Directed assembly of cell-laden hydrogels for engineering functional tissues

Tissue engineering aims to develop functionalized tissues for organ replacement or restoration. Biodegradable scaffolds have been used in tissue engineering to support cell growth and maintain mechanical and biological properties of tissue constructs. Ideally cells on these scaffolds adhere, proliferate, and deposit matrix at a rate that is consistent with scaffold degradation. However, the cellular rearrangement within these scaffolds often does not recapitulate the architecture of the native tissues. Directed assembly of tissue-like structures is an attractive alternative to scaffold-based approach for tissue engineering which potentially can build tissue constructs with biomimetic architecture and function. In directed assembly, shape-controlled microstructures are fabricated in which organized structures of different cell types can be used as tissue building blocks. To fabricate tissue building blocks, hydrogels are commonly used as biomaterials for cell encapsulation to mimic the matrix in vivo. The hydrogel-based tissue building blocks can be arranged in pre-defined architectures by various directed tissue assembly techniques. In this paper, recent advances in directed assembly-based tissue engineering are summarized as an emerging alternative to meet challenges associated with scaffold-based tissue engineering and future directions are addressed.     

Engineering towards functional tissues and organs

Engineering towards fully functional tissues and organs fall under the widespread banner of tissue engineering and regenerative biology/medicine (which includes therapeutics). This endeavor is said to evolve from the Greek mythical story¹ where Prometheus is punished by Zeus who assigned a long winged eagle to feed on Prometheus’s liver, for stealing fire from the Gods. It is said that Prometheus’s liver grew as much it was eaten. Thus, conceptualizing tissue engineering and regenerative medicine. This in its very early stages was investigated by Alexis Carrel and Charles Lindbergh at the Rockefeller Institute for Medical Research² and was subsequently clinically implemented in 1954 in renal transplantation medicine in the United States by Joseph Murray and colleagues.3 Both Carrel and Murray were awarded Nobel Prizes for their findings and contributions.⁴ Although these are hallmarks for tissue engineering and regenerative medicine, there are many obstacles and to date we are still grappling with some of these hurdles whist others have to a great extent been understood, and could be argued as being to some degree resolved. Some obstacles we face today range from (1) the ability to grow specialized and unspecialized cells outside the human body in practical quantities while retaining their native functionality, (2) biomaterials (advancing the development of synthetic materials for enhanced bioactivity), (3) advanced approaches for forming three-dimensional fully functional tissues and or organs to (4) controlled vascularization, which are a few examples amongst many.     

Two-phase bioreactor system for cell-laden hydrogel assembly

Bottom-up approach is a potentially useful tool for hydrogel assembly of cell-laden individual building blocks. In this article, we assembled individual building blocks of photocrosslinkable microgels in a rapid and controlled manner. Individual building blocks of poly(ethylene glycol) (PEG) microgels with square and hexagonal shapes were fabricated by using a photolithography technique. Individual building blocks of PEG microgels were assembled on a hydrophobic mineral oil phase in a bioreactor with a magnetic stirrer. The hydrophobic mineral oil minimized the surface free energy to assemble hydrophilic PEG microgels on a two-phase oil-aqueous solution interface. We used the hydrophobic effect as a driving force for the hydrogel assembly. Various types of the hydrogel assembly were generated by controlling the stirring rate. As stirring speed increased, the percentage of linear, branched, and closely packed hydrogel assembly was increased. However, the percentage of random assembly was reduced by increasing stirring rate. The stirring time also played an important role in controlling the types of hydrogel assembly. The percentage of linear, branched, and closely packed hydrogel assembly was improved by increasing stirring time. Therefore, we performed directed cell-laden hydrogel assembly using a two-phase bioreactor system and optimized the stirring rate and time to regulate the desired types of hydrogel assembly. Furthermore, we analyzed cell viability of hydrogel linear assembly with square shapes, showing highly viable even after secondary photocrosslinking reaction. This bioreactor system-based hydrogel assembly could be a potentially powerful approach for creating tissue microarchitectures in a three-dimensional manner.     

Strategies for organ level tissue engineering

The field of tissue engineering has made considerable strides since it was first described in the late 1980s. The advent and subsequent boom in stem cell biology, emergence of novel technologies for biomaterial development, and further understanding of developmental biology have contributed to this accelerated progress. However, continued efforts to translate tissue engineering strategies into clinical therapies have been hampered by the problems associated with scaling up laboratory methods to produce large, complex tissues. The significant challenges faced by tissue engineers include the production of an intact vasculature within a tissue-engineered construct and recapitulation of the size and complexity of a whole organ. Here we review the basic components necessary for bioengineering organs – biomaterials, cells and bioactive molecules–and discuss various approaches for augmenting these principles to achieve organ level tissue engineering. Ultimately, the successful translation of tissue-engineered constructs into everyday clinical practice will depend upon the ability of the tissue engineer to “scale up” every aspect of the research and development process.     

Sequential assembly of cell-laden hydrogel constructs to engineer vascular-like microchannels

Microscale technologies, such as microfluidic systems, provide powerful tools for building biomimetic vascular-like structures for tissue engineering or in vitro tissue models. Recently, modular approaches have emerged as attractive approaches in tissue engineering to achieve precisely controlled architectures by using microengineered components. Here, we sequentially assembled microengineered hydrogels (microgels) into hydrogel constructs with an embedded network of microchannels. Arrays of microgels with predefined internal microchannels were fabricated by photolithography and assembled into 3D tubular construct with multi-level interconnected lumens. In the current setting, the sequential assembly of microgels occurred in a biphasic reactor and was initiated by swiping a needle to generate physical forces and fluidic shear. We optimized the conditions for assembly and successfully perfused fluids through the interconnected constructs. The sequential assembly process does not significantly influence cell viability within the microgels indicating its promise as a biofabrication method. Finally, in an attempt to build a biomimetic 3D vasculature, we incorporated endothelial cells and smooth muscle cells into an assembled construct with a concentric microgel design. The sequential assembly is simple, rapid, cost-effective, and could be used for fabricating tissue constructs with biomimetic vasculature and other complex architectures.     

Tissue engineering of a collagen-based vascular media

The property of vasoactivity is important for both resistance vessels and larger arteries. Evaluation of smooth muscle cell phenotype is often done in place of functional testing in engineered tissues, assuming a direct correlation between cell phenotype and tissue contractile force. In this study we look at a large panel of vasoactive agents to determine the functionality of our collagen-based tissue. The engineered vascular media elicited a measurable change in force in response to seven of the nine agents used. As part of this characterization, TGF-β₁ and TNF-α were used to promote a more contractile and synthetic cell phenotype respectively. Both smooth muscle α-actin and vasoconstriction were evaluated in ring sections. Due to large differences in cell-compaction and cell distribution in the tissues, no correlation was found between α-actin expression and contractile strength. This highlights the need for functional testing of engineered tissue and the importance of cell-matrix interactions in vasoactivity.     

Bioconjugation of hydrogels for tissue engineering

Success of tissue engineered constructs in regenerative medicine is limited by the lack of cellmatrix interactions to guide devleopment of the seeded cells into the desired tissue. This review highlights the most exciting developments in bioconjugation of synthetic hydrogels targeted to tissue engineering. Application of conjugation techniques has resulted in the synthesis of novel biomimetic cell-responsive hydrogels to control the cascade of cell migration, adhesion, survival, differentiation, and maturation to the desired lineage concurrent with matrix remodeling. The future outlook includes developing conjugated patterned hydrogel matrices, developing novel hydrogel matrices to support self-renewal and pluripotency of embryonic and adult stem cells, and merging 3D printing with bioconjugation to fabricate hydrogels with anatomical arrangement of cells and biomolecules.     

Development of hydrogels for regenerative engineering

The aim of regenerative engineering is to restore complex tissues and biological systems through convergence in the fields of advanced biomaterials, stem cell science, and developmental biology. Hydrogels are one of the most attractive biomaterials for regenerative engineering, since they can be engineered into tissue mimetic 3D scaffolds to support cell growth due to their similarity to native extracellular matrix. Advanced nano‐ and micro‐technologies have dramatically increased the ability to control properties and functionalities of hydrogel materials by facilitating biomimetic fabrication of more sophisticated compositions and architectures, thus extending our understanding of cell‐matrix interactions at the nanoscale. With this perspective, this review discusses the most commonly used hydrogel materials and their fabrication strategies for regenerative engineering. We highlight the physical, chemical, and functional modulation of hydrogels to design and engineer biomimetic tissues based on recent achievements in nano‐ and micro‐technologies. In addition, current hydrogel‐based regenerative engineering strategies for treating multiple tissues, such as musculoskeletal, nervous and cardiac tissue, are also covered in this review. The interaction of multiple disciplines including materials science, cell biology, and chemistry, will further play an important role in the design of functional hydrogels for the regeneration of complex tissues.     

Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for adipose tissue regeneration

An injectable, biodegradable and glucose-responsive hydrogel derived from natural polysaccharide derivatives was synthesized to deliver adipogenic factor of insulin in vitro for adipose tissue engineering. The biodegradable hydrogel based N-succinyl-chitosan (SCS) and aldehyde hyaluronic acid (AHA) with covalently conjugated glucose oxidase and catalase. The gelation is attributed to the Schiff-base reaction between amino and aldehyde groups of SCS and AHA, respectively. The morphologies and compressive modulus of the freeze-dried hydrogels demonstrated that the incorporated insulin and enzymes results in the formation of a tighter network structure in composite hydrogels. The immobilized enzymes triggered conversion of glucose reduces the pH value of the microenvironment, and results in hydrolysis and increasing swelling of the network basing on Schiff-base cross-linking. The pH inside the hydrogel, kept in PBS solution at pH 7.4 and 37oC, linearly dropped from 7.40 to 7.17 during 4 h of initial period, then slowly increased to 7.36 after 24 h. Correspondingly, the swelling ratio increased from 20.8 to 28.6 at 37oC in PBS with 500 mg/dL glucose. In PBS buffer with 500mg/dL glucose, about 10.8 % of insulin was seen to be released from the hydrogel after 8 h of incubation. The results demonstrated that the adipogenic factor of insulin would be released from this biodegradable hydrogel device into the local microenvironment in a controlled fashion by the swelling of hydrogel network. These preliminary studies indicate that the biodegradable and glucose-responsive hydrogel may have potential uses in adipose tissue engineering applications.     

Upslope movements and large scale expansions: the taxonomy and biogeography of the Coenonympha arcania –C. d arwiniana –C. gardetta butterfly species complex

Sibling species groups are suitable models for the understanding of inter‐ and intraspecific processes in taxonomy and biogeography. We analysed 262 individuals from the Alps of the Coenonympha arcania/gardetta species complex by allozyme electrophoresis. These taxa showed high variance amongst populations (F_ST: 0.391) and strong intertaxon genetic differentiation (F_CT: 0.376). Although morphologically similar, Coenonympha gardetta and Coenonympha arcania clearly differ in their genetic characteristics; the morphologically intermediate taxa Coenonympha darwiniana darwiniana and Coenonympha darwiniana macromma are genetically well distinguished from each other and the two other taxa. Coenonympha arcania and C. d. macromma most probably share a common ancestor and evolved by cladogenesis, whereas the taxonomic situation of C. d. darwiniana is still unresolved: This taxon might be the result of hybridization between C. arcania and C. gardetta or it might have a common ancestor together with C. gardetta. We suggest species rank for all four taxa. The distribution of genetic diversity of these populations and the differentiation amongst populations suggest rather different biogeographical scenarios: C. arcania most probably is of Mediterranean origin with postglacial range expansion northwards; C. gardetta survived the last ice age in peripheral refugia of the Alps and has spread all over this high mountain system in the postglacial; C. darwiniana and C. macromma survived the Würm in geographic proximity to their actual distribution areas and only have performed moderate uphill translocations during postglacial warming. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 159 , 890–904.     

Distribution of prostaglandin ω-hydroxylases in different tissues

The conversion of prostaglandins E₂ and F_2α to their 19- and 20-hydroxy metabolites by various tissues has been measured by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring. A number of different tissues of the pregnant rabbit possess prostaglandin 20-hydroxylase activity (lung > liver > fetal placenta > maternal placenta ≈ uterus > renal cortex > renal medulla ≈ placental membranes). With the exception of the liver, prostaglandins E₂ and F_2α are metabolized at equal rates by the 20-hydroxylases of different tissues. Only lung and liver microsomes possess high levels of prostaglandin 20-hydroxylase in non-pregnant rabbits and only liver microsomes have appreciable 19-hydroxylase activity. Pulmonary prostaglandin 20-hydroxylase is induced in male rabbits by treatment with progesterone. On the basis of substrate specificity studies and the effects of a cytochrome P-450 inhibitor, SKF-525A, the prostaglandin 20-hydroxylases of lung and liver microsomes from pregnant rabbits appear to be different enzymes. In pregnant rats and hamsters, liver and kidney are the only tissues in which we detected prostaglandin ω-hydroxylase activity.     

Directed assembly of a bacterial quorum

Many reports have elucidated the mechanisms and consequences of bacterial quorum sensing (QS), a molecular communication system by which bacterial cells enumerate their cell density and organize collective behavior. In few cases, however, the numbers of bacteria exhibiting this collective behavior have been reported, either as a number concentration or a fraction of the whole. Not all cells in the population, for example, take on the collective phenotype. Thus, the specific attribution of the postulated benefit can remain obscure. This is partly due to our inability to independently assemble a defined quorum, for natural and most artificial systems the quorum itself is a consequence of the biological context (niche and signaling mechanisms). Here, we describe the intentional assembly of quantized quorums. These are made possible by independently engineering the autoinducer signal transduction cascade of Escherichia coli (E. coli) and the sensitivity of detector cells so that upon encountering a particular autoinducer level, a discretized sub-population of cells emerges with the desired phenotype. In our case, the emergent cells all express an equivalent amount of marker protein, DsRed, as an indicator of a specific QS-mediated activity. The process is robust, as detector cells are engineered to target both large and small quorums. The process takes about 6 h, irrespective of quorum level. We demonstrate sensitive detection of autoinducer-2 (AI-2) as an application stemming from quantized quorums. We then demonstrate sub-population partitioning in that AI-2-secreting cells can ‘call'' groups neighboring cells that ‘travel'' and establish a QS-mediated phenotype upon reaching the new locale.     

Albumin-based hydrogels for regenerative engineering and cell transplantation

Albumin, the most abundant plasma protein in mammals, is a versatile and easily obtainable biomaterial. It is pH and temperature responsive, dissolvable in high concentrations and gels readily in defined conditions. This versatility, together with its inexpensiveness and biocompatibility, makes albumin an attractive biomaterial for biomedical research and therapeutics. So far, clinical research in albumin has centered mainly on its use as a carrier molecule or nanoparticle to improve drug pharmacokinetics and delivery to target sites. In contrast, research in albumin-based hydrogels is less established albeit growing in interest over recent years. In this minireview, we report current literature and critically discuss the synthesis, mechanical properties, biological effects and uses, biodegradability and cost of albumin hydrogels as a xeno-free, customizable, and transplantable construct for tissue engineering and regenerative medicine.     

Rheological properties of cross-linked hyaluronan-gelatin hydrogels for tissue engineering

Hydrogels that mimic the natural extracellular matrix (ECM) are used in three-dimensional cell culture, cell therapy, and tissue engineering. A semi-synthetic ECM based on cross-linked hyaluronana offers experimental control of both composition and gel stiffness. The mechanical properties of the ECM in part determine the ultimate cell phenotype. We now describe a rheological study of synthetic ECM hydrogels with storage shear moduli that span three orders of magnitude, from 11 to 3 500 Pa, a range important for engineering of soft tissues. The concentration of the chemically modified HA and the cross-linking density were the main determinants of gel stiffness. Increase in the ratio of thiol-modified gelatin reduced gel stiffness by diluting the effective concentration of the HA component.     

Helical spring template fabrication of cell‐laden microfluidic hydrogels for tissue engineering

Cell‐laden microfluidic hydrogels find great potential applications in microfluidics, tissue engineering, and drug delivery, due to their ability to control mass transport and cell microenvironment. A variety of methods have been developed to fabricate hydrogels with microfluidic channels, such as molding, bioprinting, and photopatterning. However, the relatively simple structure available and the specific equipment required limit their broad applications in tissue engineering. Here, we developed a simple method to fabricate microfluidic hydrogels with helical microchannels based on a helical spring template. Results from both experimental investigation and numerical modeling revealed a significant enhancement on the perfusion ability and cell viability of helical microfluidic hydrogels compared to those with straight microchannels. The feasibility of such a helical spring template method was also demonstrated for microfluidic hydrogels with complex three‐dimensional channel networks such as branched helical microchannels. The method presented here could potentially facilitate the development of vascular tissue engineering and cell microenvironment engineering. Biotechnol. Bioeng. 2013; 110: 980–989. © 2012 Wiley Periodicals, Inc.     

Diels-Alder Click cross-linked hyaluronic acid hydrogels for tissue engineering

Hyaluronic acid (HA) is a naturally occurring polymer that holds considerable promise for tissue engineering applications. Current cross-linking chemistries often require a coupling agent, catalyst, or photoinitiator, which may be cytotoxic, or involve a multistep synthesis of functionalized-HA, increasing the complexity of the system. With the goal of designing a simpler one-step, aqueous-based cross-linking system, we synthesized HA hydrogels via Diels-Alder "click" chemistry. Furan-modified HA derivatives were synthesized and cross-linked via dimaleimide poly(ethylene glycol). By controlling the furan to maleimide molar ratio, both the mechanical and degradation properties of the resulting Diels-Alder cross-linked hydrogels can be tuned. Rheological and degradation studies demonstrate that the Diels-Alder click reaction is a suitable cross-linking method for HA. These HA cross-linked hydrogels were shown to be cytocompatible and may represent a promising material for soft tissue engineering.