study was to determine if such tissues exhibit depth-dependent compressive properties, and how these depth-varying properties were correlated with cell and matrix composition of the tissue. The compressive moduli of fetal and newborn bovine articular cartilage increased with depth (p<0.05) by a factor of 4-5 from the top 0.1 mm (28+/-13 kPa, 141+/-10 kPa, respectively) to 1 mm deep into the tissue. Likewise, the glycosaminoglycan and collagen content increased with depth (both p<0.001), and correlated with the modulus (both p<0.01). In contrast, tissue-engineered cartilage formed by either layering or mixing cells from the superficial and middle zone of articular cartilage exhibited similarly soft regions at both construct surfaces, as exemplified by large equilibrium strains. The properties of immature cartilage may provide a template for developing tissue-engineered cartilage which aims to repair cartilage defects by recapitulating the natural development and growth processes. These results suggest that while depth-dependent properties may be important to engineer into cartilage constructs, issues other than cell heterogeneity must be addressed to generate such tissues.

Animals↗

Modeling the mechanics of tissue-engineered human heart valve leaflets.

Mathematical models can provide valuable information to assess and evaluate the mechanical behavior of tissue-engineered constructs. In this study, a structurally based model is applied to describe and analyze the mechanics of tissue-engineered human heart valve leaflets. The results from two orthogonal uniaxial tensile tests are used to determine the model parameters of the constructs after two, three and four weeks of culturing. Subsequently, finite element analyses are performed to simulate the mechanical response of the engineered leaflets to a pressure load. The stresses in the leaflets induced by the pressure load increase monotonically with culture time due to a decrease in the construct's thickness. The strains, on the other hand, eventually decrease as a result of an increase in the elastic modulus. Compared to native porcine leaflets, the mechanical response of the engineered tissues after four weeks of culturing is more linear, stiffer and less anisotropic.

Biomechanical Phenomena↗

Tissue engineering of the anterior cruciate ligament using a braid-twist scaffold design.

The anterior cruciate ligament (ACL) is the most commonly injured intra-articular ligament of the knee. The insufficient vascularization of this tissue prevents it from healing completely after extreme tearing or rupture, creating a need for ACL grafts for reconstruction. The limitations of existing grafts have motivated the investigation of tissue-engineered ACL grafts. A successful tissue-engineered graft must possess mechanical properties similar to the ACL; to date no commercially available synthetic graft has achieved this. To accomplish this goal we have combined the techniques of polymer fiber braiding and twisting to design a novel poly L-lactic acid (PLLA) braid-twist scaffold for ACL tissue engineering. The scaffold is designed to accurately mimic the biomechanical profile and mechanical properties of the ACL. In this study, braid-twist scaffolds were constructed and compared to braided scaffolds and twisted fiber scaffolds. The addition of fiber twisting to the braided scaffold resulted in a significant increase in the ultimate tensile strength, an increase in ultimate strain, and an increase in the length of the toe region in these constructs over scaffolds that were braided. Based on the findings of this study, the braid-twist scaffold studied was found to be a promising construct for tissue engineering of the ACL.

Anterior Cruciate Ligament↗

Engineering primary metabolic pathways of industrial micro-organisms.

Metabolic engineering is a powerful tool for the optimisation and the introduction of new cellular processes. This is mostly done by genetic engineering. Since the introduction of this multidisciplinary approach, the success stories keep accumulating. The primary metabolism of industrial micro-organisms has been studied for long time and most biochemical pathways and reaction networks have been elucidated. This large pool of biochemical information, together with data from proteomics, metabolomics and genomics underpins the strategies for design of experiments and choice of targets for manipulation by metabolic engineers. These targets are often located in the primary metabolic pathways, such as glycolysis, pentose phosphate pathway, the TCA cycle and amino acid biosynthesis and mostly at major branch points within these pathways. This paper describes approaches taken for metabolic engineering of these pathways in bacteria, yeast and filamentous fungi.

Bacteria↗

Nonviral transposon&#x2011;engineered stem cells characterization: dose&#x2011;dependency between vector copy number and transgene expression.

Genetically engineered stem cells hold substantial promises for advancing regenerative medicine, yet ensuring their genomic safety remains a critical challenge. A key safety concern is vector copy number (VCN), which defines the number of integrated transgene copies per genome. Although ddPCR is used to assess VCN in virally transduced cells, its application in transposon&#x2011;engineered systems is limited. In this study, we extended VCN determination to non&#x2011;viral, transposon&#x2011;engineered stem cells. In alignment with FDA recommendations, the primary objective was to establish a robust and quantitative framework for interim VCN determination at the time of lot release. Specifically, we demonstrate that reliable interim VCN estimates increase in a dose&#x2011;dependent manner with increasing plasmid input. In addition, strong linear correlations between VCN and both EGFP median fluorescence intensity (MFI) and gene&#x2011;of&#x2011;interest (GOI) protein expression validate the accuracy of this framework. Furthermore, comparison of two distinct GOIs revealed gene&#x2011;specific differences in expression efficiency. Together, these findings validate a standardized VCN determination workflow that quantitatively links plasmid dose, genomic integration, and functional transgene expression. This workflow provides a systematic characterization of engineered cells, offering comprehensive information to support downstream risk&#x2011;based analyses to ensure the genomic safety and stability of the final cell product.

Transgenes↗

Evaluation of engineering properties for the use of leached brown coal ash in soil covers.

The need to engineer cover systems for the successful rehabilitation or remediation of a wide variety of solid wastes is increasing. Some common applications include landfills, hazardous waste repositories, or mine tailings dams and waste rock/overburden dumps. The brown coal industry of the Latrobe Valley region of Victoria, Australia, produces significant quantities of coal ash and overburden annually. There are some site-specific acid mine drainage (AMD) issues associated with overburden material. This needs to be addressed both during the operational phase of a project and during rehabilitation. An innovative approach was taken to investigate the potential to use leached brown coal ash in engineered soil covers on this overburden dump. The basis for this is two-fold: first, the ash has favourable physical characteristics for use in cover systems (such as high storage capacity/porosity, moderately low permeability, and an ability to act as a capillary break layer generating minimal leachate or seepage); and second, the leachate from the ash is mildly alkaline (which can help to mitigate and reduce the risk of AMD). This paper will review the engineering issues involved in using leached brown coal ash in designing soil covers for potentially acid-forming overburden dumps. It presents the results of laboratory work investigating the technical feasibility of using leached brown coal ash in engineered solid waste cover systems.

Coal Mining↗

Tissue engineering for the hand surgeon: a clinical perspective.

Hand surgeons may be faced with tissue shortages for reconstruction after trauma, tumor resection, or congenital deficiency. Tissue engineering is a developing scientific field that combines the principles of life sciences and engineering in developing biologic substitutes that will restore, maintain, or improve tissue function. This article reviews the general principles of tissue engineering as applied to musculoskeletal tissues including nerve, bone, tendon, skin, vessels, and cartilage and focuses on the application of tissue engineering that is relevant to clinical hand surgery.

Blood Vessel Prosthesis↗

Correlated evolutionary pressure at interacting transcription factors and DNA response elements can guide the rational engineering of DNA binding specificity.

Understanding the molecular mechanisms of the specific interaction between transcription factor proteins and DNA is key to comprehend the regulation of gene expression and to develop technologies to engineer transcription factors. Thus far, although there have been several attempts to elucidate protein-DNA interaction through amino acid-base recognition codes, sequence based profiles, or physical models of interaction, the greatest successes in engineering DNA binding specificity remain experimental. Here we present the first systematic evidence of correlated evolutionary pressure at interacting amino acid residues and DNA base-pairs in transcription factors, and show that it can be used to rationally engineer DNA binding specificity. The correlation is between the relative evolutionary importance of protein residues and DNA bases, measured, respectively, in terms of the Evolutionary Trace (ET) rank and information entropy. The evolutionarily most important residues interact with the most conserved base-pairs within the response element while residues of least importance interact with the most variable base-pairs. The correlation averages 0.74 over 12 unrelated families of transcriptional regulators, including nuclear hormone receptors, basic helix-loop-helix, ETS- and homeo-domain family. To test the predictive power of this correlation, we targeted a mutational swap of top-ranked ET residues in a transcription factor, LRH-1. This redirects LRH-1 binding as predicted and showed that, in this case, evolutionary importance and binding specificity are coupled sufficiently strongly for the Evolutionary Trace to guide the computational design of DNA binding specificity. This establishes the existence of evolutionary importance correlation at protein-DNA interfaces, and demonstrates that it is a useful principle for the rational engineering of binding specificity.

Animals↗

Engineering zinc finger protein transcription factors: the therapeutic relevance of switching endogenous gene expression on or off at command.

Modulating gene expression directly at the DNA level represents a novel approach to control cellular processes. In this respect, zinc finger protein DNA-binding domains can be engineered to target virtually any gene. Coupling of a transcription activation or repression domain to these zinc fingers permits regulating gene expression at will, providing a platform of unlimited therapeutic applications. In this review, steps involved in the engineering of zinc finger protein transcription factors are described. In addition, an overview of endogenous genes successfully targeted for modulating expression by engineered zinc finger protein transcription factors is given. So far, research has mainly focused on targeting genes involved in cancer and angiogenesis, with encouraging evaluation in vivo and progression into a clinical trial. Altogether, engineered zinc finger proteins offer a new and exciting direction in the field of medical research with promising prospects.

Animals↗

Adjacent tissues (cartilage, bone) affect the functional integration of engineered calf cartilage in vitro.

OBJECTIVE: An in vitro model was used to test the hypothesis that culture time and adjacent tissue structure and composition affected chondrogenesis and integrative repair in engineered cartilage. METHOD: Engineered constructs made of bovine calf chondrocytes and hyaluronan benzyl ester non-woven mesh were press-fitted into adjacent tissue rings made of articular cartilage (AC), devitalized bone (DB), or vital bone (VB) and cultured in rotating bioreactors for up to 8 weeks. Structure (light and electron microscopy), biomechanical properties (interfacial adhesive strength, construct compressive modulus), biochemical composition (construct glycosaminoglycans (GAG), collagen, and cells), and adjacent tissue diffusivity were assessed. RESULTS: Engineered constructs were comprised predominately of hyaline cartilage, and appeared either closely apposed to adjacent cartilage or functionally interdigitated with adjacent bone due to interfacial deposition of extracellular matrix. An increase in culture time significantly improved construct adhesive strength (P<0.001), modulus (P=0.02), GAG (P=0.04) and cellularity (P<0.001). The type of adjacent tissue significantly affected construct adhesion (P<0.001), modulus (P<0.001), GAG (P<0.001) and collagen (P<0.001). For constructs cultured in rings of cartilage, negative correlations were observed between ring GAG content (log transformed) and construct adhesion (R2=0.66, P<0.005), modulus (R2=0.49, P<0.05) and GAG (R2=0.44, P<0.05). Integrative repair was better for constructs cultured adjacent to bone than cartilage, in association with its solid architectural structure and high GAG content, and best for constructs cultured adjacent to DB, in association with its high diffusivity. CONCLUSIONS: Chondrogenesis and integrative repair in engineered cartilage improved with time and depended on adjacent tissue architecture, composition, and transport properties.

Animals↗