Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number R15AR064481, the National Science Foundation (CMMI-1404716), as well as the Arkansas Biosciences Institute.

1. Production of Extracellular Matrix Using Sacrificial Hollow Fiber Membranes
CAUTION: N-methyl-2-pyrrolidone is an irritating solvent and reproductive toxicant. Exposure to NMP may cause irritation to the skin, eyes, nose, and throat. Solvent-resistant personal protective equipment should be used when handling NMP. Use of NMP should be performed within a fume hood.
<ol>
	<li>Preparation of polysulfone polymer solution for hollow fiber membranes
	<ol>
		<li>Weigh 70 g of po...

<ol>
	<li>Cobb, W. S., Kercher, K. W., Heniford, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+argument+for+lightweight+polypropylene+mesh+in+hernia+repair.">The argument for lightweight polypropylene mesh in hernia repair.</a> Surgical Innovation. 12 (1), 63-69 (2005).</li><li>Morais, J. M., Papadimitrakopoulos, F., Burgess, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials/Tissue+Interactions:+Possible+Solutions+to+Overcome+Foreign+Body+Response.">Biomaterials/Tissue Interactions: Possible Solutions to Overcome Foreign Body Response.</a> The AAPS Journal. 12 (2), 188-196 (2010).</li><li>Simon, P., 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=Early+failure+of+the+tissue+engineered+porcine+heart+valve+SYNERGRAFT&#174;+in+pediatric+patients.">Early failure of the tissue engineered porcine heart valve SYNERGRAFT&#174; in pediatric patients.</a> European Journal of Cardio-Thoracic Surgery. 23 (6), 1002-1006 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FDA strengthens requirements for surgical mesh for the transvaginal repair of pelvic organ prolapse to address safety risks. , (2016).</li><li>Kartus, J., Movin, T., Karlsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-site+morbidity+and+anterior+knee+problems+after+anterior+cruciate+ligament+reconstruction+using+autografts.">Donor-site morbidity and anterior knee problems after anterior cruciate ligament reconstruction using autografts.</a> Arthroscopy: The Journal of Arthroscopic &amp; Related Surgery. 17 (9), 971-980 (2001).</li><li>Lu, H., Hoshiba, T., Kawazoe, N., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+extracellular+matrix+scaffolds+for+tissue+engineering.">Autologous extracellular matrix scaffolds for tissue engineering.</a> Biomaterials. 32 (10), 2489-2499 (2011).</li><li>Wolchok, J. C., Tresco, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+cell+derived+extracellular+matrix+constructs+using+sacrificial+open-cell+foams.">The isolation of cell derived extracellular matrix constructs using sacrificial open-cell foams.</a> Biomaterials. 31 (36), 9595-9603 (2010).</li><li>Roberts, K., Schluns, J., Walker, A., Jones, J. D., Quinn, K. P., Hestekin, J., Wolchok, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+derived+extracellular+matrix+fibers+synthesized+using+sacrificial+hollow+fiber+membranes.">Cell derived extracellular matrix fibers synthesized using sacrificial hollow fiber membranes.</a> Biomedical Materials. 13 (1), (2017).</li><li>Hurd, S. A., Bhatti, N. M., Walker, A. M., Kasukonis, B. M., Wolchok, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+biological+scaffold+engineered+using+the+extracellular+matrix+secreted+by+skeletal+muscle+cells.">Development of a biological scaffold engineered using the extracellular matrix secreted by skeletal muscle cells.</a> Biomaterials. 49, 9-17 (2015).</li><li>Kasukonis, B. M., Kim, J. T., Washington, T. A., Wolchok, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+infusion+bioreactor+for+the+accelerated+preparation+of+decellularized+skeletal+muscle+scaffolds.">Development of an infusion bioreactor for the accelerated preparation of decellularized skeletal muscle scaffolds.</a> Biotechnology Progress. 32 (3), 745-755 (2016).</li><li>Murad, S., 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=Regulation+of+collagen+synthesis+by+ascorbic+acid.">Regulation of collagen synthesis by ascorbic acid.</a> Proceedings of the National Academy of Sciences of the United States of America. 78 (5), 2879-2882 (1981).</li><li>Zhang, 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=Tissue-specific+extracellular+matrix+coatings+for+the+promotion+of+cell+proliferation+and+maintenance+of+cell+phenotype.">Tissue-specific extracellular matrix coatings for the promotion of cell proliferation and maintenance of cell phenotype.</a> Biomaterials. 30 (23-24), 4021-4028 (2009).</li><li>Feng, C. Y., Khulbe, K. C., Matsuura, T., Ismail, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progresses+in+polymeric+hollow+fiber+membrane+preparation,+characterization+and+applications.">Recent progresses in polymeric hollow fiber membrane preparation, characterization and applications.</a> Separation and Purification Technology. 111, 43-71 (2013).</li><li>Domb, A. J., Kost, J., Wiseman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Biodegradable Polymers. , (1998).</li></ol>

The authors declare that they have no competing financial interests.

The processes described enable the production of bulk ECM biomaterials in vitro using hollow fiber membranes cast by a dry-jet wet spinning system allowing for inexpensive bulk production of membranes as well as standard cell culture equipment. While the membranes fabricated in this protocol are intended for use in cell culture, the system described can also be adapted for the production of membranes for separation purposes, with pore size distribution and hollow fiber dimensions tunable by varying spinneret dim...

Implantable surgical scaffolds are a fixture of&#160;wound repair,with over one million synthetic polymer meshes implanted worldwide each year for abdominal wall repair alone1. However, following implantation the synthetic materials polymers traditionally used in the fabrication of these scaffolds tend to provoke a foreign body response, resulting in inflammation deleterious to the function of the implant and scarring of tissue2. Further, as the predominant synthetic mesh materials (i.e., polypropylene) are not appreciably remodeled by the body, they are generally applicable to tissues where scarring can be tolerated, limiting their clinical usefulness toward the treatment of tissues with higher-order function such as muscle. While there are many surgical mesh products which have been applied with clinical success, recent manufacturer recalls of synthetic surgical meshes and complications from interspecies tissue implants highlight the importance of maximizing implant biocompatibility, prompting the FDA to tighten regulations on surgical mesh manufacturers3,4. Implantation of scaffolds derived from patients&#39; own tissues reduces this immune response, but can result in significant donor-site morbidity5. Extracellular matrix (ECM) scaffolds produced in vitro are a possible alternative, as decellularized ECM scaffolds exhibit excellent biocompatibility, particularly in the case of autologous ECM implants6.
Because of the limited availability of patient tissue to harvest for autologous implantation and the risk of impeding function at the donor site, the ability to produce ECM scaffolds in vitro from the culture of human cell lines or, if possible, a patient&#39;s own cells is an attractive alternative. The primary challenges in the manufacture of substantial amounts of ECM in vitro is the sequestration of these difficult-to-capture molecules. In previous work, we have demonstrated that ECM can be produced by culturing ECM-secreting fibroblasts in sacrificial polymeric foams that are dissolved after the culture period to yield ECM which can be decellularized for implantation7,8,9,10. As ECM produced in foams tend to adopt the internal architecture of the foams, hollow fiber membranes (HFMs) were explored as a sacrificial scaffold for production of threads of ECM. Described herein are methods tasked for lab scale manufacturing of cell culture quality hollow fiber membranes and the extraction of bulk extracellular matrix fibers from the same following a period of fibroblast culture. This static culture approach is readily adoptable by laboratories containing standard mammalian cell culture equipment. ECM produced by this approach could be applied toward a variety of clinical applications.

<table>
	<tbody>
		<tr>
			<td>1/32 inch thick silicone rubber</td>
			<td>Grainger</td>
			<td>B01LXJULOM</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL Scintillation Vials, Borosilicate glass, Disposable - VWR</td>
			<td>VWR</td>
			<td>66022-004</td>
			<td>With attached white urea cap and cork foil liner</td>
		</tr>
		<tr>
			<td>3 inch by 1 inch microscopy slides</td>
			<td>VWR</td>
			<td>75799-268</td>
			<td></td>
		</tr>
		<tr>
			<td>4C refrigerator</td>
			<td>Thermo Fisher Scientific</td>
			<td>FRGG2304D</td>
			<td>Any commercial 4C refrigerator will suffice.</td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>VWR</td>
			<td>21008-178</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plates</td>
			<td>VWR</td>
			<td>10062-892</td>
			<td>Alternative brands may be used</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>VWR</td>
			<td>E646</td>
			<td>Alternative brands may be used</td>
		</tr>
		<tr>
			<td>Bore vessel</td>
			<td>McMaster-Carr</td>
			<td>89785K867</td>
			<td>6 ft 316 steel tubing</td>
		</tr>
		<tr>
			<td>Bovine Plasma Fibronectin</td>
			<td>Thermo Fisher Scientific</td>
			<td>33010018</td>
			<td>Comes as 1 mg of lyophilized protein</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>VWR/Amresco</td>
			<td>97062-590</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Incubator w/ CO2</td>
			<td></td>
			<td></td>
			<td>Any appropriate CO2-supplied mammalian cell incubator will suffice.</td>
		</tr>
		<tr>
			<td>Disposable Serological Pipets, Glass - Kimble Chase</td>
			<td>VWR</td>
			<td>14673-208</td>
			<td>Alternative brands may be used</td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330032</td>
			<td>Warm in water bath at 37&#176;C for 30 minutes prior to use</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td>DN25-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dope vessel</td>
			<td>McMaster-Carr</td>
			<td>89785K867</td>
			<td>6 ft 316 steel tubing</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>BDH1160</td>
			<td>Dilute to 70% for sterilization</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, US origin - Gibco</td>
			<td>Thermo Fisher Scientific</td>
			<td>26140079</td>
			<td>Mix with growth media at 10% concentration (50mL in 500mL media)</td>
		</tr>
		<tr>
			<td>Four 1/4-inch to 1&#34; reducing unions</td>
			<td>Swagelok</td>
			<td>SS-1610-6-4</td>
			<td>One reducing union for each inlet and outlet of each vessel</td>
		</tr>
		<tr>
			<td>Freeze-dryer/lyophilizer</td>
			<td>Labconco</td>
			<td>117 (A65312906)</td>
			<td>Any lyophilizer will suffice.</td>
		</tr>
		<tr>
			<td>Hexagonal Antistatic Polystyrene Weighing Dishes - VWR</td>
			<td>VWR</td>
			<td>89106-752</td>
			<td>Any weigh boat will suffice</td>
		</tr>
		<tr>
			<td>Hollow fiber membrane immersion bath</td>
			<td></td>
			<td></td>
			<td>34L polypropylene tubs may be used or large bath containers can be fabricated from welded steel sheets</td>
		</tr>
		<tr>
			<td>Hollow Fiber Membrane Spinneret</td>
			<td>AEI</td>
			<td>http://www.aei-spinnerets.com/specifications.html</td>
			<td>Made to order. Inner diameter = 0.8 mm, outer diameter = 1.6 mm</td>
		</tr>
		<tr>
			<td>Hot plate/stirrer</td>
			<td>VWR</td>
			<td>97042-634</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TGF-&#946;1</td>
			<td>PeproTech</td>
			<td>100-21</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid 2-phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM) - Gibco</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td>Mix with growth media at 1% concentration (5mL in 500mL media)</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>VWR/Alfa Aesar</td>
			<td>AA12315-A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Minus 80 Freezer</td>
			<td>Thermo Fisher Scientific</td>
			<td>UXF40086A</td>
			<td>Any commercial -80C freezer will suffice.</td>
		</tr>
		<tr>
			<td>N2 gas cylinders (two)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIH/3T3 cells</td>
			<td>ATCC</td>
			<td>CRL-1658</td>
			<td>Alternative fibrogenic cell lines may be used.</td>
		</tr>
		<tr>
			<td>N-methyl-2-pyrrolidone</td>
			<td>VWR</td>
			<td>BDH1141</td>
			<td>Alternative brands may be used</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin Solution - Gibco</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Mix with growth media at 0.1% concentration (0.5 mL in 500mL media)</td>
		</tr>
		<tr>
			<td>Polysulfone</td>
			<td>Sigma-Aldrich</td>
			<td>428302</td>
			<td>Any polysulfone with an average Mw of 35,000 daltons may be used</td>
		</tr>
		<tr>
			<td>Portable Pipet-Aid Pipetting Device - Drummond</td>
			<td>VWR</td>
			<td>53498-103</td>
			<td>Alternative brands may be used</td>
		</tr>
		<tr>
			<td>PTFE tubing (1/4-inch inner diameter)</td>
			<td>McMaster-Carr</td>
			<td>52315K24</td>
			<td>Alternative brands may be used.</td>
		</tr>
		<tr>
			<td>Rat skeletal muscle fibroblasts</td>
			<td></td>
			<td></td>
			<td>Independently isolated from rat skeletal muscle. Alternative fibrogenic cell lines may be used.</td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Sigma-Aldrich</td>
			<td>R4642</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone sheet</td>
			<td>McMaster-Carr</td>
			<td>1460N28</td>
			<td></td>
		</tr>
		<tr>
			<td>Take-up motor</td>
			<td>Greartisan</td>
			<td>B071GTTSV3</td>
			<td>200 RPM DC Motor</td>
		</tr>
		<tr>
			<td>Tris HCl</td>
			<td>VWR/Amresco</td>
			<td>97063-756</td>
			<td></td>
		</tr>
		<tr>
			<td>Two needle valves</td>
			<td>Swagelok</td>
			<td>SS-1RS4</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of extracellular matrix fibers via sacrificial hollow fiber membrane cell culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

Successful production of extracellular matrix from sacrificial scaffolds is contingent on appropriate scaffold fabrication, cell culture, and solvent rinse procedures. Fabrication of the hollow fiber membranes is performed using a dry-jet wet-spinning system assembled from commercially available components (Figure 1) which uses extrusion of polymer solution through the annulus of a commercially available steel spinneret (inner diameter = 0.8 mm, outer diamete...

Watch this Scientific Journal Video about Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture at JoVE.com

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

This protocol allows for the production of filaments of bulk extracellular matrix from cultured cell lines, allowing for research into cell matrix interactions, as well as research into the potential of this biomaterial for wound repair. These synthetic fibers can avoid the foreign body response directed against synthetic materials, while still drawing from the rich repertoire of textile fabrication techniques to create a wide range of woven implantables. Demonstrating the procedure will be Cassandra Reed, a graduate student from my lab.Before seeding the cells, autoclave the hollow fiber membranes at 121 degrees Celsius for 30 minutes, followed by treatment with 50 milliliters of bovine plasma fibronectin in PBS at 37 degrees Celsius in 5%carbon dioxide for one hour. At the end of the incubation, use sterile micro scissors to cut the fibers to six centimeters or less, and use a one milliliter syringe equipped with a 21 gauge needle to seed one times 10 to the fifth fibroblast cells directly into the lumina of the hollow fiber membranes. Place six seeded fibers into a six inch diameter Petri dish, and briefly incubate the seeded fibers at 37 degrees Celsius in 5%carbon dioxide.After five minutes, transfer the fibers from the incubator into a new six centimeter Petri dish containing fibroblast culture medium supplemented with L-ascorbic acid, L-ascorbic acid 2 phosphate, and transforming growth factor beta 1 for up to three weeks in the cell culture incubator. At the end of the incubation, use forceps o transfer the cultured fibers into individual scintillation vials, and tilt each vial to allow the addition of up to five milliliters of N-Methyl-2-pyrrolidone down the side of the vials. Then use a serological pipette to slowly aspirate the N-Methyl-2-pyrrolidone from each fiber, and transfer the fibers into new scintillation vials for a second immersion in fresh N-Methyl-2-pyrrolidone.After the third immersion, rinse the resulting extracellular matrix threads three times in deionized water in a similar manner, and place a three-inch-long, one-inch wide 1/23rd-inch-thick piece of silicone rubber with a central eight-by-four millimeter rectangular mold onto a standard three-by-one inch glass microscope slide. After autoclaving, lay each extracellular matrix fiber side-by-side in the eight-by-four millimeter silicone mold until there is no visible open space. Place the mold into a 50 milliliter conical tube, and freeze the fibers at negative 80 degrees Celsius until completely frozen.For decellularization, incubate the mold in 1%sodium dodecyl sulfate for 24 hours at room temperature on a rocker with gentle agitation. The next day, rinse the extracted extracellular matrix with three washes in three milliliters of PBS per wash, and fill the mold with freshly prepared DNAs/RNAs digestion buffer for a six hour incubation at four degrees Celsius. At the end of the digestion, aspirate the digestion solution and rinse the mold three times in sterile PBS as demonstrated.After the third wash, incubate the scaffolds in 10%Penicillin-Streptomycin in PBS at four degrees Celsius overnight before freezing in a 50 milliliter conical tube at negative 80 degrees Celsius for about one hour. When the mesh has completely frozen, lyophilize the decellularized extracellular mesh overnight, or until completely dry, and transfer the lyophilized scaffolds to a sterile container within a bio safety hood and store at four degrees Celsius until use. Here a transverse cross-section of a polysulfone hollow fiber membrane fabricated using this protocol is shown, exhibiting outer and inner layers of finger-like pores characteristic of an asymmetric membrane.After fibroblast cell seeding and culture, N-Methyl-2-pyrrolidone rinsing as demonstrated, translucent threads of extracellular matrix are produced. The extracellular matrix producing cells remain viable inside the hollow fiber membranes throughout the entire three week culture period. The extracted matrix has a translucent appearance when hydrated, exhibiting an off-white color and fibrous appearance with a gross longitudinal alignment upon mesh assembly and lyophilization.The most critical step is the dissolving process of the cultured hollow fiber membranes in the N-Methyl-2-pyrrolidone solvent in order to extract the extracellular matrix. The material produced by this method can be used for fabricating tissue engineered implants for investigating the repair of tissues in pre-clinical studies. The N-Methyl-2-pyrrolidone used in this protocol is a skin and eye irritant, and therefore it is recommended that you handle the chemical using nitrile gloves, goggles, a lab coat, and handle it within a fume hood.

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Bioengineering

7.3K visualizações.  University of Arkansas. Este protocolo permite a produção de filamentos de matriz extracelular a granel a partir de linhas de células cultivadas, permitindo pesquisas sobre interações matriciais celulares, bem como pesquisas sobre o potencial deste biomamaterial para reparação de feridas. Essas fibras sintéticas podem evitar a resposta do corpo estranho direcionada contra materiais sintéticos, enquanto ainda se dedem do rico repertório de técnicas de fabricação têxtil para criar uma ampla gama de impla...