Name: fabrication of size-controlled and emulsion-free chitosan-genipin microgels for tissue engineering applications
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Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institute of Health under award numbers R03AR068087 and R21AR071585 and by the Boettcher Foundation (#11219) to MDK. CBE was supported by NIH/NCATS Colorado CTSA Grant Number TL1 TR001081.

All animal procedures were approved by the University of Colorado Denver Institutional Animal Care and Use Committee. 6-week old male Sprague-Dawley rats were used for the present study. The rat growth plate injury model was created following a previously published report30.
1. Preparation of the chitosan polymer
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	<li>Obtain purified and lyophilized low molecular weight (LMW) chitosan from commercially available sources (see Table of Mate...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+physeal+closure+following+distal+tibia+physeal+fractures:+a+new+radiographic+predictor.">Premature physeal closure following distal tibia physeal fractures: a new radiographic predictor.</a> Journal of Pediatric Orthopaedics. 23 (6), 733-739 (2003).</li><li>Shaw, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+approaches+for+the+treatment+of+pediatric+physeal+injuries.">Regenerative medicine approaches for the treatment of pediatric physeal injuries.</a> Tissue Engineering Part B: Reviews. 24 (2), 85-97 (2018).</li><li>Dabash, S., Prabhakar, G., Potter, E., Thabet, A. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+delivery+of+anti-VEGF+from+injectable+hydrogel+systems+provides+a+prolonged+decrease+of+endothelial+cell+proliferation+and+angiogenesis+in+vitro.">Sustained delivery of anti-VEGF from injectable hydrogel systems provides a prolonged decrease of endothelial cell proliferation and angiogenesis in vitro.</a> RSC Advances. 8 (16), 8999-9005 (2018).</li><li>Fletcher, N. A., Babcock, L. R., Murray, E. A., Krebs, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+delivery+of+antibodies+from+injectable+hydrogels.">Controlled delivery of antibodies from injectable hydrogels.</a> Materials Science and Engineering: C. 59, 801-806 (2016).</li><li>Fletcher, N. A., Von Nieda, E. L., Krebs, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-interactive+alginate-chitosan+biopolymer+systems+with+tunable+mechanics+and+antibody+release+rates.">Cell-interactive alginate-chitosan biopolymer systems with tunable mechanics and antibody release rates.</a> Carbohydrate Polymers. 175, 765-772 (2017).</li><li>Erickson, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+degradation+rate+of+alginate-chitosan+hydrogels+influences+tissue+repair+following+physeal+injury.">In vivo degradation rate of alginate-chitosan hydrogels influences tissue repair following physeal injury.</a> Journal of Biomedical Materials Research Part B: Applied Biomaterials. , 34580 (2020).</li><li>Erickson, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-VEGF+antibody+delivered+locally+reduces+bony+bar+formation+following+physeal+injury+in+rats.">Anti-VEGF antibody delivered locally reduces bony bar formation following physeal injury in rats.</a> Journal of Orthopaedic Research. , 24907 (2020).</li><li>Lee, M. A., Nissen, T. P., Otsuka, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+a+murine+model+to+investigate+the+molecular+process+of+transphyseal+bone+formation.">Utilization of a murine model to investigate the molecular process of transphyseal bone formation.</a> Journal of Pediatric Orthopaedics. 20 (6), 802-806 (2000).</li><li>Planka, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+and+mesenchymal+stem+cells+with+chondrocytes+in+prevention+of+partial+growth+plate+arrest+in+pigs.">Nanotechnology and mesenchymal stem cells with chondrocytes in prevention of partial growth plate arrest in pigs.</a> Biomedical Papers. 156 (2), 128-134 (2012).</li><li>Yu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbit+model+of+physeal+injury+for+the+evaluation+of+regenerative+medicine+approaches.">Rabbit model of physeal injury for the evaluation of regenerative medicine approaches.</a> Tissue Engineering Part C: Methods. 25 (12), 701-710 (2019).</li><li>Xian, C. J., Zhou, F. H., McCarty, R. C., Foster, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramembranous+ossification+mechanism+for+bone+bridge+formation+at+the+growth+plate+cartilage+injury+site.">Intramembranous ossification mechanism for bone bridge formation at the growth plate cartilage injury site.</a> Journal of Orthopaedic Research. 22 (2), 417-426 (2004).</li><li>Erickson, C. B., Shaw, N., Hadley-Miller, N., Riederer, M. S., Krebs, M. D., Payne, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+tibial+growth+plate+injury+model+to+characterize+repair+mechanisms+and+evaluate+growth+plate+regeneration+strategies.">A rat tibial growth plate injury model to characterize repair mechanisms and evaluate growth plate regeneration strategies.</a> Journal of Visualized Experiments. 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Microgels have been widely researched in recent years due to their high level of applicability for various purposes, such as drug delivery or cell encapsulation9. The ease of manufacturing of micro-scale biomaterial constructs is of significant relevance in tissue engineering, as it allows researchers to develop hydrogel-based strategies at a specific size and time scale. However, most methods for fabricating chitosan microgels require expensive equipment and reagents, emulsions, or cytotoxic solv...

The growth plate, also known as the physis, is the cartilage structure located at the end of long bones that mediates growth in children. If the growth plate becomes injured, repair tissue known as a &#34;bony bar&#34; can form, which interrupts normal growth and can cause growth defects or angular deformities. Epidemiological data have shown that 15%-30% of all childhood skeletal injuries are related to the growth plate. Bony bar formation occurs in up to 30% of these injuries, making growth plate injuries and their associated treatment a significant clinical manifestation issue1,2,3,4. When bony bar formation occurs, the most common treatment avenue involves resectioning the bony bar and inserting an interpositional material, such as silicon or adipose tissue5. However, patients that undergo bony bar resection surgery often have a poor prognosis for full recovery, as there is currently no treatment that can fully repair an injured growth plate6,7,8. In light of these shortcomings, there is a critical need for effective strategies for treating growth plate injuries, both in preventing the formation of a bony bar and regenerating healthy physeal cartilage tissue.
Hydrogel microparticles, or microgels, have recently gained interest as injectable scaffolds that can provide sustained release of therapeutics9. Due to their high tunability and biocompatibility, microgels are also well-suited for bioactive factor or cell encapsulation. Microgels can be made of various materials, ranging from synthetic polymers, such as polyethylene glycol (PEG), to natural polymers like alginate or chitosan10,11,12.&#160;Chitosan has been shown to have several beneficial effects for tissue engineering, such as its ability to destabilize the outer membrane of gram-negative bacteria, thereby offering inherent antimicrobial activity13,14. Additionally, chitosan is cost-effective, cell-interactive, and easily modified using its amine-containing structure. Chitosan-based microgels promise a biomaterial strategy for drug delivery and material signaling that can promote tissue regeneration while preventing bacterial infection. However, chitosan microgels are often fabricated with a wide range of techniques that require special equipment, emulsion techniques, or cytotoxic solvent rinses. For example, some studies have fabricated chitosan microgels with emulsion-based methods, but these protocols require solvent rinses and cytotoxic crosslinkers, potentially negating their translation to clinical settings15,16. Other studies have used microfluidics or electrospray approaches to fabricate chitosan microgels, which require special equipment, preparation, and training17,18. Chitosan microgels are also commonly made with a dropwise process of crosslinker into chitosan solution; however, this method is highly dependent on solution viscosity, polymer concentration, and flow rate, making it difficult to control the size and dispersity of the microgels19,20. Conversely, the method for microgel fabrication described herein requires no specialist equipment or solvent rinses, making these microgels viable for fabrication in nearly any lab or setting. Therefore, these microgels represent highly relevant biomaterials for a quick, cost-effective, and easy-to-produce drug delivery vehicle for many applications.
By modulating a microgel&#39;s composition and material characteristics, researchers can gain precise control over the cellular microenvironment, thus directing cell behavior in a material-dependent manner. Microgels can be employed on their own or combined with bulk biomaterial systems to impart specific functionalities, such as the extended release of bioactive factors or precise special signaling for native or exogenous cells. Biomaterials and microgels have emerged as attractive treatment avenues for growth plate injuries. Significant effort has been dedicated to developing alginate and chitosan-based biomaterials to treat growth plate injuries21,22,23,24,25. Due to the dynamic temporal nature of growth plate ossification and bone elongation, the mechanism of bony bar formation is not fully understood.&#160;Therefore, several animal models have been developed to better elucidate the mechanisms of endochondral ossification and bony bar formation, such as in rats, rabbits, and sheep26,27,28. One such model is a rat growth plate injury model, which uses a drill-hole defect in the rat tibia to produce a bony bar in a predictable and reproducible manner and mimics human injuries across all three zones of the growth plate29,30. Several biomaterial-based strategies for treating growth plate injuries have been tested using this model. Additionally, two different methods for fabricating chitosan microgels have been developed, which can be used as an injectable biomaterial system that releases therapeutics in a sustained manner10,31. These microgels have been employed in a rat physeal injury model, and they showed improved cartilage regeneration31 when releasing SDF-1a and TGF-b3. The techniques provided in this protocol describe methods developed to fabricate these chitosan microgels, which can then be employed in a wide variety of tissue engineering applications. For example, recent studies have used thermo- or magento-responsive chitosan microgels for controlled oncological drug delivery applications32,33.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>SigmaAldrich</td>
			<td>AX0073</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Luer-Lock Syringe</td>
			<td>Fisher Scientific</td>
			<td>14-823-16E</td>
			<td></td>
		</tr>
		<tr>
			<td>B&#252;chner Funnel</td>
			<td>Fisher Scientific</td>
			<td>FB966F</td>
			<td>100 mm diameter</td>
		</tr>
		<tr>
			<td>Chitosan (low molecular weight)</td>
			<td>SigmaAldrich</td>
			<td>448869</td>
			<td>75-80% deacetylation</td>
		</tr>
		<tr>
			<td>Dialysis Membrane Tubing</td>
			<td>Fisher Scientific</td>
			<td>08-670-5C</td>
			<td>3500 MWCO</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>SigmaAldrich</td>
			<td>493538</td>
			<td></td>
		</tr>
		<tr>
			<td>Genipin</td>
			<td>SigmaAldrich</td>
			<td>G4796</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell 150i Incubator</td>
			<td>ThermoFisher</td>
			<td>50116047</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>13-374-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human SDF-1a</td>
			<td>Peprotech</td>
			<td>300-28A</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human TGF-b3</td>
			<td>Peprotech</td>
			<td>100-36E</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Filter Paper Grade 540</td>
			<td>SigmaAldrich</td>
			<td>Z241547</td>
			<td>8 mm pore size</td>
		</tr>
		<tr>
			<td>Whatman Filter Paper Grade 541</td>
			<td>SigmaAldrich</td>
			<td>WHA1541055</td>
			<td>22 mm pore size</td>
		</tr>
		<tr>
			<td>Whatman Filter paper Grade 542</td>
			<td>SigmaAldrich</td>
			<td>WHA1542185</td>
			<td>2.7 mm pore size</td>
		</tr>
		<tr>
			<td>Wire Mesh Sieve</td>
			<td>McMaster-Carr</td>
			<td>9317T86</td>
			<td>No. 100 Mesh</td>
		</tr>
	</tbody>
</table>

fabrication of size-controlled and emulsion-free chitosan-genipin microgels for tissue engineering applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

Successful fabrication of chitosan microgels relies on the crosslinking reaction between genipin and chitosan, specifically involving the amines on the chitosan polymer chains. In contrast to other microgel fabrication techniques, this method does not require emulsions or solvent rinses and can be quickly and easily conducted with inexpensive equipment. A hallmark indicator for successful microgel fabrication is the distinct color change from off-white to dark blue after the chitosan and genipin have been mixed. The cros...

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Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...

This protocol allows convenient fabrication of chitosan microgels without toxic solvents that can be used for a variety of tissue engineering and drug delivery applications. This technique does not require special equipment or training and does not require toxic emulsion techniques or solvent rinses, making it highly biocompatible and translatable to a clinical setting. We have applied this technique as a biomaterial strategy for treating growth plate injuries.However, we believe that this technique will also be useful in other regenerative medicine applications, too. This technique can be applied to other regenerative medicine applications that would benefit from an injectable, biodegradable, biomaterial scaffold system with the potential for sustained drug release. Begin by adding acetic acid and purified chitosan to a 10-milliliter Luer lock syringe to form a 6%weight-by-volume chitosan solution.Using a female female Luer lock connector, connect two Luer lock syringes, then mix the solution back and forth until the chitosan in has fully dissolved in the ascetic acid. Now add 100 microliters of the genipin solution to the chitosan-containing syringe and mix back and forth between the syringes for 30 seconds, then eject the mixture from the syringe onto a 35-millimeter Petri dish. Cover the Petri dish with paraffin film and incubate it at 37 degrees Celsius overnight in a humidified atmosphere.Use a spatula to break the hydrogel into smaller pieces, then place a filter of the desired mesh size into the back of a clean 10-milliliter syringe. Transfer the broken gel pieces into the syringe fitted with the filter and add 6 milliliters of double distilled water. Connect the syringe via a Luer lock connector to another clean 10-millimeter syringe.Force the gel and water mixture through the syringe with the filter to create microgels. After the first filtration, open the syringe containing the filter and add the mixture back into this syringe. Force the mixture through the filter again.Now, transfer the filtered gel mixture to a 50-milliliter conical tube and add double distilled water to bring the volume to 20 milliliters. Vortex the solution to get a homogenous solution. Centrifuge the microgels at 100 times g for 5 minutes at room temperature.After centrifugation, remove the upper aqueous phase and resuspend the microgels in 10 milliliters of 70%ethanol, then vortex the microgels and place them under UV light for one hour to sterilize. Now, centrifuge the microgels at 1000 times g for 5 minutes at room temperature. Discard the ethanol and rinse 3 times with double distilled water.Resuspend the microgel pellets in an equal volume of double distilled water. With the increase in pH, the microgels showed a decrease in swelling as depicted by the change in Feret diameter. Also, the size of the microgel particles depends on the pore size of the filter used.Number 200 mesh produced small particles, while number 100 mesh gave rise to large particles. The presence of microgels at the injured site promoted cartilage regeneration. Alcian blue hematoxylin staining showed that microgels injected at the injured tissue prevented early bony bar formation and microgels loaded with bioactive agents SDF-1a and TGF-B3 promoted cartilage formation.It is important to ensure that the wire mesh filter is placed correctly into the syringe so that effective filtering can take place. It is also important to repeat the filtration a few times to ensure a homogenous distribution of microgel size. These microgels can be applied to a wide variety of tissue engineering applications that require a biomaterial scaffold substrate that has the potential for sustained release of therapeutics.

fabrication-size-controlled-emulsion-free-chitosan-genipin-microgels

Research

JoVE Journal

Bioengineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...