most cell what are porous to water and other materials

• Journal Listing
• Tissue Eng Part B Rev
• PMC2946907

Tissue Eng Part B Rev. 2010 Aug; sixteen(4): 371–383.

Controlling the Porosity and Microarchitecture of Hydrogels for Tissue Engineering

Nasim Annabi, Thou.S.,^{1, *} Jason W. Nichol, Ph.D.,^{2,, iii, *} Xia Zhong, M.S.,¹ Chengdong Ji, M.B.E.,^ane Sandeep Koshy, B.A.Sc.,^{two,, iii} Ali Khademhosseini, Ph.D.,^{2,, 3} and Fariba Dehghani, Ph.D. ¹

Nasim Annabi

¹Schoolhouse of Chemic and Biomolecular Applied science, University of Sydney, Sydney, Australia.

Jason W. Nichol

^twoDepartment of Medicine, Center for Biomedical Engineering, Brigham and Women's Hospital, Harvard Medical Schoolhouse, Boston, Massachusetts.

³Harvard-MIT Partitioning of Health Sciences and Technology, Massachusetts Establish of Applied science, Cambridge, Massachusetts.

Xia Zhong

¹School of Chemic and Biomolecular Engineering, University of Sydney, Sydney, Commonwealth of australia.

Chengdong Ji

^aneSchool of Chemic and Biomolecular Engineering, Academy of Sydney, Sydney, Commonwealth of australia.

Sandeep Koshy

^twoDepartment of Medicine, Middle for Biomedical Engineering, Brigham and Women's Infirmary, Harvard Medical School, Boston, Massachusetts.

^threeHarvard-MIT Division of Wellness Sciences and Technology, Massachusetts Plant of Technology, Cambridge, Massachusetts.

Ali Khademhosseini

²Section of Medicine, Center for Biomedical Engineering, Brigham and Women'southward Hospital, Harvard Medical School, Boston, Massachusetts.

^threeHarvard-MIT Partition of Health Sciences and Technology, Massachusetts Found of Engineering, Cambridge, Massachusetts.

Fariba Dehghani

ⁱSchool of Chemical and Biomolecular Engineering, Academy of Sydney, Sydney, Australia.

Received 2009 Sep 24; Accustomed 2010 Jan 29.

Abstruse

Tissue engineering holds great promise for regeneration and repair of diseased tissues, making the evolution of tissue engineering scaffolds a topic of great interest in biomedical research. Because of their biocompatibility and similarities to native extracellular matrix, hydrogels have emerged equally leading candidates for engineered tissue scaffolds. However, precise control of hydrogel properties, such as porosity, remains a challenge. Traditional techniques for creating majority porosity in polymers have demonstrated success in hydrogels for tissue engineering; withal, often the conditions are incompatible with direct cell encapsulation. Emerging technologies have demonstrated the power to control porosity and the microarchitectural features in hydrogels, creating engineered tissues with structure and office similar to native tissues. In this review, we explore the various technologies for controlling the porosity and microarchitecture inside hydrogels, and demonstrate successful applications of combining these techniques.

Introduction

In nearly scaffolding materials, the porosity of the scaffolds plays an important role in directing tissue formation and function.^one–3 A substantial corporeality of scaffold porosity is often necessary to let for homogeneous cell distribution and interconnection throughout engineered tissues. In addition, increased porosity can have a benign effect on the diffusion of nutrients and oxygen, especially in the absenteeism of a functional vascular organisation.^two Hydrogels have been used as scaffolds for tissue engineering science applications because of their similarities with extracellular matrix (ECM), excellent biological performance, and inherent cellular interaction capability. Hydrogels are crosslinked macromolecular networks formed by hydrophilic polymers swollen in water or biological fluids.⁴ Upon implantation, hydrogel porosity allows for local angiogenesis to occur, which is a central requirement for vascularized tissues. The caste of porosity volition also take a substantial consequence on the mechanical backdrop, with the stiffness of the scaffold decreasing equally porosity increases,^v and the mechanical characteristics varying greatly with fluid flux caused by deformation.⁶ The porosity and pore architecture in terms of porosity and pore interconnectivity play a significant role in prison cell survival, proliferation, and migration to fabricate functional hydrogel, and secrete ECM.^7,8 The pore interconnectivity allows for cell ingrowth, vascularization, and nutrient improvidence for cell survival.^ix–11 The extent of ECM secretion also increases by increasing the pore size.^eight It was plant that in genipin crosslinked gelatin hydrogels with smaller pores, the tendency was tilted toward prison cell growth rather than of ECM secretion,⁸ resulting in overconfluence during the eye and belatedly stages of differentiation; consequently, the extent of ECM secretion decreased compared to that within gelatin hydrogels with larger pores.⁸ The average pore size of the hydrogels profoundly affects the growth and penetration of cells in the 3D structure of hydrogels. Without using an intrinsic capillary network, the maximal thickness of engineered tissue is approximately 150–200 μm because of insufficient oxygen and nutrient transport within the deeper compartments of the biomaterial.¹² In improver, mean pore size has been shown to impact the amount of contraction a graft will undergo after implantation. An average pore diameter of 20–125 μm was required for contraction-inhibiting action to be observed in collagen–glycosaminoglycan graft copolymers used for dermal repair.¹³ The effect of implant pore size on tissue regeneration is emphasized by experiments demonstrating the optimum pore size of five μm for neovascularization, v–15 μm for fibroblast ingrowth, 20–125 μm for regeneration of adult mammalian pare, 100–350 μm for regeneration of os, xl–100 μm for osteoid ingrowth, and 20 μm for the ingrowth of hepatocytes.¹⁴ Fibrovascular tissues also require pore sizes greater than 500 μm for rapid vascularization and survival transplanted cells.¹⁵ The microscale features of individual and clusters of pores combine to create the hydrogel microarchitecture, controlling many aspects of cellular orientation, assemblage, and function.^2,16 Thus, command of scaffold porosity and microarchitecture plays a key role in regulating engineered tissue backdrop.¹⁷

Control of these intricate hydrogel features is important toward guiding the development of the resulting engineered tissues. Techniques to control the overall porosity of hydrogels include solvent casting/particle leaching, freeze-drying, gas foaming, and electrospinning. Combinations of these methods have been used to fabricate porous hydrogels for many tissue engineering applications. In addition, more advanced control of specific pore features and microarchitecture has been achieved through diverse micropatterning¹⁸ and micromolding^xix–22 techniques. With these techniques it is possible not but to specifically control individual and group pore architecture, but too to take the next footstep to create microvascular features to improve integration within host tissues. In this review we will describe the potential and limitations of these methods to control bulk porosity as well as the microarchitectural features of channels and capillary networks.

Macroscale Porosity and Microarchitecture

Solvent casting/particle leaching

Solvent casting/particle leaching begins with the dispersion of a porogen with controlled particle size into a polymer solution. The appropriate technique is used to solidify the polymer, producing a polymer–porogen network.^23,24 The solute particles are subsequently leached, or dissolved away by immersing the material in a selective solvent, resulting in the formation of a porous network.

A wide variety of porogens have been employed for this technique depending on the hydrogel and awarding. Historically, salt particles are most commonly used because they are inexpensive, widely available, piece of cake to handle, and stable under an assortment of processing conditions. Alternative porogen materials, including sugars,²⁵ paraffin,²⁶ and gelatin,²⁷ take also been employed with hydrogels. The nature of the porogen has been demonstrated to play an important role in the interconnectivity of pores and in turn the jail cell–scaffold interactions after seeding.²⁸ Porogen geometry has besides been shown to influence the construction of the porous network formed using particle leaching.²⁹ In detail, it has been demonstrated that spherical particles outcome in more interconnected pores than cubic particles at the same final porosity.²⁹

Using table salt leaching, poly(ethylene glycol) (PEG)–poly(ɛ-caprolactone) (PCL)–based hydrogels were produced using NaCl as a porogen (pore size distribution: 180–400 μm) and dimethyl sulfoxide as a solvent.³⁰ After leaching with distilled h2o, a highly porous and interconnected matrix with enhanced swelling properties was formed and was demonstrated to be capable of facilitating efficient prison cell seeding of rabbit chondrocytes.³⁰ Similar processing techniques using NaCl have recently been applied to oligo[(PEG) fumarate],³¹ alginate-g-poly(N-isopropylacrylamide),³² poly(ii-hydroxyethyl methacrylate),³³ and other hydrogel systems.

In another variation, salt leaching has been used to create polyester scaffold templates that can exist utilized for fabrication of macroporous hydrogels. In i study, a PEG-poly(lysine) prepolymer was bandage and cured around a salt-leached poly(lactic acid-co-glycolic acid) (PLGA) scaffold.³⁴ The PLGA network was degraded using sodium hydroxide (NaOH), leaving backside a macroporous PEG-poly(lysine) hydrogel.³⁴ Endothelial cells were seeded and shown to found tubules with dimensions that were consequent with the pore size, suggesting that the scaffold structure provided concrete direction for vessel germination.³⁴

Particle leaching has recently been used to fabricate porous components in microfluidic devices.³⁵ A living radical photopolymer system and salt leaching technique were combined to generate porous polymer networks in microfluidic channels. The microchannel was produced past living radical photopolymer, whereas porosity was generated later salt leaching.³⁵ Valve systems were made and it was shown that porous valves volition smashing and close much more rapidly than nonporous valves fabricated with similar materials.³⁵

A major advantage of the solvent casting method is that pore size and overall porosity tin can be tuned by changing the particle size and concentration within the prepolymer, respectively. Additionally, this technique can be performed feasibly on a small-scale scale, making it widely bachelor and well suited for use during the development stages of a new hydrogel arrangement. However, using the solvent casting method there is picayune control over the orientation and the caste of interconnectivity of pores. Owing to limitations associated with removing solid particles from the hydrogel, this technique is unremarkably restricted to the fabrication of commonly thin hydrogel sheets (typically less than 500 μm) that must after be assembled into a larger construct. It should be noted that many particle leaching methods rely on the employ of cytotoxic organic solvents that require lengthy drying times to assure complete removal and are not compatible with cell viability.

Freeze-drying

Freeze-drying, besides known as lyophilization, has been used extensively for the fabrication of porous hydrogels for tissue engineering. This method uses rapid cooling to produce thermodynamic instability within a arrangement and crusade phase separation. The solvent is so removed by sublimation under vacuum³⁶ leaving behind voids in the regions it previously occupied.

This technique was applied to produce porous collagen–chitosan hydrogels.³⁷ After preparation of a 9:1 collagen:chitosan blend, glutaraldehyde solution was added to crosslink the mixture before freeze-drying. The freezing temperature before lyophilization was shown to have an impact on the characteristics of the resulting hydrogel. In item, samples that were frozen at −20°C and −80°C resulted in open pore structures afterwards lyophilization, whereas parallel sheet structure was obtained at −196°C (in liquid N₂).³⁷ Moreover, the swelling ratio macerated from 4400% to 2000% every bit the temperature decreased from −20°C to −196°C due to the reduction of contact expanse.³⁷ Both in vitro and in vivo characterizations confirmed that the fabricated hydrogels seeded by preadipocytes cells were biocompatible, induced vascularization, and formed adipose tissue.³⁷

A modified freeze-drying procedure has been used to fabricate agarose hydrogels with linear pores (Fig. 1A, B).³⁸ This method utilizes a freezing step that involves exposing just 1 terminate of a pillar of agarose to a block of dry ice immersed inside a puddle of liquid nitrogen.³⁸ The resulting uniaxial temperature slope caused ice crystals to form that were oriented in the management of the gradient.³⁸ Upon removal of water past lyophilization, a highly linear network of porous channels was formed with dimensions suitable for cell infiltration.³⁸ Scaffolds fabricated with this method were able to guide axonal regeneration in a spinal string injury model (Fig. 1C–Due east).³⁹

Morphology and in vivo performance of freeze-stale agarose hydrogel scaffolds. Scanning electron microscopy (SEM) images of longitudinal (A) and cross-sectional (B) orientations show linear porosity. Axonal penetration after 1 month in vivo in a spinal cord injury model in the absence (C) and presence (D) of brain-derived neurotrophic factor. The all-time sample attained shows axons completely crossing the construct (Eastward). Calibration bar: 100 μm. Adapted with permission from Stokols et al. ³⁹

Since the quenching procedure used in freeze-drying techniques is generally rapid, the phase separation occurring during the initial freezing may non exist complete, resulting in the formation of a polymer-lean stage that may still contain suboptimal amounts of polymer.⁴⁰ To circumvent this problem, an alternating method involving the use of repeated freeze–thaw cycles may be employed. Upon repeating the freezing process, further phase separation of the polymer-lean stage inside the pores occurs, forming a new, more diluted, polymer-lean stage and a more concentrated polymer-rich phase assuasive the germination of larger pores. This technique was used to fabricate poly(vinyl alcohol) (PVA) hydrogels using a wheel consisting of a twenty h freeze step at −22°C followed by a 4h thawing step at 25°C.^twoscore

Freeze-drying techniques are suitable for a broad range of hydrogel materials, including natural^41–43 and synthetic hydrophilic polymers,^44,45 to produce interconnected porous structures. However, these methods encounter difficulty in precisely tuning pore size since the hydrogel compages formed using this method are extremely sensitive to the kinetics of the thermal quenching procedure. Other issues associated with this technique are the low structural stability and generally weak mechanical properties of the made materials. The freeze-drying process often results in the formation of a surface skin considering the matrix may collapse at the scaffold–air interface due to the interfacial tension caused by solvent evaporation.⁴⁶ In addition, freeze-drying is energy intensive and requires a relatively long processing time for complete removal of solvent.²³

To address these issues, an alternative freeze gelation procedure has been utilized to produce porous hydrogels for chitosan and alginate, respectively.⁴⁶ In this report, a frozen chitosan solution (−20°C) was immersed in a precooled NaOH/ethanol solution to accommodate its pH to allow for gelation of chitosan below its freezing point.⁴⁶ Since the polymer was already gelled in the frozen country, the solvent could exist removed with drying at room temperature without the formation of a surface pare. A highly interconnected porous hydrogel with the pores that ranged from 60 to 150 μm and porosity of 90% was produced using this technique.⁴⁶ In vitro studies indicated that seeded rat osteoblast-like cells were able to adhere, spread, and proliferate both inside and on the surface of the resulting hydrogel.⁴⁶ Compared with conventional freeze-drying, this method has improved energy efficiency, and is amenable to scale up.⁴⁶

Gas foaming

Conventional gas foaming

Gas foaming utilizes the nucleation and growth of gas bubbles dispersed throughout a polymer to generate a porous structure.⁴⁷ The gas bubbling can either be formed past foaming/bravado agent via chemical reaction,⁴⁸ or be released from a presaturated gas–polymer mixture at a high pressure.⁴⁷ A foaming/bravado agent is a substance that is mixed into the prepolymer and generates a gas when it chemically decomposes. The most ordinarily used foaming agent for fabricating porous hydrogels is sodium bicarbonate attributable to its ability to generate CO₂ in mildly acidic solutions. As an example, sodium bicarbonate has been used to fabricate an acrylic acrid–acryl amide porous hydrogel by crosslinking acrylic acid and acryl amide with N,N′-methylenebisacrylamide.⁴⁹ The resultant hydrogel showed an interconnected construction with pore size ranging from 100 to 250 μm and 100% equilibrium swelling ratio.⁴⁹

Sodium bicarbonate has recently been used as a blowing agent to create macroporous hydrogels from photocrosslinkable PEG diacrylate covalently linked to the peptide sequence RGD to promote cell adhesion on this otherwise cell-repellant hydrogel textile. This scaffold was found to support the adhesion and long-term viability of homo mesenchymal stem cells and facilitated mineralization when exposed to osteogenic medium.^fifty Interestingly, human being mesenchymal stalk cells were likewise capable of bounden to porous PEG-based gels even in the absence of the RGD adhesion sequence, suggesting that structure of the scaffold was sufficient for cell attachment.⁵⁰

Ammonium bicarbonate is another gas bravado agent that has been used to produce interconnected pores within hydrogels via its decomposition to CO_ii and NH₃. As an example, ammonium bicarbonate was added during the irradiation of carboxymethylcellulose–sodium and polyacrylamide to induce porosity⁵¹ and was shown to enhance the swelling ratio in comparison with hydrogels prepared in the absence of a blowing amanuensis.

Attributable to the wide availability of the most common bravado agents, this method provides an cheap platform to introduce porosity into hydrogel materials. The blowing agents used are generally cell friendly, and this technique can be employed without the use of organic solvent, making it well suited for tissue engineering applications.

Gas foaming with dumbo gas CO₂

A dense gas is a fluid to a higher place or close to its critical temperature and pressure, which demonstrates physical properties intermediate to those of a true gas or liquid phase. Dumbo gas CO₂ has a relatively depression critical temperature (T _c: 31°C) and is an bonny candidate for biomaterial processing because information technology is inert, nontoxic, and inexpensive.^23,52 This gas has been widely used as a foaming agent to induce porosity in the structure of several common hydrophobic polymers such equally poly(lactic acrid) (PLA), PLGA, and PCL.^23,52–55 All the same, dense gas CO₂ by and large has low solubility in hydrophilic polymers. Diverse techniques such as CO_ii–water emulsion templating^56–60 or the employ of a cosolvent system take been developed to amend the ability of a dense gas to diffuse into a hydrophilic polymer and produce porosity.^61,62 Using dense gas CO₂ to generate porosity eliminates the utilize of surfactant or foam stabilizer that is required in conventional gas foaming techniques.^50,51

Emulsion templating involves forming a loftier internal stage emulsion (HIPE) that is composed of an external phase consisting of a curable polymer and an internal phase fabricated of minute droplets. The structure of the external stage is locked in past reaction-induced phase separation such every bit sol–gel chemistry or free-radical polymerization.^threescore Subsequent removal of the emulsion droplets allows highly porous and interconnected materials (polyHIPES) to be obtained.⁶⁰ Conventional emulsion templating techniques require large amounts of organic solvent to generate the internal phase (more 75%), which may exist hard to remove after curing,⁵⁶ resulting in cell cytotoxicity. CO₂–water HIPEs, using supercritical CO₂ as the internal droplet phase and an aqueous solution every bit the external phase, have been considered to produce emulsion-templated materials without the use of an organic solvent.⁵⁹ Supercritical CO₂–water emulsion templating techniques have been used to produce highly porous crosslinked hydrogels using many naturally occurring biopolymers such equally dextran,⁵⁶ chitosan,⁵⁹ and alginate⁵⁷ and synthetic polymers, including PVA, blended PVA/PEG,⁵⁹ and CaCO_three/polyacrylamide composites.^threescore

CO_ii–water emulsion polymerization templating has been used with the polysaccharide dextran to class highly interconnected and thin-walled porous hydrogels.⁵⁶ In this procedure, perfluoropolyether was employed as a surfactant to stabilize the supercritical CO₂–water emulsion and potassium peroxydisulfate as an initiator for radical polymerization to produce an interconnected porous dextran hydrogel.⁵⁶ Variation of the book fraction of CO_two did non take a significant effect on pore size.⁵⁶ It was constitute that every bit the volume fraction of CO₂ increased, the walls between side by side pores became thinner and the pores had a more than polyhedrical shape. An increase in the surfactant concentration led to more open up interconnected structure. The use of nonbiodegradable surfactant and a mean pore size less than 26 μm may not permit this technique to exist used for cell civilisation applications.

Physically crosslinked alginate hydrogels have been produced using the CO₂–water emulsion templating technique.⁵⁷ In this method, supercritical CO₂ simultaneously served as the templating amanuensis as well equally inducing acidity, which acquired the release of calcium ions from their chelated class, leading to crosslinking of the alginate and germination of a porous hydrogel.⁵⁷ The fabricated alginate hydrogels displayed an open up and interconnected pore network in the range of 23.nine–250 μm depending on the surfactant concentration and CO_ii fraction.⁵⁷

A dense gas hydrogel germination procedure was designed to fabricate highly porous biopolymeric hydrogels such as elastin-based hydrogels using different crosslinking agents, including glutaraldehyde⁶³ and hexamethylene diisocyanate.⁶⁴ In the latter process, α-elastin solution in phosphate-buffered saline or dimethyl sulfoxide was mixed with the crosslinking agent and pressurized with CO_ii to threescore bar for at least 30 min to crosslink.⁶⁴ Subsequent depressurization induced big channels in the 3D structures of the hydrogels (Fig. 2C). The average pore size of hydrogels fabricated past using two% (5/v) hexamethylene diisocyanate increased from 3.ix ± 0.8 to 79.8 ± 54.8 μm when pressure was increased from 1 bar to 60 bar (Fig. 2A, C). The α-elastin hydrogels facilitated fibroblast growth and proliferation in the 3D structures (Fig. 2D).⁶³

α-Elastin hydrogels made using high-pressure CO_ii. SEM images of α-elastin hydrogels fabricated at atmospheric pressure (A) and 60 bar (B, C) [top surface (B), cross department (C)]. Fibroblast cells were shown to attach on the scaffold surface (D). Adjusted with permission from Annabi et al. ⁶⁴

A cosolvent is ofttimes used to amend the improvidence of a dense gas into a hydrophilic polymer to produce porous hydrogels. In a contempo study, a biodegradable polymer such as collagen and gelatin as well as a solvent such as ethanol or diluted acid were placed in a high pressure sleeping room.⁶² The vessel was then pressurized with a supercritical fluid at a predetermined temperature and pressure to allow the supercritical fluid to dissolve into the polymer with the aid of the solvent.⁶² Finally, the pressure was released and a porous structure was obtained. The size and morphology of the porous hydrogel can be controlled by adjusting the operating pressure and temperature.⁶²

The use of supercritical CO₂ allows low temperatures to be maintained within the polymer, which is favorable if temperature-sensitive growth factors are to be straight incorporated into the material. The requirement of a reactor that can handle loftier pressures and access to supercritical CO₂ limits wide access to this processing technique. Information technology is critical to determine the result of high-pressure CO₂ on the biopolymers such as proteins. Loftier-pressure CO₂ has been used in micronization, impregnation, and fabrication of hydrogels using proteins such every bit insulin,^65,66 catalase,⁶⁷ recombinant human deoxyribonuclease,⁶⁸ elastin,⁶⁹ trypsin, and lysozymes.^65,seventy The biological action and structural perturbations induced during the dense gas process were constitute to be poly peptide specific. The biochemical integrity of proteins such as elastin,⁶⁹ lysozyme, and insulin⁶⁵ was preserved upon exposure to dumbo gas CO₂. The biological activity of proteins with isoelectric points to a higher place three such every bit recombinant human deoxyribonuclease⁶⁸ and β-galactosidase⁷¹ was a office of CO_two operating weather condition.

Electrospinning

Electrospinning has garnered meaning involvement in the field of tissue engineering because of its ability to fabricate interconnected porous scaffolds. The basic premise of electrospinning is the apply of an externally applied electric field to draw fibers from a charged polymer solution held past surface tension at the end of a capillary tube. The polymer is charged by applying a high voltage and is then drawn as a thin jet toward an oppositely charged collector plate by electrostatic forcefulness. Every bit the jet travels through the air, the solvent evaporates and jet diameter decreases substantially.⁷² The fibers are typically collected on a stationary ground plate, but a rotating pulsate tin can exist used as the collector to reach a preferred orientation.^73,74 The resulting cloth properties such equally fiber diameter, porosity, and morphology can be controlled by various parameters such as practical voltage, viscosity, solution conductivity, and temperature.⁷⁵ This technique can exist used to produce fibers in the micro- and nanometer range.

Electrospinning has been used to fabricate porous hydrogels of PVA and polyacrylic acid.⁷⁶ Using this method, a PVA and polyacrylic acid solution mixture at different ratios was placed in a glass capillary with the 0.iv mm inner diameter tip, which was tilted downward at an angle between 0° and 30° depending on the solution viscosity.⁷⁶ An ultrafine (submicron) fibrous hydrogel was formed after the electrospinning procedure. The bulk of the interfiber pores were connected, which is desirable for tissue engineering applications to let cell–cell interaction and migration.⁷⁶ The swelling behavior of the fibrous hydrogel membrane was found to exist dependent on the environmental pH and could be enhanced past application of an electric field.⁷⁶

The techniques of electrospinning and salt-leaching were combined to form a macroporous hyaluronic acid (HA)–collagen hydrogel with fibers on the nanometer calibration.⁷⁷ In this process, HA and collagen were dissolved in a NaOH/N,N-dimethyl formamide solvent mixture.⁷⁷ During the electrospinning process, NaCl particles were simultaneously deposited into the electrospun fibers to induce interfiber porosity. Later subsequent chemical crosslinking and salt leaching, a porous HA-based hydrogel was produced.⁷⁷ In vitro studies demonstrated that the hydrogel could support adhesion, proliferation, and memory of in vivo morphology of bovine chondrocyte cells (Fig. three).⁷⁷

Morphology and in vitro assessment of hyaluronic acid (HA)–based scaffolds. SEM images of electrospun salt-leached HA-based scaffolds at 95:five (A) and 80:20 (B) ratios of HA:collagen using sodium hydroxide and N,North-dimethyl formamide every bit a mixed solvent. Laser scanning confocal microscopy (C) and SEM (D) images showed that chondrocytes maintained their typical rounded morphology on an 80:twenty HA:collagen scaffold subsequently three days. Adapted with permission from Kim et al. ⁷⁷ Colour images available online at world wide web.liebertonline.com/10.

Many natural polymers such as collagen, silk fibroin, and fibrinogen have been processed using electrospinning into fine nonwoven mats with fibers in the micro- and nanometer range for tissue engineering applications.^74,78–82 Various prison cell types have been reported to attach, proliferate, and differentiate inside these matrices, demonstrating their utility in tissue engineering.⁷³ However, significant challenges that even so be in using this technique with hydrogels include an inability to fabricate complex 3D hydrogel shapes, poor mechanical properties, and express control over the porosity and pore size.⁸³

Traditional methods for hydrogel fabrication normally involve procedures or chemicals that are not desirable for jail cell viability. Low force per unit area and dehydration in freeze-drying techniques, the utilize of surfactants in dumbo gas templating techniques, organic solvents, and cytotoxic crosslinkers are examples of these annoying conditions. Typically, cells tin be seeded into these materials after creation; nonetheless, the inability to encapsulate the cells during the initial fabrication could diminish the ability to reach heterogeneous jail cell distribution.⁸⁴ For example, if the pores are too pocket-size for cells to penetrate, or if the pores are comparatively interconnected, information technology may non exist possible to seed cells throughout a hydrogel-based scaffold after formation, whereas this is readily achievable if the fabrication technique is cytocompatible. Direct encapsulation is possible in hydrogels that are created through such techniques every bit temperature alter,²² change in ion concentration,^85,86 or UV crosslinking.²¹ Although cell viability can be maintained long-term later on creation using these methods, oftentimes these techniques exercise not allow for control over pore size and distribution equally compared with more cytotoxic techniques. A balance must be reached in terms of heterogeneous cell seeding, pore size and distribution, and the hydrogel concrete properties for each intended application.

Inducing porosity in hydrophilic–hydrophobic hybrid hydrogels

A common challenge in many of the techniques described previously for the fabrication of porosity in hydrogels is to improve the mechanical stability of the 3D structure. Hydrophilic hydrogels often exhibit poor mechanical properties, especially in their swollen state, which can be an obstruction for their broad applications in tissue regeneration. I technique is the improver of biodegradable hydrophobic polymers to raise the mechanical properties of purely hydrophilic hydrogels. For example, the mechanical properties of collagen, elastin, and gelatin hydrogels fabricated by electrospinning were dramatically increased with the addition of 10% PCL without utilise of any chemical crosslinker.⁸⁷

Ane of the major issues in the fabrication of porosity in hybrid hydrogels is the phase separation caused by intrinsic immiscibility of hydrophilic and hydrophobic polymers. Before the creation of porosity by several methods such as solvent casting/particulate leaching, freeze-drying, gas foaming, and electrospinning, a homogeneous mixture of the polymer components must be formed. Methods including intimate mixing and interpenetrating polymer network (IPN) via sequential or simultaneous reaction of both polymers take been used to mix the 2 phases.

Mechanical stirring can be used for intimate mixing of the polymer solutions. A homogeneous unmarried stage is readily acquired by simple mechanical stirring when both compounds are soluble in i solvent. Using this technique, chitosan/PCL porous hydrogels were prepared by dissolving both compounds in an acidic solutions followed past freeze-drying (Fig. 4A, B).⁸⁸ The hydrogel exhibited splendid support for cellular action in both 2D and 3D. Mechanical stirring and an emulsifier have been used to create emulsions of 2 immiscible phases before the fabrication of porosity in hydrogels. Porous PVA-PCL hybrid hydrogels with enhanced mechanical properties were successfully made by creating an emulsion followed by freeze-drying and crosslinking.⁸⁸ In this study, PVA and PCL solutions were well mixed past using mechanical stirring to create homogeneous cream. The foam was immediately frozen at −70°C and lyophilized at −85°C for 24 h to acquire a highly porous 3D hydrogel with open and interconnected pores ranging from thirty to 300 μm.⁸⁸ The compressive loading of this hydrogel was enhanced at least twofold past increasing the concentration of PCL from xxx to 50 wt%. Fibroblasts and chondrocyte cells were shown to grow well in 3D porous composite hydrogels fabricated from PVA-PCL (1:1 weight ratio), underlining their first-class properties for tissue regeneration applications.⁸⁹

SEM images of chitosan-poly(ɛ-caprolactone) hydrogel scaffold formed by freeze-drying. Hydrogels of pure chitosan (A) and chitosan blended with fifty wt% poly(ɛ-caprolactone) (B). Solutions in 25 vol.% acetic acid were frozen at −196°C and lyophilized at −80°C. Adapted with permission from Sarasam et al. ⁸⁷

The creation of an IPN is an efficient method to reinforce porous hydrogels. In one study, nanosized particles of PLA was added to a solution of N-isopropyl acrylamide and a crosslinker to form a porous hydrogel by in situ polymerization and crosslinking.⁸⁹ PLA nanospheres were attached to the network matrix of poly N-isopropyl acrylamide hydrogels and generated enlarged porous structure. The presence of these large pores enhanced the swelling ratio at room temperature.⁹⁰ An IPN construction of porous-hybrid hydrogel of poly(ethyl methacrylate) as a hydrophobic and poly(2-hydroxyethyl acrylate) every bit a hydrophilic network was made by using sequential polymerization.⁹⁰ A hydrophobic poly(ethyl methacrylate) network was prepared by simultaneous polymerization and crosslinking of ethyl methacrylate (EMA) monomers in ethanol, followed by polymerization and crosslinking of two-hydroxyethyl acrylate (HEA) monomers. Herein ethanol served as both a solvent and a porogen to induce porosity in this hybrid hydrogel.^91,92

The combination of solvent casting/particulate leaching and freeze-drying has been used to produce porous hybrid hydrogels. A hybrid method for the fabrication of a homogenous mixture of porous collagen hydrogel and biodegradable polymers such as poly(glycolic acid), PLA, and PLGA has been reported.^91,92 A porous structure of hydrophobic polymer was first formed by solvent casting/particulate leaching technique and then immersed in a collagen solution. The mixture was placed under vacuum to fill the pores with collagen solution and then freeze-dried to fabricate microporosity into the matrix followed past crosslinking.²² The integration of these techniques was used to fabricate a highly porous hybrid hydrogel with well-defined 3D structures.

Creating an interconnected, highly porous hydrogel structure with splendid mechanical strength and the desired pore size is critical for tissue applied science. Hybrid hydrogels can be used to melody the desired properties of hydrogel for a specific awarding, making them desirable for use in many engineered tissues. The average pore sizes that can be achieved by using different techniques to induce porosity in hydrogels are compared in Table 1.

Table 1.

Pore Size Achieved Using Different Processes for Hydrogel Fabrication

Process	Polymer	Pore size (μm)	Ref.
Conventional gas foaming	AAm	100–250	49
	PEGDA	100–600	50
CO2–water emulsion templating	Dextran	6.25–seven	56
	Chitosan, PVA, PVA/PEG	3–xv	59
	Alginate	23.9–250	57
	CaCO3/PAM	4.vii–4.9	60
Dense gas CO2+crosslinker	Elastin	eighty	64
Dumbo gas CO2+cosolvent	Gelatin	80–120	62
Porogen leaching	PEG/PCL	180–400	thirty
	OPF	100–500	31
	Alginate-g-poly(N-isopropylacrylamide)	100–300	32
	PHEMA	200–500	33
		45–106	35
	PEG-poly(lysine)	250–500	34
Freeze-drying	Collagen/chitosan	50	37
	Agarose	71–187	38
	Chitosan, alginate	60–150	46
	Gelatin	40–500	viii,43
	PVA/PCL	thirty–300	88
	Chitosan/PCL	ten–100	87
Electrospinning	Gelatin/PCL	20–fourscore	83

Microscale Command of Porosity and Microarchitecture

Fabrication of microchannels

To further control and improve diffusion and transport in hydrogels, researchers have used multiple techniques to create microchannels within hydrogel structures. Although hydrogels ofttimes have a high caste of hydration with diffusion properties similar to that in the majority surrounding fluid, microchannels have been shown to be effective in farther improving the mass ship capabilities of hydrogels.^22,93 A common method for producing microfluidic channels inside hydrogels is the use of soft lithography micromolding.⁹⁴ Briefly, a photomask containing the desired pattern is printed and used in combination with an SU-8 photoresist-coated silicon wafer to create a template. Polydimethylsiloxane (PDMS) is and so poured onto the SU-8 pattern, cured, and removed to generate a PDMS mold. The SU-8 template tin can typically be used multiple times to create multiple PDMS molds or stamps. Some polymers can be molded directly on the SU-viii primary, fugitive the production of a PDMS replica. Otherwise, the polymer of involvement is poured onto the PDMS postage stamp and cured. For example, PEG diacrylate on a PDMS mold may be polymerized using UV calorie-free in the presence of a photoinitiator. The PEG-PDMS combination can then be dissociated with hydration and mild agitation. These surface channel patterns tin can be made into microchannels past curing another solid hydrogel layer on top of the patterned surface to enclose the channels. Microscale channels accept been successfully created using PDMS micromolding with PEG-DA and methacrylated HA,²² whereas channels have been created direct on SU-eight masters using thermal gelation of agarose.²²

Creation of microchannels in cell-laden hydrogels has been shown to improve jail cell viability as well as orientation and alignment. Diffusion studies demonstrated that prison cell viability and function were improved in cells that were closer to the perfused channels, presumably considering of increased nutrient exchange.⁹⁵ The combination of controlled pore size throughout a poly(2-hydroxyethyl methacrylate) hydrogel, created with spherical sacrificial elements, with photopatterned microchannels led to improved elongation, spreading, and fibrillar formation of C2C12 myoblasts.⁹⁶ Similarly, focused photoablation achieved using pulsed lasers in PEGylated fibrinogen hydrogels successfully created microchannels of controlled size that were shown to drive the directional growth of neurites.⁹⁷ Creation of microchannels to command both food send and cellular alignment, combined with controlled pore size in the bulk material, brings hydrogel-based tissues closer to the appearance and function of native tissues.

Rapid prototyping or solid free-form fabrication is another family of techniques that has recently been demonstrated as potentially useful for the product of patterned hydrogels for tissue engineering. A detailed consideration of this engineering is beyond the scope of this review; even so, the reader is directed to a review of this technology and its advantages published elsewhere.^three,98 These approaches utilise calculator-aided blueprint to automatically generate 3D structures using a multifariousness of methods.⁹⁸ A rapid prototyping technique that has constitute favor in the fabrication of hydrogels containing microchannels is stereolithography. Briefly, stereolithography is a liquid-based technique that utilizes layer-by-layer curing of a photosensitive prepolymer solution.⁹⁹ A laser scanner is used to photopolymerize a thin layer of liquid prepolymer, which is located to a higher place a computer-controlled phase. After 2D photopatterning of a single polymer layer, the stage is moved downwards to embrace the top of the cured polymer with fresh liquid prepolymer, and the process is repeated to generate a 3D object layer-by-layer.

As a proof of principle, this technique was used to create prison cell-laden methacrylated-PEG hydrogels with simple ring shapes.⁹⁹ It was shown that high cell viability could be maintained even when two–3 min were needed to fabricate each layer.¹⁰⁰ Others have extended this work to generate complex shapes at loftier resolution.¹⁰⁰ Imbedded channels were made with multiple bifurcations that could potentially serve as artificial microvasculature for tissue-engineered constructs.¹⁰⁰ Multiple lumen conduit structures were formed with the aim of guiding nerve regeneration (Fig. 5A, B). Information technology was demonstrated that microbeads could be precisely placed within the made structures, demonstrating the ability to generate single constructs from multiple materials containing dissimilar growth factors or cell types (Fig. 5C, D).^101,102

Multilumen poly(ethylene glycol) hydrogel conduits fabricated using stereolithography. Isometric (A, C) and acme (B, D) views of a poly(ethylene glycol) hydrogel channels with potential in nerve regeneration. The conduits in (C) and (D) incorporate dark-green and blue fluorescent particles in the outer and inner region, respectively, demonstrating the ability to fabricate constructs from multiple materials. Scale bars represent i mm. Reprinted with permission from Arcaute et al. ¹⁰⁰ Color images bachelor online at www.liebertonline.com/10.

Stereolithography is capable of delivering loftier-resolution features that are limited primarily by the diameter of the laser used. It also allows the directly incorporation of cells during the fabrication of scaffolds, which is advantageous as it allows for a uniform cell distribution within the construct. The relatively high cost of equipment necessary for stereolithography has limited its use among researchers. Additionally, this technique is as well only uniform with photocrosslinkable polymers limiting the potential fabric selection. Layer-by-layer assembly using this method is relatively fourth dimension consuming, potentially limiting the technique to more robust cell types.

Interconnected microvascular networks

Although private microchannels tin meliorate nutrient send and tissue function, the greater goal is to create intact microvascular networks in hydrogel-based tissues to improve tissue function and integration with the host vasculature. Synthetic materials such as self-assembling peptide gels,^103,104 as well as natural hydrogels such as collagen^105,106 take been used to study in vitro capillary morphogenesis.

Recent piece of work has used micromolded gelatin channels as a sacrificial chemical element to create perfusable microvascular networks in collagen and fibrin hydrogels.^xvi The 3D gelatin channels were micromolded in PDMS, and then encapsulated in collagen or fibrin, and placed in a standard 37°C incubator, causing the gelatin to cook away, leaving backside a series of connected microchannels ranging from 6 to 50 μm in bore (Fig. 6). Subsequent experiments determined that seeded endothelial cells would line the channels and provide typical barrier function against perfused particles as expected in vivo, making this technique useful every bit an in vitro model of microvascular part, as well as a potential technique for creating vascularized, hydrogel-based engineered tissues.

Use of sacrificial gelatin to create perfusable microvascular networks. Gelatin patterns are cast in polydimethylsiloxane molds and and then encapsulated past collagen (A, B) or fibrin (C, D), and the gelatin is removed via melting at 37°C. Perfusable, one-layer cell-laden structures were created using collagen (A, B) and fibrin (C), and multilayer structures were also demonstrated (D). Reprinted with permission from Golden and Tien.¹⁰⁵ Scale confined are 200 μm for panels A, C, D, 25 μm for panel B, and 50 μm for panel A inset. Color images available online at www.liebertonline.com/ten.

Control of microarchitectural features

One ultimate goal of microengineering techniques for tissue fabrication is the precise control of not but porous structures, merely of the microarchitectural features inside the construct equally well. By recreating specific microarchitectural motifs, tissue engineers aim to optimize cell viability, morphology, and part.^107,108 Using basic micromolding techniques, researchers have demonstrated the ability to create complex structures with microscale pore and tissue structures.¹⁰⁹ Agarose was formed into rods, tori, and honeycomb, or multiple connected tori, and H35 hematoma or human fibroblasts were seeded on these hydrogel structures and immune to self-gather inside the hydrogels. The resulting structures demonstrated the ability to permit cells to grade their own structures equally dictated past the prescribed microarchitectural features and pores.

Using microfabrication techniques researchers have besides demonstrated the ability to restate native micro- and macroscale features to create engineered tissues with biomimetic appearance and function. For example, it has been demonstrated that the subsequent polymerization of prison cell-laden hydrogels, using dissimilar photomasks, tin can be used to build tissues layer past layer (Fig. 7A). Private hepatic features are photopolymerized sequentially to create dual-layered tissues, surrounded by an ultrastructural honeycomb structure similar to the native sinusoid of the liver. Viability and hepatic cell part were improved in micropatterned constructs as compared to unpatterned controls (Fig. 7B, C), demonstrating the importance of recreating the native microarchitectural features. This technique demonstrates how the combination of dictating the microarchitectural features and pore construction simultaneously tin can atomic number 82 to improved cell viability and function.

Schematic of layer-past-layer hepatic tissue formation. Using incremental spacers combined with alternate photomasks allows for the layer-by-layer creation of cell-laden tissues in specific microarchitectural organisation (A). Perfusion of micropatterned hepatic tissues demonstrated pregnant improvements in albumin and urea secretion over unpatterned controls (B, C). Reprinted with permission from Tsang et al. ¹⁰⁹ Color images available online at www.liebertonline.com/ten.

Conclusions and Hereafter Directions

Hydrogels hold substantial promise for creating functional engineered tissues, providing a pregnant need to control hydrogel porosity and microarchitecture. Traditional polymer-processing techniques, such as porogen leaching and gas foaming, have demonstrated the ability to create hydrogels with uniform porosity throughout the scaffold with high accuracy. Combining these traditional processes with more recent microfabrication techniques has brought the field closer to the ultimate goal of consummate control over microarchitecture and porosity in engineered tissues. With continued research in these and other advanced techniques such every bit cell and organ printing, hydrogel-based tissue engineering will go along to make advances toward clinical restoration of tissue function. There are considerable challenges to overcome before clinical application of circuitous ex vivo–engineered tissues can become a reality, such as command over mechanical backdrop, cell alignment, and behavior and integration with the host vasculature. However, with the techniques presented here, and new techniques in the future, the improved ability to command the porosity and microarchitecture of hydrogels will bulldoze the inquiry closer to these goals.

Acknowledgments

The Khademhosseini group is funded past the U.S. Army Engineer Research and Evolution Middle, the Plant for Soldier Nanotechnology, the National Scientific discipline Foundation, and the National Found of Health grants (HL092836, EB009196, and DE019024). The authors also acknowledge the financial back up from the Australian Enquiry Council (Grant No. DP0988545).

Disclosure Statement

No competing financial interests exist.

References

one. Peppas N. Hilt J.Z. Khademhosseini A. Langer R. Hydrogels in biology and medicine. Adv Mater Deerfield. 2006;18:1. [Google Scholar]

2. Khademhosseini A. Langer R. Microengineered hydrogels for tissue engineering. Biomaterials. 2007;28:5087. [PubMed] [Google Scholar]

3. Nichol J.Westward. Khademhosseini A. Modular tissue engineering: engineering biological tissues from the lesser upwardly. Soft Thing. 2009;5:1312. [PMC free article] [PubMed] [Google Scholar]

4. Peppas N.A. Hydrogels in Medicine and Pharmacy. Boca Raton, FL: CRC Printing; 1987. [Google Scholar]

5. Gerecht S. Townsend South.A. Pressler H. Zhu H. Nijst C.L. Bruggeman J.P. Nichol J.W. Langer R. A porous photocurable elastomer for cell encapsulation and culture. Biomaterials. 2007;28:4826. [PubMed] [Google Scholar]

half-dozen. Martin I. Obradovic B. Treppo S. Grodzinsky A.J. Langer R. Freed L.E. Vunjak-Novakovic G. Modulation of the mechanical properties of tissue engineered cartilage. Biorheology. 2000;37:141. [PubMed] [Google Scholar]

7. Mandal B. Kundu S. Cell proliferation and migration in silk fibroin 3D scaffolds. Biomaterials. 2009;30:2956. [PubMed] [Google Scholar]

viii. Lien S.M. Ko L.Y. Huang T.J. Issue of pore size on ECM secretion and jail cell growth in gelatin scaffold for articular cartilage tissue engineering. Acta Biomater. 2009;5:670. [PubMed] [Google Scholar]

ix. Griffon D.J. Sedighi G.R. Schaeffer David 5. Eurell Jo A. Johnson Ann L. Chitosan scaffolds: interconnective pore size and cartilage technology. Acta Biomater. 2006;two:313. [PubMed] [Google Scholar]

10. Kim H.J. Kim U.J. Vunjak-Novakovic G. Min B.-M. Kaplan D.L. Influence of macroporous protein scaffolds on os tissue engineering from os marrow stem cells. Biomaterials. 2005;26:4442. [PubMed] [Google Scholar]

xi. Roy T.D. Simon J.L. Ricci J.L. Rekow E.D. Thompson V.P. Parsons J.R. Performance of degradable composite bone repair products made via iii-dimensional fabrication techniques. J Biomed Mater Res. 2003;66:283. [PubMed] [Google Scholar]

12. Fidkowski C. Kaazempur-Mofrad M.R. Borenstein J. Vacanti J. Langer R. Wang Y. Endothelialized microvasculature based on a biodegradable elastomer. Tissue Eng. 2005;11:302. [PubMed] [Google Scholar]

13. Yannas I.V. Lee Eastward. Orgill D.P. Skrabut Due east.Thou. Spud K.F. Synthesis and label of a model extracellular matrix that induces partial regeneration of adult mammalian skin. Proc Natl Acad Sci USA. 1989;86:933. [PMC costless article] [PubMed] [Google Scholar]

xiv. Whang Grand. Healy K.Eastward. Elenz D.R. Nam East.K. Tsai D.C. Thomas C.H. Nuber G. Glorieux R. Travers R. Sprague S.G. Engineering os regeneration with bioabsorbable scaffolds with novel microarchitecture. Tissue Eng. 1999;5:35. [PubMed] [Google Scholar]

fifteen. Wake M.C. Patrick C.W., Jr. Mikos Antonios M. Pore morphology effects on the fibrovascular tissue growth in porous polymer substrates. Jail cell Transplant. 1994;three:339. [PubMed] [Google Scholar]

sixteen. Khademhosseini A. Langer R. Borenstein J. Vacanti J.P. Microscale technologies for tissue technology and biology. Proc Natl Acad Sci The states. 2006;103:2480. [PMC gratuitous article] [PubMed] [Google Scholar]

17. Khademhosseini A. Bettinger C. Karp J.M. Yeh J. Ling Y. Borenstein J. Fukuda J. Langer R. Interplay of biomaterials and micro-scale technologies for advancing biomedical applications. J Biomater Sci Polym Ed. 2006;17:1221. [PubMed] [Google Scholar]

18. Du Y. Lo E. Ali Southward. Khademhosseini A. Directed associates of cell-laden microgels for fabrication of 3D tissue constructs. Proc Natl Acad Sci United states. 2008;105:9522. [PMC free article] [PubMed] [Google Scholar]

19. Khademhosseini A. Eng Thou. Yeh J. Fukuda J. Blumling J., tertiary Langer R. Burdick J.A. Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment. J Biomed Mater Res A. 2006;79:522. [PubMed] [Google Scholar]

20. Khademhosseini A. Yeh J. Jon South. Eng 1000. Suh Thou.Y. Burdick J.A. Langer R. Molded polyethylene glycol microstructures for capturing cells within microfluidic channels. Lab Chip. 2004;4:425. [PubMed] [Google Scholar]

21. Yeh J. Ling Y. Karp J.M. Gantz J. Chandawarkar A. Eng Chiliad. Blumling J., 3rd Langer R. Khademhosseini A. Micromolding of shape-controlled, harvestable prison cell-laden hydrogels. Biomaterials. 2006;27:5391. [PubMed] [Google Scholar]

22. Ling Y. Rubin J. Deng Y. Huang C. Demirci U. Karp J.M. Khademhosseini A. A cell-laden microfluidic hydrogel. Lab Bit. 2007;7:756. [PubMed] [Google Scholar]

23. Quirk R.A. France R.K. Shakesheff K.M. Howdle S.M. Supercritical fluid technologies and tissue applied science scaffolds. Curr Opin Solid State Mater Sci. 2005;8:313. [Google Scholar]

24. Sheridan Thou.H. Shea L.D. Peters M.C. Mooney D.J. Bioabsorbable polymer scaffolds for tissue engineering capable of sustained growth factor delivery. J Command Release. 2000;64:91. [PubMed] [Google Scholar]

25. Horák D. Kroupová J. Slouf M. Dvorák P. Poly (2-hydroxyethyl methacrylate)-based slabs as a mouse embryonic stem jail cell support. Biomaterials. 2004;25:5249. [PubMed] [Google Scholar]

26. Draghi L. Resta S. Pirozzolo One thousand. Tanzi M. Microspheres leaching for scaffold porosity control. J Mater Sci Mater Med. 2005;sixteen:1093. [PubMed] [Google Scholar]

27. Gong Y. Zhou Q. Gao C. Shen J. In vitro and in vivo degradability and cytocompatibility of poly (50-lactic acid) scaffold fabricated by a gelatin particle leaching method. Acta Biomater. 2007;3:531. [PubMed] [Google Scholar]

28. Suh South.W. Shin J.Y. Kim J. Kim J. Beak C.H. Kim D.-I. Kim H. Jeon S.S. Choo I.-Westward. Effect of different particles on cell proliferation in polymer scaffolds using a solvent-casting and particulate leaching technique. ASAIO J. 2002;48:460. [PubMed] [Google Scholar]

29. Zhang J. Wu L. Jing D. Ding J. A comparative study of porous scaffolds with cubic and spherical macropores. Polymer. 2005;46:4979. [Google Scholar]

thirty. Park J.Due south. Woo D.K. Sun B.Thousand. Chung H.-G. Im S.J. Choi Y.M. Park K. Huh K.M. Park K.-H. In vitro and in vivo test of polyethylene glycol/poly e-caprolactone-based hydrogel scaffold for jail cell delivery awarding. J Control Release. 2007;124:51. [PubMed] [Google Scholar]

31. Dadsetan M. Hefferan T.E. Szatkowski Jan P. Mishra P.K. Macura S.I. Lu L. Yaszemski Michael J. Effect of hydrogel porosity on marrow stromal jail cell phenotypic expression. Biomaterials. 2008;29:2193. [PMC free article] [PubMed] [Google Scholar]

32. Kim J.H. Lee S.B. Kim S.J. Lee Y.Thousand. Rapid temperature/pH response of porous alginate-g-poly(N-isopropylacrylamide) hydrogels. Polymer. 2002;43:7549. [Google Scholar]

33. Horák D. Hlídková H. Hradil J. Lapcíková One thousand. Slouf G. Superporous poly(2-hydroxyethyl methacrylate) based scaffolds: grooming and characterization. Polymer. 2008;49:2046. [Google Scholar]

34. Ford M.C. Betrtram J.P. Hynes S.R. Michaud M. Li Q. Young One thousand. Segal S.S. Madri J.A. Lavik Due east.B. A macroporous hydrogel for the coculture of neural progenitor and endothelial cells to form functional vascular networks in vivo. Proc Natl Acad Sci USA. 2006;103:eight. [PMC costless article] [PubMed] [Google Scholar]

35. Simms H.Thou. Brotherton C.Thousand. Proficient B.T. Davis R.H. Anseth K.S. Bowman C.N. In situ fabrication of macroporous polymer networks within microfluidic devices by living radical photopolymerization and leaching. Lab Bit. 2005;5:151. [PubMed] [Google Scholar]

36. Thomson R.C. Wake M.C. Yaszemski Yard.J. Mikos A.Chiliad. Biodegradable polymer scaffolds to regenerate organs. Adv Polym Sci. 1995;122:245. [Google Scholar]

37. Wu Ten. Black L. Santacana-Laffitte 1000. Patrick C.W., Jr. Training and cess of glutaraldehyde-crosslinked collagen-chitosan hydrogels for adipose tissue applied science. J Biomed Mater Res Office A. 2007;81:59. [PubMed] [Google Scholar]

38. Stokols South. Tuszynski M.H. The fabrication and characterization of linearly oriented nerve guidance scaffolds for spinal cord injury. Biomaterials. 2004;25:5839. [PubMed] [Google Scholar]

39. Stokols Southward. Tuszynski M.H. Freeze-dried agarose scaffolds with uniaxial channels stimulate and guide linear axonal growth following spinal cord injury. Biomaterials. 2006;27:443. [PubMed] [Google Scholar]

40. Ricciardi R. D'Errico G. Auriemma F. Ducouret G. Tedeschi A.G. De Rosa C. Laupretre F. Lafuma F. Short time dynamics of solvent molecules and supramolecular system of poly (vinyl booze) hydrogels obtained past freeze/thaw techniques. Macromolecules. 2005;38:6629. [Google Scholar]

41. Jin R. Moreira Teixeira L.S. Dijkstra P.J. Karperien M. Zhong Z. Feijen J. Fast in-situ formation of dextran-tyramine hydrogels for in vitro chondrocyte culturing. J Command Release. 2008;132:24. [Google Scholar]

42. Lv Q. Hu K. Feng Q. Cui F. Fibroin/collagen hybrid hydrogels with crosslinking method: preparation, properties, and cytocompatibility. J Biomed Mater Res A. 2008;84:198. [PubMed] [Google Scholar]

43. Kang H.West. Tabata Y. Ikada Y. Fabriction of porous gelatin scaffolds for tissue engineering. Biomaterials. 1999;xx:1339. [PubMed] [Google Scholar]

44. Lee Y.-Chiliad. Kang H.-S. Kim M.-S. Son T.-I. Thermally crosslinked anionic hydrogels equanimous of poly (vinyl alcohol) and poly (gamma-glutamic acid): preparation, characterization, and drug permeation behavior. J Appl Polym Sci Symp. 2008;109:3768. [Google Scholar]

45. Lin West.C. Yu D.G. Yang One thousand.C. Blood compatibility of novel poly([gamma]-glutamic acrid)/polyvinyl alcohol hydrogels. Colloids Surf B Biointerfaces. 2006;47:43. [PubMed] [Google Scholar]

46. Ho K.-H. Kuo P.-Y. Hsieh H.-J. Hsien T.-Y. Hou L.-T. Lai J.-Y. Wang D.-Thousand. Training of porous scaffolds by using freeze-extraction and freeze-gelation methods. Biomaterials. 2004;25:129. [PubMed] [Google Scholar]

47. Lips P.A.M. Velthoen I.W. Dijkstra P.J. Wessling M. Feijen J. Gas foaming of segmented poly(ester amide) films. Polymer. 2005;46:12737. [Google Scholar]

48. Caykara T. Kucuktepe S. Turan Eastward. Swelling characteristics of thermo-sensitive poly[(2-diethylaminoethyl methacrylate)-co-(Northward,N-dimethylacrylamide)] porous hydrogels. Polym Int. 2007;56:532. [Google Scholar]

49. Huh G.Thou. Baek N. Park K. Enhanced swelling rate of poly(ethylene glycol)-grafted superporous hydrogels. J Bioact Compat Polym. 2005;20:231. [Google Scholar]

50. Keskar V. Marion N.West. Mao J.J. Gemeinhart R.A. In vitro evaluation of macroporous hydrogels to fabricate stalk cell infiltration, growth, and mineralization. Tissue Eng Part A. 2009;15:1695. [PMC free article] [PubMed] [Google Scholar]

51. Abd El-Rehim H.A. Hegazy Eastward.-Due south.A. Diaa D.A. Characterization of super-absorbent material based on carboxymethylcellulose sodium salt prepared past electron axle irradiation. J Macromol Sci Pure. 2006;43:101. [Google Scholar]

52. Tai H. Popov V.K. Shakesheff Thousand.M. Howdle S.One thousand. Putting the fizz into chemical science: applications of supercritical carbon dioxide in tissue engineering, drug delivery and synthesis of novel cake copolymers. Biochem Soc Trans. 2007;35:516. [PubMed] [Google Scholar]

53. Barry J.J.A. Silva Chiliad.M.C.G. Popov Five.1000. Shakesheff K.K. Howdle Southward.M. Supercritical carbon dioxide: putting the fizz into biomaterials. Philos Trans A Math Phys Eng Sci. 2006;364:249. [PMC complimentary article] [PubMed] [Google Scholar]

54. Barry J.J.A. Gidda H.S. Scotchford C.A. Howdle S.Chiliad. Porous methacrylate scaffolds: supercritical fluid fabrication and in vitro chondrocyte responses. Biomaterials. 2004;25:3559. [PubMed] [Google Scholar]

55. Cansell F. Aymonier C. Loppinet-Serani A. Review on materials scientific discipline and supercritical fluids. Curr Opin Solid Land Mater Sci. 2003;7:331. [Google Scholar]

56. Palocci C. Barbetta A. La Grotta A. Dentini M. Porous biomaterials obtained using supercritical CO₂-water emulsions. Langmuir. 2007;23:8243. [PubMed] [Google Scholar]

57. Partap S. Rehman I. Jones J.R. Darr J.A. Supercritical carbon dioxide in water emulsion-templated synthesis of porous calcium alginate hydrogels. Adv Mater Deerfield. 2006;xviii:501. [Google Scholar]

58. Tan B. Lee J.-Y. Cooper A.I. Synthesis of emulsion-templated poly(acrylamide) using CO₂-in-water emulsions and poly (vinyl acetate)-based block copolymer surfactants. Macromolecules. 2007;40:1945. [Google Scholar]

59. Lee J.-Y. Tan B. Cooper A.I. CO_two-in-water emulsion-templated poly(vinyl alcohol) hydrogels using poly(vinyl acetate)-based surfactants. Macromolecules. 2007;40:1955. [Google Scholar]

60. Bing Z. Lee J.Y. Choi S.W. Kim J.H. Training of porous CaCO₃/PAM composites by CO₂ in water emulsion templating method. Eur Polym J. 2007;43:4814. [Google Scholar]

61. Chen C.-F. Chang C.-S. Chen Y.-P. Lin T.-Due south. Su C.-Y. Lee S.-Y. Applications of supercritical fluid in alloplastic bone graft: a novel method and in vitro tests. Ind Eng Chem Res. 2006;45:3400. [Google Scholar]

62. Shih H.-h. Lee Thousand.-r. Lai H.-m. Tsai C.-c. Chang Y.-c. Method of making porous biodegradable polymers, Usa 6673286. 2004.

63. Annabi N. Mithieux S.K. Weiss A.S. Dehghani F. The fabrication of elastin-based hydrogels using high pressure CO₂. Biomaterials. 2009;30:ane. [PubMed] [Google Scholar]

64. Annabi N. Mithieux Southward.M. Boughton E.A. Ruys A.J. Weiss A.Southward. Dehghani F. Synthesis of highly porous crosslinked elastin hydrogels and their interaction with fibroblasts in vitro. Biomaterials. 2009;30:4550. [PubMed] [Google Scholar]

65. Winters M.A. Knutson B.L. Debenedetti P.Grand. Sparks H.G. Przybycien T.M. Stevenson C.L. Prestrelski S.J. Precipitation of proteins in supercritical carbon dioxide. J Pharm Sci. 1996;85:586. [PubMed] [Google Scholar]

66. Yeo Southward.D. Debenedetti P.One thousand. Patro S.Y. Przybycien T.M. Secondary structure characterization of microparticulate insulin powders. J Pharm Sci. 1994;83:1651. [PubMed] [Google Scholar]

67. Yeo S.D. Lim G.B. Debenedetti P.One thousand. Bernstein H. Formation of microparticulate protein powders using a supercritical fluid antisolvent. Biotechnol Bioeng. 1993;41:341. [PubMed] [Google Scholar]

68. Bustami R.T. Chan H.K. Sweeney T. Dehghani F. Foster N.R. Generation of fine powders of recombinant human deoxyribonuclease using the aerosol solvent extraction system. Pharm Res. 2003;20:2028. [PubMed] [Google Scholar]

69. Dehghani F. Annabi N. Valtchev P. Mithieux S.M. Weiss A.S. Kazarian Southward.Grand. Tay F.H. Issue of dumbo gas CO₂ on the coacervation of elastin. Biomacromolecules. 2008;ix:1100. [PubMed] [Google Scholar]

70. Striolo A. Favaro A. Elvassore N. Bertucco A. Noto V.D. Evidence of conformational changes for protein films exposed to high-pressure CO_ii by FT-IR spectroscopy. J Supercrit Fluids. 2003;27:283. [Google Scholar]

71. LeClair Ellis J. Tomasko D.L. Dehghani F. Novel dense CO₂ technique for beta-galactosidase immobilization in polystyrene microchannels. Biomacromolecules. 2008;ix:1027. [PubMed] [Google Scholar]

72. Reneker D.H. Chun I. Nanometer diameter fibers of polymer, produced by electrospinning. Nanotechnology. 1996;vii:216. [Google Scholar]

74. Matthews J.A. Wnek G.E. Simpson D.Thousand. Bowlin G.L. Electrospinning of collagen nanofibers. Biomacromolecules. 2002;3:232. [PubMed] [Google Scholar]

75. Pham Q.P. Sharma U. Mikos A.M. Electrospinning of polymeric nanofibers for tissue applied science applications: a review. Tissue Eng. 2006;12:1197. [PubMed] [Google Scholar]

76. Li 50. Hsieh Y.-Fifty. Ultra-fine polyelectrolyte hydrogel fibers from poly (acrylic acid)/poly (vinyl alcohol) Nanotechnology. 2005;sixteen:2852. [Google Scholar]

77. Kim T.K. Chung H.J. Park T.G. Macroporous and nanofibrous hyaluronic acid/collagen hybrid scaffold fabricated past concurrent electrospinning and deposition/leaching of table salt particles. Acta Biomater. 2008;4:1611. [PubMed] [Google Scholar]

78. Li W.-J. Laurencin C.T. Caterson E.J. Tuan R.S. Ko F.K. Electrospun nanofibrous construction: a novel scaffold for tissue engineering. J Biomed Mater Res. 2002;lx:613. [PubMed] [Google Scholar]

79. Yang F. Murugan R. Wang S. Ramakrishna Southward. Electrospinning of nano/micro calibration poly (Fifty-lactic acid) aligned fibers and their potential in neural tissue engineering. Biomaterials. 2005;26:2603. [PubMed] [Google Scholar]

80. Chua Thousand.-N. Lim Westward.-S. Zhang P. Lu H. Wen J. Ramakrishna S. Leong K.W. Mao H.-Q. Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold. Biomaterials. 2005;26:2537. [PubMed] [Google Scholar]

81. Li W.-j. Danielson K.Thou. Alexander P.K. Tuan R.S. Biological response of chondrocytes cultured in iii-dimensional nanofibrous poly(eastward-caprolactone) scaffolds. J Biomed Mater Res A. 2003;67:1105. [PubMed] [Google Scholar]

82. Pham Q.P. Sharma U. Mikos A.G. Electrospun poly (east-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration. Biomacromolecules. 2006;7:2796. [PubMed] [Google Scholar]

83. Heydarkhan-Hagvall Southward. Schenke-Layland Thousand. Dhanasopon A.P. Rofail F. Smith H. Wu B.M. Shemin R. Beygui R.E. Maclellan W.R. Three-dimensional electronspun ECM-based hybrid scaffolds for cardiovascular tissue engineering science. Biomaterials. 2008;29:2907. [PMC free article] [PubMed] [Google Scholar]

84. Peppas N.A. Hilt J.Z. Khademhosseini A. Langer R. Hydrogels in biological science and medicine: from molecular principles to bionanotechnology. Adv Mater Deerfield. 2006;18:1345. [Google Scholar]

85. Zhang S. Fabrication of novel biomaterials through molecular self-assembly. Nat Biotechnol. 2003;21:1171. [PubMed] [Google Scholar]

86. Nichol J.W. Engelmayr Thou.C., Jr. Cheng M. Freed L.E. Co-civilisation induces alignment in engineered cardiac constructs via MMP-2 expression. Biochem Biophys Res Commun. 2008;373:360. [PMC free article] [PubMed] [Google Scholar]

87. Sarasam A.R. Samli A.I. Hess L. Ihnat K.A. Madihally S.V. Blending chitosan with polycaprolactone: porous scaffolds and toxicity. Macromol Biosci. 2007;seven:1160. [PubMed] [Google Scholar]

88. Mohan North. Nair P.D. Polyvinyl alcohol-poly (caprolactone) semi IPN scaffold with implication for cartilage tissue engineering. J Biomed Mater Res B Appl Biomater. 2007;84:584. [PubMed] [Google Scholar]

89. Zhang X.-Z. Chu C.-C. Zhuo R.-X. Using hydrophobic additive as pore-forming agent to prepare macroporous PNIPAAm hydrogels. J Polym Sci A Polym Chem. 2005;43:5490. [Google Scholar]

ninety. Gallego Ferrer G. Soria Melia J.G. Hernandez Canales J. Meseguer Duenas J.Yard. Romero Colomer F. Monleon Pradas M. Gomez Ribelles J.L. Pissis P. Polizos Grand. Poly(2-hydroxyethyl acrylate) hydrogel confined in a hydrophobic porous matrix. Colloid Polym Sci. 2005;283:681. [Google Scholar]

91. Chen G. Ushida T. Tateishi T. Hybrid biomaterials for tissue engineering: a preparative method for PLA or PLGA-collagen hybrid sponges. Adv Mater Deerfield. 2000;12:455. [Google Scholar]

92. Chen G. Ushida T. Tateishi T. A biodegradable hybrid sponge nested with collagen microsponges. J Biomed Mater Res. 2000;51:273. [PubMed] [Google Scholar]

93. Brigham M.D. Bick A. Lo E. Bendali A. Burdick J.A. Khademhosseini A. Mechanically robust and bioadhesive collagen and photocrosslinkable hyaluronic acid semi-interpenetrating networks. Tissue Eng Function A. 2008;15:1645. [PMC free article] [PubMed] [Google Scholar]

94. Khademhosseini A. Suh K.Y. Jon S. Eng G. Yeh J. Chen G.J. Langer R. A soft lithographic approach to fabricate patterned microfluidic channels. Anal Chem. 2004;76:3675. [PubMed] [Google Scholar]

95. Bryant S.J. Cuy J.L. Hauch Grand.D. Ratner B.D. Photo-patterning of porous hydrogels for tissue engineering. Biomaterials. 2007;28:2978. [PMC free commodity] [PubMed] [Google Scholar]

96. Sarig-Nadir O. Livnat N. Zajdman R. Shoham S. Seliktar D. Light amplification by stimulated emission of radiation photoablation of guidance microchannels into hydrogels directs cell growth in three dimensions. Biophys J. 2009;96:4743. [PMC free commodity] [PubMed] [Google Scholar]

97. Yang S. Leong One thousand.-F. Du Z. Chua C.-K. The blueprint of scaffolds for employ in tissue engineering. Part II. Rapid prototyping techniques. Tissue Eng. 2002;8:1. [PubMed] [Google Scholar]

98. Hollister S.J. Porous scaffold design for tissue engineering science. Nat Mater. 2005;4:518. [PubMed] [Google Scholar]

99. Dhariwala B. Chase Due east. Boland T. Rapid prototyping of tissue-engineering constructs, using photopolymerizable hydrogels and stereolithography. Tissue Eng. 2004;10:1316. [PubMed] [Google Scholar]

100. Arcaute Thou. Mann B. Wicker R. Stereolithography of three-dimensional bioactive poly (ethylene glycol) constructs with encapsulated cells. Ann Biomed Eng. 2006;34:1429. [PubMed] [Google Scholar]

101. Sieminski A.L. Hebbel R.P. Gooch K.J. The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro. Exp Cell Res. 2004;297:574. [PubMed] [Google Scholar]

102. Sieminski A.L. Hebbel R.P. Gooch K.J. Improved microvascular network in vitro past man blood outgrowth endothelial cells relative to vessel-derived endothelial cells. Tissue Eng. 2005;11:1332. [PubMed] [Google Scholar]

103. Sieminski A.L. Was A.S. Kim One thousand. Gong H. Kamm R.D. The stiffness of three-dimensional ionic self-assembling peptide gels affects the extent of capillary-similar network formation. Cell Biochem Biophys. 2007;49:73. [PubMed] [Google Scholar]

104. Sieminski A.Fifty. Semino C.E. Gong H. Kamm R.D. Principal sequence of ionic cocky-assembling peptide gels affects endothelial cell adhesion and capillary morphogenesis. J Biomed Mater Res A. 2008;87:494. [PubMed] [Google Scholar]

105. Golden A.P. Tien J. Fabrication of microfluidic hydrogels using molded gelatin as a sacrificial chemical element. Lab Chip. 2007;7:720. [PubMed] [Google Scholar]

106. Chrobak K.M. Potter D.R. Tien J. Formation of perfused, functional microvascular tubes in vitro. Microvasc Res. 2006;71:185. [PubMed] [Google Scholar]

107. Dean D.M. Napolitano A.P. Youssef J. Morgan J.R. Rods, tori, and honeycombs: the directed cocky-assembly of microtissues with prescribed microscale geometries. FASEB J. 2007;21:4005. [PubMed] [Google Scholar]

108. Napolitano A.P. Chai P. Dean D.1000. Morgan J.R. Dynamics of the self-assembly of complex cellular aggregates on micromolded nonadhesive hydrogels. Tissue Eng. 2007;xiii:2087. [PubMed] [Google Scholar]

109. Tsang V.L. Chen A.A. Cho 50.M. Jadin Yard.D. Sah R.L. DeLong S. Due west J.50. Bhatia South.North. Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels. FASEB J. 2007;21:790. [PubMed] [Google Scholar]

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2946907/

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