Bu eser iç bölümü ve çevre mühendisliği ve Mühendislik Fakültesi, University of Illinois Urbana Champaign tarafından desteklenmiştir. Dar ve derin koyak Kinra ve Kavita Kinra dostluk Kavya Mendu lisansüstü çalışmaları desteklemek için anıyoruz. Elektron mikroskobu soruşturma tarama Frederick Seitz malzeme araştırma laboratuvarı ve University of Illinois Urbana Champaign, Beckman Enstitüsü tesislerinde gerçekleştirilmiştir.

Not: Burada açıklanan protokol Illinois kurumsal hayvan bakım ve kullanım Komitesi ve hayvan bakımı kuralları izler.
1. numune tedarik
<ol><li>Taze hasat sığır kemiklerine bir mezbaha tarım USDA sertifikalı Amerika Birleşik Devletleri bölümünden toplamak ve onları soğutucuda plastik hava sıkı torbalarda taşıma. Not: burada koordinatörlüğünde giden bu çalışma için 24-30 ay yaşlı, taşralı ve ağırlığını yaklaşık 1.000 - 1.100 pound hayvanlardan kemiklerine toplanmıştır.</li>
	<li>Uyluk -, 20 ° C numune hazırlama prosedürü başlayana dondur. Bu sıcaklık kemiklerine taze12,13,14tutar.</li>
</ol>2. kesme, temizlik ve numuneler katıştırma
<ol><li>Yaklaşık 2 saat oda sıcaklığında su ile bir kap içinde donmuş kemiklerine çözülme.</li>
	<li>Numuneler tek tip kesiti kortikal kemik ile üretmek için tablo üst elmas şerit testere kullanarak orta diaphysis bölgesinden çoklu diskler yaklaşık 10-15 mm kalınlığında kesin.</li>
	<li>Bir diseksiyon kit herhangi bir yumuşak doku veya kortikal kemiğe bağlı eti kaldırmak için kullanın.</li>
	<li>Adım üzerinde düşük hızlı bir elmas wafering bıçak kullanarak 2.2 kemiklerine kesitleri elde kesik ıslak koşullarda kemiğin boyuna ekseni boyunca birden çok kabaca tetrahedron bölüm elde etmek için gördüm. Not: Burada, yalnızca numune hazırlama ve çizik kısa – yapılan testler boyuna örnekler ele alınmıştır. Ancak, kesim yönü hariç, hazırlık yordamı enine yönlendirme için aynı kalır.</li>
	<li>20 dk ultrasonik bir süpürge bir süre %1,5 Anyonik temizleyici kullanarak hazırlanan solüsyona ve %5 çamaşır suyu numuneler temiz.</li>
	<li>
Akrilik Reçine kortikal kemik örnekleri katıştır (burada polimetil metakrilat (PMMA)) işleme ve istikrar için kolaylaştırmak için.
	<ol>
<li>Numuneler katıştırmak için önce bir yayın aracı ile kalıp duvarları kat. Sonra mix Akrilik Reçine ve sertleştirici PMMA üreticisi tarafından verilen talimatlara göre bir ölçek.</li>
		<li>Kesme kortikal kemik örneklerin her kalıp kaşıyacak biri yüzeyi ile içine yerleştirin aşağı doğru bakacak. Akrilik Reçine karışımı dökün içine bu numune sahipleri hazır. Numuneler en fazla 4-5 saat süresince tedavi edelim.</li>
	</ol>
</li>
	<li>5 mm kalınlığında diskler katıştırılmış örneklerden kesmek, düşük hızlı kullanarak kaşıyacak biri için yüzey açığa gördüm ve numuneler çapının metal (alüminyum) diskler üzerine monte 34 mm ve yükseklik 5 mm siyanoakrilat yapıştırıcı kullanarak.</li>
	<li>Hanks dengeli tuzlu çözüm (HBSS içinde) batırılmış bir ölçüm örnekleri şal ve 4 ° C'de daha fazla kullanım15,16kadar buzdolabında bekletin.</li>
</ol>3. Bileme ve parlatma iletişim kuralları
Not: önceden küçük uzunluklu ölçeklerde yüksek hassasiyetli test için gerekli bir pürüzsüz ve tesviye örneklerin yüzeydir. Önceki iletişim kuralları parlatma13,17 neden önemli yanlışlık ölçümdeki önde gelen büyük bir yüzey pürüzlülüğü içinde. Düşük ortalama yüzey pürüzlülüğü, 100 ulaşmada sorun yatıyor nm, geniş bir alan 3 x 8 mm2 yüzey üzerinde.
<ol><li>400 grit ve 600 grit silikon karbid kağıtları 1 dk ve 5 min için sırasıyla kullanarak oda sıcaklığında sığır kemik numuneler bilenmesi. Değirmeni-Perdah 100 rpm ve 150 rpm, temel hızında sırasıyla korumak.</li>
	<li>Makine her adımı için 15 dk süresince sığır kemik örnekleri, oda sıcaklığında 800 ile 1,200 grit kağıt üzerinde eziyet. Değirmeni-Perdah 150 devir/dakika, baş hız 60 rpm ve yük 1 lb çalıştırmanın temel hızı korumak.</li>
	<li>3 µm, 1 µm ve 0.25 µm elmas süspansiyon çözümleri sert, delikli, dokuma olmayan kumaş üzerine aynı sırada 90 dk her oda sıcaklığında bir süre kullanarak örnekler Lehçe. Çalışma yük Parlatıcı 300 rpm ve 60 rpm, temel ve baş hızları ile 1 lb her adımda için sırasıyla korumak.</li>
	<li>0,05 µm Alümina süspansiyon çözüm bir yumuşak, sentetik Rayonu kumaş üzerine 100 rpm ve 60 rpm, temel ve baş hızına sahip 90 dk 1 lb az bir süre sırasıyla, aynı zamanda oda sıcaklığında kullanarak numune Lehçe.</li>
	<li>De-iyonize su ile bir ölçek örnekleri koymak ve Bileme ve parlatma kalıntı temiz ve Çapraz bulaşma önlemek için 2 dk arasında birbirini izleyen her adım için bir ultrasonik banyo kabı sok.</li>
	<li>Optik mikroskobu ve SEM görüntüleri kullanarak yüzey özelliklerine bakın. Not: gösterildiği gibi Resim 1, osteonları, Haversian kanalları, çimento hatları, interstisyel bölgeler ve lacunae sığır kemik numuneler üzerinde gözlendi. Bu görüntüleme yöntemleri kortikal kemik örnekleri gözenekli, heterojen ve Anizotropik doğasını ortaya çıkarmak. Ayrıca, Gelişmiş yüzey sınav örneklerin cilalı yüzey kalitesini değerlendirmek için gerçekleştirildi. Temsil edici bir cilalı yüzey Şekil 2' de gösterilmiştir.</li>
</ol>4. mikro deri testi
Not: Mikro kazıma testi mikro sıfırdan Sınayıcısı (şekil 3) kullanma parlatılmış sığır kortikal kemik numuneler üzerinde gerçekleştirilir. Bir elmas Rockwell uç 200 µm ve tepe açısı 120 ° uç yarıçaplı çalışma için kullanılır. Araç bir lineer ilerici yükte 30 N. uygulanması sağlar Ayrıca, araç yatay yük, penetrasyon derinliği ve Akustik emisyon tırmalamak nedeniyle oluşturulan ölçmek için yüksek doğruluk sensörleri ile donatılmıştır. Araç sıfırdan yivli panoramalar yakalayabilirsiniz.
<ol><li>Kortikal kemik örnekleri test önce polikarbonat referans malzeme3kullanarak Rockwell uç uç kalibre edin.</li>
	<li>Kortikal kemik örneği Sahne Alanı'nda yerleştirin ve deri testi mikro sıfırdan tester modülüne entegre kurmak optik mikroskop kullanarak siteyi seçin.</li>
	<li>Bir başlangıç yük 30 mN ve sonunda yükü 30 N. ile doğrusal bir ilerici yük uygulamak Yükleme hızı 60 N/dk ve sıfırdan uzunluğu 3 mm olarak ayarlanması gerekir.</li>
	<li>Kazıma testi dizi kısa boyuna (şekil 3büzerinde) yapmak şekil 3' te gösterildiği gibi sığır kemik örnekleri.</li>
	<li>HBSS ile numune yüzey onları nemli tutmak için her üç-dört kazıma testi bir dizi sonra ıslak.</li>
	<li>Doğrusal olmayan kırılma mekaniği2modelleme bağlı deri testi verilerinizi çözümlemek.</li>
</ol>

<ol>
	<li>Akono, A., Reis, P., Ulm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scratching+as+a+fracture+process:+From+butter+to+steel.">Scratching as a fracture process: From butter to steel.</a> Phys Rev Lett. 106 (20), 204302-204304 (2011).</li><li>Akono, A. T., Randall, N. X., Ulm, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+determination+of+the+fracture+toughness+via+microscratch+tests:+application+to+polymers,+ceramics,+and+metals.">Experimental determination of the fracture toughness via microscratch tests: application to polymers, ceramics, and metals.</a> J of Mat Res. 27 (02), 485-493 (2012).</li><li>Akono, A. T., Ulm, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+technique+for+characterizing+the+fracture+toughness+via+scratch+test+experiments.">An improved technique for characterizing the fracture toughness via scratch test experiments.</a> Wear. 313 (1-2), (2014).</li><li>Akono, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetic+size+effect+law+at+the+microscopic+scale:+Application+to+progressive-load+scratch+testing.">Energetic size effect law at the microscopic scale: Application to progressive-load scratch testing.</a> J of Nanomech and Micromech. 6 (2), (2016).</li><li>Kataruka, A., Mendu, K., Okeoghene, O., Puthuvelil, J., Akono, A. -. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cracking+during+nanoindentation+and+its+use+in+the+measurement+of+fracture+toughness.">Cracking during nanoindentation and its use in the measurement of fracture toughness.</a> MRS Proceedings. 356, 663-668 (1994).</li><li>Islam, A., Dong, X. N., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+modeling+of+a+nanoscratch+test+for+determination+of+in+situ+toughness+of+bone.">Mechanistic modeling of a nanoscratch test for determination of in situ toughness of bone.</a> J of the Mech Bhvr of Biomed Mat. 5 (1), 156-164 (2012).</li><li>McAlden, R. W., McGeogh, J. A., Barker, M. B., Court-Brown, C. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+tough+is+bone?+Application+of+elastic–plastic+fracture+mechanics+to+bone.">How tough is bone? Application of elastic–plastic fracture mechanics to bone.</a> Bone. 40 (2), 479-484 (2007).</li><li>Ritchie, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+conflicts+between+strength+and+toughness.">The conflicts between strength and toughness.</a> Nat Mater. 10 (11), 817-822 (2011).</li><li>Kim, K. T., Ba&#382;ant, Z. P., Yu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-uniqueness+of+cohesive-crack+stress-separation+law+of+human+and+bovine+bones+and+remedy+by+size+effect+tests.">Non-uniqueness of cohesive-crack stress-separation law of human and bovine bones and remedy by size effect tests.</a> Intrnl J of Frac. 181 (1), 67-81 (2013).</li><li>Bazant, Z. P., Planas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fracture and size effect in concrete and other quasibrittle materials. 16, (1997).</li></ol>

Yazarlar ifşa gerek yok.

Mikro kazıma testi karma mod kırık3neden. Ayrıca, sonda daha derin kazılar gibi kısa boyuna sığır kortikal kemik örnekleri kırık işlemler etkinleştirilir. 3 mm uzun çizik için probed Prizmatik yaklaşık 3600 µm uzun, 600 µm geniş ve 480 µm derin birimdir. Bu büyük hacimli bir homojenize öngörmede içinde yardımcı oldu. Doğrusal olmayan kırılma mekaniği manken üzerinde Jdayalı kırık direnç ayıklamak için bize etkin-ayrılmaz hesaplama...

Bu çalışmada, bir roman mikro sıfırdan tekniği1,2,3,4kullanarak kırık kırılmaya karşı dayanıklılık (lamel düzeyi) microscale mesoscale (osteonları) sığır kompakt kemiği ölçmek, 5. Çatlamak başlatma ve çatlamak yayma gibi kemik kırığı işlemlere doğrudan uzunluğu ölçekler farklı yapısal bileşenlerinin ve organizasyon hiyerarşisinin farklı düzeylerde tarafından etkilenmektedir. Bu nedenle, kemik kırığı daha küçük uzunluk ölçeklerde değerlendirilmesi için verimli bir temel anlayış kemik kırılganlık esastır. Bir yandan, üç sayılık bükme gibi geleneksel testleri kompakt gerginlik ve kivrimi sınamaları yaygın olarak sığır femur ve tibia kırığı karakterizasyonu makroskopik ölçek6,7, için yürütülen 8. Öte yandan, kırık tokluk mikroskobik ölçekte ölçmek için önerilen9Vicker'ın girinti kırık oldu. Mikro girinti Radyal çatlaklar oluşturmak için Vicker'ın uç kullanılarak gerçekleştirildi. Ayrıca, Oliver Pharr nanoindentation kırık tokluk yöntemi kullanarak bir keskin küp köşe uç10gerçekleştirildi.
Yukarıdaki temel nanoindentation kırık tokluk çalışmalarda böylece üretilen çatlaklar uzunlukları gözlemci tarafından ölçüldü ve yarı deneysel bir model kırık tokluk hesaplamak için kullanıldı. Ancak, bu yöntemler irreproducible, öznel ve sonuçları son derece gözlemcinin beceri optik mikroskobu kullanılarak veya elektron mikroskobu tarama çatlamak uzunlukları ölçmek için ihtiyaç nedeniyle bağlı. Ayrıca, kazıma testi nano ölçekte yapılmıştır, ancak gücü çatlaklar nedeniyle azalma için hesaba katmaz ve11kusurları gibi temel matematiksel model fizik tabanlı değildir. Böylece, bir boşluk bilgi var: bir yöntem mikroskobik düzeyde kırığı değerlendirmesi için temel bir fizik tabanlı mekanik modeli. Bu boşluğu bilgi mikro kazıma testi uygulanması kemik ilk domuz numuneler5üzerinde odaklanarak kompakt için motive. Çalışma artık daha fazla sığır kemik anlamaya biçimde genişletilmiştir.
Örneklerin iki farklı yönelimleri mümkündür: boyuna enine ve kısa boyuna. Boyuna enine özellikleri femur boyuna eksenine dikey kırık karşılık gelir. Oysa, kısa boyuna karşılık gelen kırık özelliklerine uyluk kemiği5boyuna ekseni boyunca. Bu çalışmada, sığır kortikal kemik kemik kırığı direnci kısa boyuna yönde karakterize etmek için sıfırdan test uygulanır.

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			<td>Acrylic Plastic Casting</td>
			<td>Electron Microscopy Sciences</td>
			<td>24210-02</td>
			<td>Polymethyl Methacrylate</td>
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			<td>CarbiMet SiC Abrasive Paper 400 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
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			<td>Grinding - Abrasive Papers</td>
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			<td>CarbiMet SiC Abrasive Paper 600 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>36080600</td>
			<td>Grinding - Abrasive Papers</td>
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			<td>MicroCut Discs 800 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>36080800</td>
			<td>Grinding - Abrasive Papers</td>
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			<td>MicroCut Discs 800 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>16081200</td>
			<td>Grinding - Abrasive Papers</td>
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			<td>Texmet P For 8&#39;&#39; Wheel PSA</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>407638</td>
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			<td>8&#39;&#39; Microcloth PSA</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>407518</td>
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			<td>Meta Di Supreme Polycrystalline Diamond Suspension, 3 &#181;m</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>406631</td>
			<td>Polishing suspension</td>
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			<td>Meta Di Supreme Polycrystalline Diamond Suspension, 1 &#181;m</td>
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			<td>406630</td>
			<td>Polishing suspension</td>
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			<td>Meta Di Supreme Polycrystalline Diamond Suspension, 0.25 &#181;m</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>406629</td>
			<td>Polishing suspension</td>
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			<td>MasterPrep Polishing Suspension, 0.05&#181;m</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>40-6377-032</td>
			<td>Polishing suspension</td>
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			<td>HBSS, calcium, magnesium, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>14025126</td>
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fragility assessment of bovine cortical bone using scratch tests

Organizasyon beş farklı düzeyde olan karmaşık bir hiyerarşik malzeme kemiktir. Yaşlanma gibi faktörler ve Osteoporoz gibi hastalıkların kemik kırığı eğilimli yapma, kırılganlık artar. Kemik kırığı bizim toplumda büyük sosyo-ekonomik etkisi sayesinde, kemik hiyerarşik her düzeyde mekanik performansını değerlendirmek yeni yollar için bir ihtiyaç vardır. Her ne kadar sertlik ve güç tüm ölçeklerde-nano - mikro, Mezo-, probed ve makroskopik – kırığı değerlendirmesi defa makroskopik test için sınırlı olmuştur. Bu sınırlama kemik kırığı anlayışımızı kısıtlar ve laboratuar ve klinik çalışmalar kapsamını sınırlar. Bu araştırmada, mikroskobik kemiğinden mikro kazıma testi doğrusal olmayan kırılma mekaniği ile kombine kullanarak mezoskopik uzunluğu ölçekler için kırılma direncini araştırıyoruz. Testler sığır kemik numuneler üzerinde kısa boyuna yönlendirme yapılmaktadır. Bir titiz deneysel protokol geliştirilmiş ve çok sayıda (102) testleri kırık tokluk kemik Mikroyapı ile ilişkili heterojenite için muhasebe sırasında kortikal kemik örneklerin değerlendirmek için yapılır.

Atomik kuvvet mikroskobu cilalı yüzey pürüzlülüğü ölçmek için kullanıldı. Yüzey pürüzlülüğü bir büyüklük daha ilgi özellik yüzey daha küçük ise kural olarak örnek iyi cilalanmış bir nitelendirir. Bu durumda, ölçülen yüzey pürüzlülüğü 60 nm 40 µm x 40 µm alanı üzerinde açıkça bu ölçütünde düşüyor.
Şekil 4 penetrasyon karşı kuvvet temsilcisi ç...

Watch this Scientific Journal Video about Fragility Assessment of Bovine Cortical Bone Using Scratch Tests at JoVE.com

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Kazıma testi kullanarak sığır kemik kırılganlığını değerlendirilmesi

Bu çalışmada, mikroskobik kazıma testi kullanarak alt-meso düzeyde sığır kortikal kemik kırığı kırılmaya karşı dayanıklılık değerlendiriyor. Bu orijinal, objektif, titiz, ve kırık tokluk makroskopik ölçek altında soruşturma tekrarlanabilir yöntemi önerdi. Potansiyel uygulamalar değişiklikler nedeniyle Osteoporoz gibi hastalıkların kemik kırılganlığını inceliyorlar.

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

fragility-assessment-of-bovine-cortical-bone-using-scratch-tests

Research

JoVE Journal

Bioengineering

9.2K Görüntüleme.  University of Illinois at Urbana Champaign. Bu çalışmada, mikroskobik kazıma testi kullanarak alt-meso düzeyde sığır kortikal kemik kırığı kırılmaya karşı dayanıklılık değerlendiriyor. Bu orijinal, objektif, titiz, ve kırık tokluk makroskopik ölçek altında soruşturma tekrarlanabilir yöntemi önerdi. Potansiyel uygulamalar değişiklikler nedeniyle Osteoporoz gibi hastalıkların kemik kırılganlığını inceliyorlar.

Video: Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Kemik Rejenerasyon için İnsan perivasküler Kök Hücre kullanın

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Biyomühendislik

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

Organ Kültür bozulmamış Sığır Kuyruğu İntervertebral Disklerin Hazırlanması

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

Miyokard Yapı, Fonksiyon Vivo Kantitatif Değerlendirilmesi yılında Perfüzyon ve Canlılık Kardiyak Mikro-bilgisayarlı Tomografi ile

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Modified Drop Tower Impact Tests for American Football Helmets

A modified National Operating Committee on Standards for Athletic Equipment (NOCSAE) test method for American football helmet drop impact test standards is presented that would provide better assessment of a helmet's on-field impact performance by including a faceguard on the helmet. In this study, a merger of faceguard and helmet test standards is proposed. The need for a more robust systematic approach to football helmet testing procedures is emphasized by comparing representative results of the Head Injury Criterion (HIC), Severity Index (SI), and peak acceleration values for different helmets at different helmet locations under modified NOCSAE standard drop tower tests. Essentially, these comparative drop test results revealed that the faceguard adds a stiffening kinematic constraint to the shell that lessens total energy absorption. The current NOCSAE standard test methods can be improved to represent on-field helmet hits by attaching the faceguards to helmets and by including two new helmet impact locations (Front Top and Front Top Boss). The reported football helmet test method gives a more accurate representation of a helmet's performance and its ability to mitigate on-field impacts while promoting safer football helmets.

This article provides a novel technique to assess the performance characteristics of American football helmets by inclusion of faceguards during NOCSAE Standard drop tests. Additionally, two more impact locations are proposed to be added to the NOCSAE certification.

Amerikan Futbolu Kasklar için Modifiye Drop Tower Darbe Testleri

A modified National Operating Committee on Standards for Athletic Equipment (NOCSAE) test method for American football helmet drop impact test standards ...

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral surface obtained from mechanical testing. This optical method requires a texture of many contrasting fiduciary marks on a solid white background for accurate tracking of surface deformation as loading is applied to the specimen. Immediately prior to testing, the surface of interest in the camera view is painted with a water-based white primer and allowed to dry for several minutes. Then, a black paint is speckled carefully over the white background with special consideration for the even size and shape of the droplets. Illumination is carefully designed and set such that there is optimal contrast of these marks while minimizing reflections through the use of filters. Images were obtained through high speed video capture at up to 12,000 frames/s. The key images prior to and including the fracture event are extracted and deformations are estimated between successive frames in carefully sized interrogation windows over a specified region of interest. These deformations are then used to compute surface strain temporally during the fracture test. The strain data is very useful for identifying fracture initiation within the femur, and for eventual validation of proximal femur fracture strength models derived from Quantitative Computed Tomography-based Finite Element Analysis (QCT/FEA).

In this protocol, the femur surface strains are estimated during fracture testing using the digital image correlation technique. The novelty of the method involves application of a high-contrast stochastic speckle pattern on the femur surface, carefully specified illumination, high speed video capture, and digital image correlation analysis for strain calculations.

Kadavra Femur kortikal suşları kullanarak dijital görüntü korelasyon test kırık sırasında tahmin etmek için bir yöntem

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Biyolojik uyumluluğu Biyomalzeme kemik rejenerasyon için profil sayfası

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

Kemik İkmal Malzemelerinde Kemik Büyümesi ve Neaskülarizasyonunun Değerlendirilmesi İçin Tavşanda Kemik Büyütmenin Kalvarial Modeli

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

Biplanar Videoradyografi kullanarak 3D In-vivo Omuz Kinematik ölçümü

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Mühendislik Nöral Benzeri Dokular için Murine Kortikal Astrositlerin 3D Biyobaskı

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

3D Ko-Kültür Testi: Kanser İlaç Taraması için Vaskülarize Osteojenik Nişler

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

Yazar Spot Işığı: Bir Görüntüleme Mikroplakasında Prekast Poli (Etilen Glikol) (PEG) Hidrojeller Kullanılarak Vaskülarize Osteojenik Kemik İliği Nişinin Basit Kurulması  

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

Bir görüntüleme mikroplakasında önceden dökülmüş poli (etilen glikol) (PEG) hidrojeller kullanılarak vaskülarize osteojenik kemik iliği nişinin basit bir şekilde kurulması

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...