Name: a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders
Uploaded: 2014-06-08T23:30:00.000+00:00
Duration: 11 min 47 s
Description: Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

Research was partly supported by NIH research grants R01 AR062173 and SHC 250862 to IEA. ES is the recipient of NIH T32 CTSC predoctoral fellowship.

SRANKL MC DNA 1. Hidrodinamik Gönderme <ol><li> Fare Tail Ven yoluyla hidrodinamik Teslim <ol><li> Kuyruk damarından enjekte edilmesinden önce fare tartılır. Fare vücut ağırlığının ~% 10 bir toplam hacim içinde sRANKL veya Ringer çözeltisi yeşil floresan protein (GFP) MC (ön sıcak, 37 ° C&#39;de) seyreltin. </li><li> Kan damarlarını genişletmek ve görünür yan damarlar (LVs) yapmak için enjeksiyondan önce 10 dakika boyunca bir kafeste fare ısıtın. Dehidratasyon ve hipertermi önlemek için dikkatli fare izleyin. </li><li> En kısa sürede LVs dilate ve görünür gibi, süzgeç için fare aktarmak ve hareketi dizginlemek için varil içine yeterince fişini takın. Solunum gibi normal bir aktivite için fare izleyin. Gerekirse süzgeç fişini ayarlayın. </li><li> Silin bir alkol ile kuyruğun ucundan enjeksiyon alanı 3/4 dezenfekte. Fare kuyruğu ven enjeksiyon için 27-30 G iğne kullanın. Bir elinizle sıkıca tutun kuyruk ve iğneyikuyruk toplardamarına. Şırınga üzerinde baskı uygulayın ve 5-7 saniye içinde enjeksiyon tamamlayın. </li><li> Kuyruk damarından gelen iğne kaldırmak ve kanamayı durdurmak için enjeksiyon yerinde bir pamuk ile basınç uygulayın. Solunum hızı normal hızına düşürülür sonra bir kez vivari geri fare aktarmak, 15-30 dakika boyunca fare izleyin. </li></ol></li><li> Fare Serum TRACP5b Mesaj Gen Transferi Sistemik Fare sRANKL İfade ve Nicelik Onay <ol><li> Kan damarlarını genişletmek ve LVs görünür yapmak için 5-10 dakika için bir kafes içinde fare ısıtın. Dehidratasyon ve hipertermi önlemek için dikkatli fare izleyin. </li><li> Kuyruk ucundan ileri bir site bir de fare kuyruğuna bir bıçak ile küçük bir kesi olun. </li><li> Serum ayırma tüplerine kan yaklaşık 100 ul toplayın. </li><li> 30 dakika boyunca oda sıcaklığında serum ayırma tüpleri inkübe edin. </li><li> Santrifüj 5 dakika boyunca 10,000 x g&#39;de ve serum numuneleri toplamak. Stordaha fazla analiz için -80 ° C e serum. </li><li> Bir ticari olarak temin edilebilen bir kit kullanılarak serum örneklerinde sRANKL ELISA ve fare serum TRACP5b deneyi yapın. </li></ol></li></ol> Fare BMMs 2. In vitro Osteoklast Üretimi <ol><li> İzolasyon ve BMMs ekimi <ol><li> Tibia ve femur kemikleri İzolasyonu: CO 2 ile donör fare Euthanize, ve% 70 etanol ile dezenfekte bacaklar. Sağlam femur ve tibia kemikleri kaldırmak için, kalkaneus kemik kasık kemiğinden ayır. </li><li> Kemik zarar vermeden dikkatli bir cilt ve kas çıkarın. Fosfat tamponlu tuzlu su (PBS) ihtiva eden bir Petri çanağı içinde işlenmiş femur ve tibia kemiği yerleştirin. </li><li> Kemik iliği çıkarılması: femur ve tibia kemikleri bir tarafındaki ucu kesin ve α-MEM,% 1 penisilin / streptomisin içeren yüklü bir 25 G iğne ile bir 1 ml şırınga ile bir 50 ml tüp içine, kemik iliği atılmasına (Osteoklast Kültür Orta / OCM). </li><li&gt; işlem kemik iliği: karıştırın ve 70 um naylon hücre süzgecinden geçirilerek, yeni bir 50 ml tüp, kemik iliği süspansiyon aktarın. Santrifüj 300 xg ve en hücreler,% 1 penisilin / streptomisin ve% 10 FBS (osteoklast kültür ortamı / OCM (+)) ihtiva eden α-MEM içinde yeniden askıya. </li></ol></li><li> Makrofajlar Kültür <ol><li> Hemasitometre veya otomatik hücre sayıcı kullanarak hücreleri saymak. </li><li> 96 oyuklu bir plaka içerisine yerleştirildi ve 37 ° C de inkübe cam kapak slipleri (5 mm) ve diş kemiği dilimleri 6 kuyulu bir levha ya da 3 x 10 5 hücre için oyuk başına Plaka 1 x 10 6 hücre </li><li> Aspire yapışmayan hücreleri ihtiva eden ve 48 saat boyunca 25 ng / ml fare M-CSF ihtiva eden taze OCM (+) ile 37 ° C&#39;de inkübe hücreleri tüm medya. </li><li> 25 ng / ml fare M-CSF ve osteoklastojenezi başlatmak için 30 ng / ml fare sRANKL ihtiva eden aspire tüm medya ve taze OCM ile 37 ° C&#39;de inkübe hücreleri (+) media. Gerektiği gibi her 3 günde bir ortam doldurun. </li><li> Hücrelerin büyümesineyaklaşık 6-8 gün kemik eritme kapasitesine sahip çok çekirdekli dev hücrelerin oluşturulması için. En kısa sürede, çok çekirdekli hücreler görünür gibi, ticari bir kit kullanılarak boyanan TRAP (tartarat dirençli asit fosfataz) yapmak, ve tam olgunlaşmış zaman, fonksiyonel testleri gerçekleştirmek. Phalloidin leke ve resim daha önce tarif edildiği gibi 19 elektron mikroskopi taraması (SEM) ile dentin dilimleri bağlanmış hücreler kullanılarak lamelleri F-aktin halkaları görselleştirme. </li></ol></li></ol> İnsan PBMC &#39;den 3. In vitro Osteoklast Üretimi <ol><li> İnsan PBMC İzolasyonu <ol><li> Bir 50 ml tüp içine önceden ısıtılmış Histopaque-1077 10 ml ilave edilir. </li><li> Boş lökosit filtre 50 ml tüp (PBS ile kan 1:1 oranında) içine steril PBS ile kan bankasından elde. Kan histopaque karışmadığı emin, çok yavaş histopaque çözüm üzerinde seyreltilmiş kan Katman. </li><li> 18 ° C&#39;de 20 dakika boyunca 1000 x g&#39;de santrifüjleyin örnekleri Takip etmeksantrifüj ing dikkatlice beyaz kan hücreleri içeren beyaz ince beyaz tabaka tabaka izole edilmesi ve yeni bir 50 ml&#39;lik tüpe transfer. </li><li> Hücreleri toplamak için 5 dakika boyunca 650 x g hızında tekrar PBS ve santrifüj ile hücreler seyreltin. </li><li> Medya ve hücre sayacı kullanarak hücrelerin sayısı (-) OCM süpernatant ve tekrar süspansiyon hücreleri çıkarın. </li></ol></li><li> PBMC&#39;Ierde Kaplama ve kültüre <ol><li> OCM hücrelerin tekrar (+) ve ortam levha 8 x 10 ug / ml göz başına 6 çukurlu bir levha ya da 1 x 10 6 hücre / ml her bir kuyucuk için 96 oyuklu bir plaka içerisinde 6 hücre. Gibi immünofloresan görüntüleme gibi kemik erimesi deneyleri için uygun kemik dilimleri üzerinde için gerekli olan cam kapak slipleri (5 mm) levhalar 20 hücreleri daha önce tarif edildiği. </li><li> Değişim kültür ortamı, insan M-CSF, her 2-3 günde bir ile ya da gerektiğinde hücreleri ilave etme. Daha sonra osteoclastogenes başlatmak için OCM (+) ortamı içinde 25 ng / ml insan M-CSF ile birlikte insan sRANKL 30 ng / ml ilave etmek suretiyle, 5. günde farklılaşma aşamasına geçinolduğunu. </li><li> Çok çekirdekli dev hücreler oluşturmak için yaklaşık olarak 14-21 gün için hücreler büyür. En kısa sürede çok çekirdekli hücreler olarak görünür, bu hücreler daha sonra TRAP için etiketlenebilir ve tam olarak olgunlaşmış zaman, işlevsel tahliller gerçekleştirilebilir. F-aktin halkaları phalloidin leke kullanarak lamelleri üzerinde görülebilir. Hücre morfolojisi, SEM ile dentin dilimleri bağlı hücrelerde incelenebilir. </li></ol></li></ol>

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The authors have nothing to disclose.

Kas-iskelet koşulları morbidite ve sakatlık nedenleri lider vardır ve 150&#39;den fazla hastalık ve sendromların oluşmaktadır; Bugün yaklaşık 90 milyon Amerikalıyı etkileyen. Eklem enflamasyonu ve kemik yok artrit ve osteoporoz dahil olmak üzere kas-iskelet sistemi koşulları baskın özellikleri vardır. Osteoporoz genellikle kemik kırıkları yol açan kemik bütünlüğünü zayıflatan bir durumdur. Artrit şiş, hassas ve sert kısıtlayan normal hareket olmak ve sakatlığa yol açabilir eklem ilti...

Kas-iskelet sistemi hastalıkları, ulusal ve yerel sağlık sistemleri için 1 Amerika Birleşik Devletleri ve mevcut ciddi sonuçlara milyonlarca insanı etkiler. Bu bozukluklar arasında kemik kaybı ve geniş bir tedavi ve kurtarma uzun süre gerektiren eklem fonksiyonu ile karakterize edilir. Genellikle, osteoklastların sayısı ve / veya aktivitesinde bir nispi artış, hücre osteoporozda, kemik emmek için özel ve artrit 2 görülmektedir. Fizyolojik koşullar altında, osteoklastların sayısı ve etkinliği osteoblastlar tarafından üretilen nükleer faktör κ-B ligandının (RANKL) reseptör aktivatörü düzenlenir. Osteoprotegerin (OPG), RANKL için bir yem reseptörü de 3 osteoblastlar tarafından üretilen sistemik sRANKL aşın veya OPG silinmesini içerir in vivo hayvan modelleri, osteoporoz araştırmalarında, çok değerlidir.; Bununla birlikte, bu yöntemler, transgenik farelerin 4,5 üretilmesini gerektirir. Burada, yeni bir alternatifkas-iskelet sistemi ile ilgili hastalıkların çalışma için sRANKL aşırı ifade eden bir yöntem tarif edilmektedir. Özellikle, minicircle (MC) DNA teknolojisi ve hidrodinamik dağıtım yöntemler, canlı ortam içinde sRANKL arasında gen transferini elde etmek ve sistemik 6 fare sRANKL aşırı ifade etmek için kullanılmıştır. Bu yöntem, aynı zamanda, düşük kalsiyum diyeti 8 ile ovariektomi 7 ve diyet müdahalesi şu osteoklast hormonal düzenlenmesi gibi osteoporoz ve diğer in vivo modeller, tamamlayıcıdır. Bu modeller cerrahi gerektiren ve önemli bir maliyetle 9 at, birkaç aya kadar sürebilir ancak kas-iskelet sistemi ile ilgili bozuklukların farklı yönlerini incelemek için çok yararlıdır. Ovariektomi uygulanmış olan (OVX) kemirgen modeli yumurtalıkların çıkarılması böylece insan menopoz sonrası osteoporoz 10 taklit eden estrojen eksikliğine yol açar deneysel bir hayvan modelidir. İnsan menopoz sonrası osteoporoz, östrojen yetersizliklerinin bir durumdurency kemik kırılmaları yönünden artan bir riske yol açar ve osteoporoz sadece Amerika Birleşik Devletleri&#39;nde yaklaşık sekiz milyon kadınları etkiler. OVX modeli, menopoz sonrası osteoporoz için faydalı olmasına rağmen, genel olarak osteoporoz okuyan sınırlı avantajlar sunmaktadır. Östrojen yokluğunda bu nedenle de artmış osteoklast aktivitesi 10-12 görülmektedir, osteoklast uyarılması ve osteoblast apoptoz inhibe ederek, kemik kaybını önler. Kemik erimesini yana A RANKL&#39;ın-OPG oranı dengesizlik ayrıca 13 görülmektedir. Büyüme faktörü β (TGF β), yüksek interlökin-7 (IL-7) ve TNF, IL-1 ve IL-6 14,15 dönüşüm seviyelerinin uzaması Bununla birlikte, in vivo koşullarında östrojen eksikliği de eşlik etmektedir. Bu sitokinler RANKL&#39;ın yolunun kemik yeniden düzenleyici fonksiyonları bağımsız bilinen gibi, sadece RANKL-RANK eksenine herhangi bir osteoklast aktivasyonunu atfetmek mümkün değildir. Bu yazıda anlatılan modeli viv çalışma için araştırmacılar sağlaro pro-enflamatuar sitokinler olmaksızın osteoclastogenez ve kemik kaybına RANKL-RANK eksen OVX kemirgen modelleri ile karşılaştırıldığında. Buna ek olarak, in vitro osteoklastojenezisi teknikleri kas-iskelet sistemi hastalıklarının tedavisi için potansiyel terapötik osteoklast aktivasyonunu çalışma için gerekli araçlardır. Daha önceki çalışmalar, aynı zamanda, fare makrofaj koloni uyarıcı faktör (M-CSF) ve fare sRANKL ile fare kemik iliğinden elde edilen makrofajların (BMMs) kültürlenmesi osteoclast farklılaşmasında 3,16,17 yol göstermiştir. Burada, fare kemik iliği hem de in vitro 18, insan periferal kan tek-çekirdekli hücreleri (PBMC) için çok çekirdekli osteoklast-benzeri hücreleri oluşturmak için protokolleri açıklanmıştır. Olgun terminal açıdan farklılaşmış ve tamamen işlevsel osteoklast tanımlamak için gerekli olan hücre bazlı deneyler, aynı zamanda kısaca açıklanmıştır. Bu in vitro teknikler vivo yaklaşım romanı tamamlamak ve p olarak hizmetowerful soruşturma araçlar osteoklast farklılaşmasını ve aktivasyonunu incelemek. Bu sistemleri kullanarak, bilim adamları, in vivo ve in vitro olarak osteoklastlar üretmek ve bunların proliferasyon ve aktivasyon için gerekli olan uyarıcılar ve sinyaller tespit hem de farmakolojik ve biyolojik inhibitörlerinin sonuç verirliğinin test edilmesi mümkün bulunmaktadır.

<table><tbody><tr><td>alpha-MEM</td><td>Life Technologies&amp;nbsp;</td><td>12561-056</td><td></td></tr><tr><td>Human M-CSF</td><td>Miltenyi Biotec</td><td>130-096-492</td><td></td></tr><tr><td>Mouse M-CSF</td><td>Miltenyi Biotec</td><td>130-094-643</td><td></td></tr><tr><td>Human RANK-Ligand - soluble</td><td>Miltenyi Biotec</td><td>130-094-631</td><td></td></tr><tr><td>Mouse RANK-Ligand - soluble</td><td>Miltenyi Biotec</td><td>130-094-076</td><td></td></tr><tr><td>Tailveiner Restrainer for mice</td><td>Braintree</td><td>TV-150 STD</td><td></td></tr><tr><td>Mouse TRANCE/RANK L/TNFSF11 Quantikine ELISA Kit&amp;nbsp;</td><td>R&amp;amp;D systems</td><td>MTR00</td><td></td></tr><tr><td>Acid Phosphatase, Leukocyte (TRAP) Kit</td><td>Sigma</td><td>387A</td><td></td></tr><tr><td>MouseTRAP assay&amp;nbsp;</td><td>immunodiagnostic systems</td><td>SB-TR103</td><td></td></tr></tbody></table>

a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

Burada, osteoklastojenez sitokinlerin etkilerini incelemek için bir yöntem olarak, in vitro olarak osteoklast öncü hücreleri ayırt etmek için in vivo ve hücre kültürü protokollerinde osteoklast farklılaşması için yeni bir gen transfer tekniği tarif edilmektedir. Şekil 1, farelerde GFP ve fare sRANKL MC başarılı gen transferi için temsili sonuçları gösterilmiştir. Şekil 2&#39;de, fare kemik iliği ya da parlak alan mikroskopi ile osteo...

Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Bir Roman<em&gt; In vivo</em&gt; Gen Transferi Tekniği ve<em&gt; In vitro</emİskelet Hastalıkları Kemik Kaybı Çalışmaları&gt; Hücre Tabanlı Tahliller

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

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Research

JoVE Journal

Medicine

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

11.6K Görüntüleme.  University of California, Davis. Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. 

Video: A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Roman kullanarak Spinal Kord Yaralanması sonra Fare Hind Ekstremite Kemik Kaybı Boyuna değerlendirilmesi, In vivo, Metodolojisi

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Tıp

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

İlaç Tedavi ve<em&gt; İn Vivo</em&gt; Bir Medaka Balık Osteoporoz Modelinde Osteoblast-Osteoklast Etkileşimler Görüntüleme

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Gelişim Biyolojisi

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Kemik Metastaz Modelleri

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Primer Kemik Tümörleri ve Kemik Metastazlarının Solid Tümör Grefti İmplantasyonu ile Modellenmesi

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Kanser Araştırmaları

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Ardışık<em&gt; In vivo</em&gt; Kırık Onarım sırasında Osteojenik Kök / Progenitör Hücrelerinin Görüntüleme

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

Angiogenez Multiphoton mikroskobu In Vivo genetiği değiştirilmiş 3D-PLGA/nHAp İskele tarafından grefti kritik Kemik defekti tamir için görselleştirme

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Biyomühendislik

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

için Femur Pencere Odası Modeli<em&gt; İn Vivo</emFare Kemik İliği içinde&gt; Hücre Takibi

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

İmmünoloji ve Enfeksiyon

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

Semiautomated boyuna Microcomputed tomografi tabanlı nicel yapısal analizi bir çıplak sıçan vertebra kırığı Osteoporoz ile ilgili Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

<meta charset="utf8"></head><body>Işınlanmış farelerde etkili HSCT için osteo-organoid türevli hücreler

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

<meta charset="utf8"></head><body>Terapötik Hematopoietik Kök/Progenitör Hücrelerin Toplanması için İn Vivo Osteo-organoid Yaklaşım

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Terapötik Hematopoietik Kök/Progenitör Hücrelerin Toplanması için İn Vivo Osteo-organoid Yaklaşım

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

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Roman kullanarak Spinal Kord Yaralanması sonra Fare Hind Ekstremite Kemik Kaybı Boyuna değerlendirilmesi, In vivo, Metodolojisi

İlaç Tedavi ve

Kemik Metastaz Modelleri

Primer Kemik Tümörleri ve Kemik Metastazlarının Solid Tümör Grefti İmplantasyonu ile Modellenmesi

Ardışık

Angiogenez Multiphoton mikroskobu In Vivo genetiği değiştirilmiş 3D-PLGA/nHAp İskele tarafından grefti kritik Kemik defekti tamir için görselleştirme

için Femur Pencere Odası Modeli

Semiautomated boyuna Microcomputed tomografi tabanlı nicel yapısal analizi bir çıplak sıçan vertebra kırığı Osteoporoz ile ilgili Model

Terapötik Hematopoietik Kök/Progenitör Hücrelerin Toplanması için İn Vivo Osteo-organoid Yaklaşım

Terapötik Hematopoietik Kök/Progenitör Hücrelerin Toplanması için İn Vivo Osteo-organoid Yaklaşım