The authors thank Texas A&#38;M University Engineering and Experiment Station (TEES) for financial support of this research. Lindsay Nail gratefully acknowledges support from the Texas A&#38;M University Louis Stokes Alliance for Minority Participation (LSAMP) and the National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP). Dawei Zhang thanks the Texas A&#38;M University Dissertation Fellowship.

1. Synthesizing PCL-DA Macromer
<ol>
	<li>Run the acrylation reaction.
	<ol>
		<li>Weigh 20 g of PCL-diol (Mn = ~10,000 g/mol) into a 250 ml&#160;round bottom flask equipped with a Teflon-covered magnetic stir bar.</li>
		<li>Dissolve the PCL-diol in DCM.
		<ol>
			<li>Add 120 mLlof DCM to the flask (concentration = 0.17 g/ml).</li>
			<li>Place a rubber septum loosely into the neck of the flask so as to avoid pressure build-up while also preventing evaporation of DCM.</li>
			<li>Stir solution for ~30 min&#160;at ~250 rpm to completely dissolve the polymer.</li>
		</ol>
		</li>
		<li>Add ~6.6 mg of 4-dimethylaminopyridine (DMAP) to the solution and dissolve with stirring.</li>
		<li>Place a rubber septum securely into the neck of the flask. Allow the solution to continue stirring.</li>
		<li>Through the rubber septum, gently purge the flask with N2 for ~3 min&#160;by using a positive N2 pressure needle inlet and an open needle as an outlet.</li>
		<li>Remove the N2 inlet and outlet.</li>
		<li>Add 0.56 ml&#160;(4.0 mmol) of triethylamine (Et3N) dropwise via a glass syringe equipped with a needle inserted through the rubber septum.</li>
		<li>Add 0.65 ml&#160;(8.0 mmol) of acryloyl chloride dropwise via a glass syringe equipped with a needle inserted through the rubber septum.</li>
		<li>Return the N2 inlet to the flask and allow the contents to stir under positive N2 pressure for ~30 min.</li>
		<li>Pre-heat an oil bath to 55 &#186;C.</li>
		<li>After the allotted ~30 min, remove the N2 inlet and replace the septum with a condenser.</li>
		<li>Submerge the flask into the pre-heated oil bath.</li>
		<li>Allow the contents of the flask to stir for 20 hr.</li>
		<li>After the allotted 20 hr, remove the flask from the oil bath and allow contents to cool to RT.</li>
		<li>Using a rotary evaporator, remove DCM solvent from the flask.</li>
	</ol>
	</li>
	<li>Purify the crude PCL-DA product.
	<ol>
		<li>To the flask, add ~135 ml&#160;of ethyl acetate and dissolve the crude PCL-DA.</li>
		<li>Gravity filter the solution through filter paper into a clean 250 ml&#160;round bottom flask. (Note: Solution may thicken on the filter paper, not readily passing through. If so, carefully apply mild heat with a heating gun.)</li>
		<li>Using a rotary evaporator, remove ethyl acetate solvent from the flask.</li>
		<li>To the flask, add ~140 ml&#160;of DCM and dissolve the crude PCL-DA.</li>
		<li>Transfer contents to a 500 ml&#160;separatory funnel.</li>
		<li>To the funnel, add 13.5 ml&#160;of 2 M potassium carbonate (K2CO3).</li>
		<li>Cap the funnel. Gently mix the two layers by inverting the funnel and swirling gently once or twice, taking care to release pressure via the stopcock. Repeat 3 times.</li>
		<li>Replace the cap with a layer of Parafilm and allow the mixture to separate O/N (~12 hr).</li>
		<li>Collect the bottom, organic layer into a 250 ml&#160;Erlenmeyer flask.</li>
		<li>Add ~5 g of anhydrous magnesium sulfate (MgSO4) to the flask and gently swirl.</li>
		<li>Gravity filter the mixture through qualitative filter paper and into a clean 250 ml&#160;round bottom flask.</li>
		<li>Using a rotary evaporator, remove DCM solvent from the flask.</li>
		<li>Dry under high vacuum to remove residual DCM. (Note: PCL-DA should be stored away from light.)</li>
		<li>Confirm acrylation with 1H NMR22,23.</li>
	</ol>
	</li>
</ol>
2. Preparing the SMP Scaffold (Figure 1)
<ol>
	<li>Prepare the fused salt template.
	<ol>
		<li>Use a 425 &#181;m sieve to obtain sodium chloride (NaCl) particles ~460 &#177; 70 &#181;m in diameter. (Note: Average particle size may be confirmed from scanning electron microscopy [SEM] images with ImageJ software .)14</li>
		<li>To a 3 ml&#160;glass vial (I.D. = 12.9 mm), add 1.8 g of the previously sieved NaCl.</li>
		<li>Slowly add, in four portions, 7.5 wt% (based on salt weight) DI water (0.146 g) to the vial. Mix with a metal spatula after the addition of each portion of water.</li>
		<li>Cap the vial, wrap in tissue and place vertically into a centrifuge tube. Centrifuge for 15 min&#160;at 3,220 x g.</li>
		<li>Remove the cap and let air-dry O/N (~12 hr).</li>
	</ol>
	</li>
	<li>In a new glass vial, prepare a &#8220;macromer solution&#8221; by combining 0.15 g of PCL-DA per ml&#160;of DCM. (Note: For one scaffold, ~ 1 ml&#160;of solution should be prepared.) Cap and mix the solution at high speed on a vortex mixer for ~1 min.</li>
	<li>In a new 3 ml&#160;glass vial, prepare a &#8220;photoinitiator solution&#8221; based on 10 wt% 2,2-dimethoxy-2-phenyl acetophenone (DMP) in 1-vinyl-2-pyrrolidinone (NVP). Combine 0.115 g of DMP in 1 ml&#160;of NVP. (Note: For one scaffold, ~180 &#181;l is required.) Cap, wrap in aluminum foil (to block light) and mix the solution at high speed on a vortex mixer for ~1 min. (Note: If prepared in advanced, the solution should be refrigerated and protected from light.)</li>
	<li>Wrap the vial containing the macromer solution (excluding the cap) with aluminum foil (to block light) and add via pipette 15 vol% (based on total volume of the macromer solution) of the photoinitiator solution. Cap and mix the solution at high speed on a vortex mixer for ~1 min.</li>
	<li>Wrap the vial containing the fused salt template (excluding the cap) with aluminum foil (to block light) and add via pipette the previously prepared macromer/photoinitiator solution (~0.6 ml&#160;or until the template is completely covered).</li>
	<li>Cap the vial, wrap in tissue and place vertically into a centrifuge tube. Centrifuge for 10 min&#160;at 1,260 x g to distribute the macromer solution throughout the template.</li>
	<li>Remove the aluminum foil, uncap the vial and expose to UV light (365 nm, 25 W) for 3 min. Air-dry O/N.</li>
	<li>Remove the &#8220;salt-containing scaffold&#8221; from the vial with tweezers after scoring and fracturing the top of the glass vial.</li>
	<li>In a 400 ml&#160;beaker, prepare ~200 ml&#160;of a water/ethanol solvent (1:1 vol:vol).</li>
	<li>Maintain the scaffold submerged in the water/ethanol solvent for 4 days with daily solvent changes.</li>
	<li>Remove the scaffold from the solvent and air-dry O/N.</li>
</ol>
3. Applying Polydopamine Coating to SMP Scaffold (Figure 1)
<ol>
	<li>In 400 ml&#160;beaker equipped with a Teflon-covered stir bar, prepare ~200 ml&#160;of a dopamine hydrochloride solution (2 mg/ml&#160;in 10 mM Tris buffer, pH = 8.5, 25 &#186;C). Stir at ~150 rpm.</li>
	<li>Place a disposable needle (length = 40 mm; gauge = 20) into the scaffold, ~half the distance through the scaffold. Wrap a wire around the needle hub.</li>
	<li>Submerge the scaffold (with the needle hub above the solution surface) into the stirring dopamine solution by anchoring the wire to the rim of the beaker.</li>
	<li>Degas the scaffold by placing a syringe into the needle hub and using it to pull air out of the scaffold. (Note: Degassing is complete when no more air can be removed and the solution has completely infiltrated the scaffold.)</li>
	<li>Maintain the scaffold submerged in the stirring dopamine solution for 16 hr.</li>
	<li>Remove the scaffold from the solution and remove the needle. Rinse with DI water and dry in a vacuum oven at RT for 24 hr.</li>
	<li>Place the scaffold in an 85 &#186;C oven for 1 hr.</li>
	<li>Allow the scaffold to cool to RT. The final cylindrical scaffold will be ~6 mm diameter x ~5 mm height.</li>
</ol>
4. Evaluating &#8220;Self-fitting&#8221; Behavior
<ol>
	<li>Prepare an &#8220;irregular CMF defect model&#8221; using a sheet of rigid plastic whose thickness is ~5 mm. Use a drill to create a void within the plastic sheet with an average diameter slightly less than ~6 mm, as demonstrated in Figure 2A.</li>
	<li>In a beaker, heat DI water (representing the clinical use of saline) to a temperature of ~60 &#186;C.</li>
	<li>Place the scaffold into the beaker of ~60 &#186;C water. Use tweezers to push the scaffold below the water surface, exposing all areas to the water. Continue for ~2 min or until the scaffold is noticeably softened (Figure 2B).</li>
	<li>Remove the scaffold from the beaker and immediately press (by hand) into the model defect.</li>
	<li>Allow to cool to RT (~5-10 min) (Figure 2C).</li>
	<li>Remove from the defect to observe the new, fixed temporary shape and the return of the relatively more rigid state (Figure 2D).</li>
</ol>
5. Testing Shape Memory Behavior
<ol>
	<li>Using a dynamic mechanical analyzer (DMA; e.g. a TA Instruments Q800 as used herein), run a strain-controlled cyclic-thermal mechanical compression test on a scaffold over two cycles (N) to determine shape fixity (Rf) and shape recovery (Rr) (Figure 3).
	<ol>
		<li>Equilibrate to 60 &#176;C (Thigh) for 5 min.</li>
		<li>Compress to a maximum strain (&#949;m = 50 %) at 50 %/min.</li>
		<li>Hold at &#949;m (5 min).</li>
		<li>Cool to 25 &#176;C (Tlow) and maintain for 10 min to fix the temporary shape.</li>
		<li>Remove the load.</li>
		<li>Measure the ultimate strain in the stress-free state (&#949;u).</li>
		<li>Reheat to 60 &#176;C (Thigh) and maintain for 10 min to recover the permanent shape.</li>
		<li>Measure the recovered strain (&#949;p).</li>
		<li>While still at 60 &#176;C (Thigh), start the 2nd cycle (N = 2) by compressing the scaffold to 50 % of the height recovered after the 1st cycle (N = 1).</li>
		<li>Repeat 5.1.3-5.1.8 for N = 2.</li>
		<li>Calculate Rf and Rr for N = 1 and 2 using the following equations: 
		Rf(N) = [&#949;u(N)/ &#949;m]Rr(N) = [&#949;m - &#949;p(N)]/[&#949;m- &#949;p(N-1)]</li>
	</ol>
	</li>
</ol>
6. Visualizing Pore Size and Pore Interconnectivity
<ol>
	<li>Using scanning electron microscopy (SEM; e.g. a FEI Quanta SEM as used herein), observe the pore size and pore interconnectivity.
	<ol>
		<li>Using tweezers to hold the SMP scaffold, submerge in liquid N2 for 1 min.</li>
		<li>Remove from liquid N2 and fracture along the middle of the scaffold with a clean razor blade.</li>
		<li>Using carbon tape, affix one of the SMP scaffold halves onto the sample stage with the fractured surface facing up.</li>
		<li>Sputter coat with Au-Pt (~4 nm).</li>
		<li>Capture the SEM image at a recommended accelerating voltage of 10-15 kV ( Figure 4A).</li>
	</ol>
	</li>
</ol>
7. Testing of in vitro Bioactivity
<ol>
	<li>Into a 50 ml&#160;centrifuge tube, add ~30 ml&#160;of 1X SBF24.</li>
	<li>Obtain a scaffold in its original, cylindrically-molded permanent shape. Cut the scaffold in half (across the circular edge) using a clean blade.</li>
	<li>Place an individual scaffold half into the prepared centrifuge tube and cap.</li>
	<li>Maintain the tube at 37 &#176;C in a water bath under static conditions with no SBF changes.</li>
	<li>After 14 days, remove the scaffold from the SBF and air dry for 24 hr.</li>
	<li>Using carbon tape, affix the scaffold onto the sample stage with the fractured surface facing up.</li>
	<li>Sputter coat with Au-Pt (~4 nm).</li>
	<li>Capture the SEM image at a recommended accelerating voltage of 10-15 kV (Figure 4B).</li>
</ol>

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The authors have nothing to disclose.

This protocol describes the preparation of a polydopamine-coated, PCL-based scaffold whose self-fitting behavior, as well as osteoinductivity and bioactivity, makes it of interest in the treatment of irregular CMF bone defects. Aspects of the protocol may be altered to change various scaffold features.
The protocol begins with acrylation of a PCL-diol to permit UV cure. In the reported example, the PCL-diol Mn is ~10,000 g/mol. However, by appropriately adjusting amount of acryloyl ...

Currently considered the gold standard of cranio-maxillofacial (CMF) bone defect treatments, transplantation of harvested autologous grafts is hindered by complex grafting procedures, donor site morbidity and limited availability1. A particular difficulty is shaping and fixing the rigid autograft tightly into the defect in order to obtain osseointegration and to prevent graft resorption. Tissue engineering has been investigated as an alternative strategy to autografting and synthetic bone substitutes (e.g. bone cement)2,3. Critical to the success of a tissue engineering approach is a scaffold with a specific set of properties. First, in order to achieve osseointegration, the scaffold must form close contact with adjacent bone tissue4. The scaffold should also be osteoconductive, permitting cell migration, nutrient diffusion and neotissue deposition4,5. This behavior is generally achieved with biodegradable scaffolds exhibiting a highly interconnected pore morphology. Lastly, the scaffold should be bioactive so as to promote integration and bonding with surrounding bone tissue5.
Here, we present a protocol to prepare a tissue engineering scaffold with these properties. Importantly, this scaffold exhibits the ability to &#8220;self-fit&#8221; into irregular CMF defects due to its shape memory behavior6. Thermoresponsive shape memory polymers (SMPs) are known to undergo shape change upon exposure to heat7,8. SMPs are comprised of &#8220;netpoints&#8221; (i.e. chemical or physical crosslinks) which determine the permanent shape and &#8220;switching segments&#8221; which maintain the temporary shape and recover the permanent shape. The switching segments exhibit a thermal transition temperature (Ttrans) corresponding to either the glass transition (Tg) or melt transition (Tm) of the polymer. As a result, SMPs may be sequentially deformed into a temporary shape at T &#62; Ttrans, fixed in the temporary shape at T &#60; Ttrans, and recovered to the permanent shape at T &#62; Ttrans. Thus, an SMP scaffold could achieve &#8220;self-fitting&#8221; within a CMF defect as follows6. After exposure to warm saline (T &#62; Ttrans), an SMP scaffold would become malleable, permitting a generically prepared cylindrical scaffold to be hand-pressed into an irregular defect, with shape recovery promoting expansion of the scaffold to the defect boundary. Upon cooling (T &#60; Ttrans), the scaffold would return to its relatively more rigid state, with shape fixity maintaining its new temporary shape within the defect. In this protocol, an SMP scaffold is prepared from polycaprolactone (PCL), a biodegradable polymer studied extensively for tissue regeneration and other biomedical applications9-11. For shape memory, the Tm of PCL serves as the Ttrans and varies between 43 and 60 &#186;C, depending on the molecular weight of the PCL12. In this protocol, the Ttrans (i.e. Tm) of the scaffold is 56.6&#177;0.3 &#186;C6.
In order to achieve osteoconductivity, a protocol was developed to make PCL-based SMP scaffolds with highly interconnected pores based on a solvent-casting particulate-leaching (SCPL) method6,13,14. Polycaprolactone diacrylate (PCL-DA) (Mn = ~10,000 g/mol) was utilized to permit rapid, photochemical crosslinking and was dissolved in dichloromethane (DCM) to allow solvent-casting over the salt template. Following photochemical cure and solvent evaporation, the salt template was removed by leaching into water. The average salt size regulates scaffold pore size. Importantly, the salt template was fused with water prior to solvent-casting to achieve pore interconnectivity.
Bioactivity was imparted to the SMP scaffold by the in situ formation of a polydopamine coating onto pore walls6. Bioactivity is often introduced into scaffolds by the inclusion of glass or glass-ceramic fillers15. However, these may give rise to unwanted brittle mechanical properties. Dopamine has been shown to form an adherent, thin polydopamine layer on a variety of substrates16-19. In this protocol, the SMP scaffold was subjected to a slightly basic solution (pH = 8.5) of dopamine to form a nanothick coating of polydopamine on all pore wall surfaces6. In addition to enhancing surface hydrophilicity for improved cell adhesion and spreading, polydopamine has been shown to be bioactive in terms of formation of hydroxyapatite (HAp) upon exposure to simulated body fluid (SBF)18,20,21. In a last step, the coated scaffold is exposed to heat treatment at 85 &#186;C (T &#62; Ttrans) which leads to scaffold densification. Heat treatment was previously noted to be essential for scaffold shape memory behavior, perhaps due to PCL crystalline domains reorganizing to closer proximity14.
We additionally describe the methods to characterize the self-fitting behavior within an irregular model defect, shape memory behavior in terms strain-controlled cyclic-thermal mechanical compression tests (i.e. shape recovery and shape fixity), pore morphology, and in vitro bioactivity. Strategies to tailor scaffold properties are also presented.

<table><tbody><tr><td>Polycaprolactone-diol (Mn ~ 10,000 g/mol)</td><td>Sigma-Aldrich</td><td>440752</td><td></td></tr><tr><td>Dichloromethane (DCM)</td><td>Sigma-Aldrich</td><td>D65100</td><td>Dried over 4A molecular sieves</td></tr><tr><td>4-dimethylaminopyridine (DMAP)</td><td>Sigma-Aldrich</td><td>D5640</td><td></td></tr><tr><td>Triethylamine (Et3N)</td><td>Sigma-Aldrich</td><td>T0886</td><td></td></tr><tr><td>Acryloyl chloride</td><td>Sigma-Aldrich</td><td>A24109</td><td></td></tr><tr><td>Ethyl acetate</td><td>Sigma-Aldrich</td><td>319902</td><td></td></tr><tr><td>Potassium carbonate (K2CO3)</td><td>Sigma-Aldrich</td><td>209619</td><td></td></tr><tr><td>Anhydrous magnesium sulfate (MgSO4)</td><td>Fisher</td><td>M65</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>2,2-dimethoxy-2-phenyl acetophenone (DMP)</td><td>Sigma-Aldrich</td><td>196118</td><td></td></tr><tr><td>1-vinyl-2-pyrrolidinone (NVP)</td><td>Sigma-Aldrich</td><td>V3409</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>459844</td><td></td></tr><tr><td>Dopamine hydrochloride</td><td>Sigma-Aldrich</td><td>H8502</td><td></td></tr><tr><td>Tris buffer (2mol/L)</td><td>Fisher</td><td>BP1759</td><td>Used at 10 mM concentration, pH = 8.5</td></tr><tr><td>Sieve</td><td>VWR</td><td>47729-972</td><td></td></tr><tr><td>UV-Transilluminator (365 nm, 25 W)</td><td>UVP</td><td>95-0426-02</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td></td></tr><tr><td>Dynamic Mechanical Analyzer (DMA)</td><td>TA Instruments</td><td>Q800</td><td></td></tr><tr><td>High Resolution Sputter Coater</td><td>Cressington</td><td>208HR</td><td></td></tr><tr><td>Scanning Electron Microscope (SEM)</td><td>FEI</td><td>Quanta 600</td><td></td></tr></tbody></table>

fabrication of a bioactive, pcl-based "self-fitting" shape memory polymer scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

The resulting PCL-based SMP scaffold is capable of self-fitting into a model CMF defect (Figure 2). After brief exposure to warm saline (~60 &#176;C), the cylindrical scaffold softens allowing the scaffold to be manually pressed into and expand within the model defect. After cooling to RT, the scaffold is fixed into its new temporary shape which is retained upon removal from the defect.
The shape memory behavior of an SMP scaffold is quantified by strain-controlled cyclic-ther...

Watch this Scientific Journal Video about Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold at JoVE.com

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

fabrication-bioactive-pcl-based-self-fitting-shape-memory-polymer

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JoVE Journal

Bioengineering

12.5K Views.  Texas A&M University. Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Video: Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

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 ...

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

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 ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 &#181;m and 5 &#181;m diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

This method provides the means to test different polycaprolactone fiber morphologies and topographies for the purpose of tissue engineering. Small and large fibers are fabricated with random orientations, aligned orientations, and also porous cryogenically electrospun structures and used as platforms for cell culture.