وتم تمويل هذه الدراسات، في جزء منه، من قبل مستشفى الأطفال في بوسطن صندوق جراحة المسالك البولية للإيرادات الأوقاف والمعاهد الوطنية للصحة منح NIBIB P41-EB002520 (كابلان)؛ NIDDK T32-DK60442 (فريمان)؛ NIDDK 1K99-DK083616 (Mauney). نحن نعترف الدكتور بيتر Zvara من جامعة فيرمونت للحصول على المساعدة في إنشاء تقنية لوضع أنبوب فغر المثانة وقياس المثانة.

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الإعلان عن أي تضارب في المصالح.

التقييمات Cystometric من تكوينات مادة بيولوجية بعد زرع المثانة وزيادة في النماذج الحيوانية الصغيرة يمثل خطوة هامة في تحديد التحقق من صحة أفضل الخصائص الهيكلية والميكانيكية والتصاميم مصفوفة لاستخدامها في الحالات السريرية. في هذه الدراسة، فإننا وصف الطرق الجراحية لإجراء عملية تكبير المثانة لدى الفئران والجرذان، وكذلك تقنيات cystometric لتحديد خصائص انسداد المثانة الأجهزة هندسيا لتقييم وظيفي. وقد استخدمت هذه التقنيات في أننا تجارب متعددة تشمل كلا من الفئران والجرذان، مع كل تجربة تتألف من القوارض + 30 بدون القضايا الهامة. مختبر أبحاثنا هي عبارة عن تكتل متنوع من العلماء والجراحين الأساسية الطبيب، والجراحين مع ما لا يقل عن 5-6 سنوات من الدراسات العليا التدريب الجراحية الجوانب الإجرائية لهذه التجارب. بغض النظر عن نوع المستعملة مادة بيولوجية، ودي الكبرىfference بين زيادة المثانة لدى الفئران مقابل الفئران هو حجم المثانة. نظرا لصغر حجم المثانة، وتشريح، وإدراج مادة بيولوجية أكثر صعوبة من الناحية الفنية في الماوس. للمساعدة في التصور، ويمكن استخدام المجهر الجراحي. منذ حجم المثانة لدى الفئران هو أكبر، وأنها أكثر استعدادا لحالات أكثر من إجراء واحد لابد من تنفيذها على المثانة (على سبيل المثال زيادة ووضع القسطرة فغر المثانة). بالإضافة إلى ذلك، يصف بروتوكول فوق استخدام أنابيب PE-50 للجرذ 13، ومع ذلك، القسطرة حجم أكبر، وتصل إلى PE-100 المستخدمة قد تم، وخاصة بالنسبة للدراسات طويلة الأجل 14. في الفئران، ويمكن الاستفادة من عيار أصغر مثل أنابيب PE-10 15،16، ولكن ينبغي أن يوضع في الاعتبار أن أنابيب مرنة أصغر حجما وأكثر قد لا نقل تغيرات الضغط على محول بدقة. كذلك، فإن طريقة بديلة لتأمين القسطرة على ظهر (الخطوة 8 * أعلاه) ويتم ذلك في ميلم بسبب حجمها أصغر الجسم وإبرة طرف كليل وقبعة الرابع هي مرهق للغاية. وعيب هذا هو الحاجة للتخدير لاستخراج نهاية القسطرة في كيس تحت الجلد قبل قياس المثانة. وقد أظهرت الدراسات أن في الأيام الأولى الأولي (0-4 أيام) بعد موضع القسطرة، قياس المثانة وكشف الضغوط العالية وفرط نشاط المثانة مع وحدات التخزين يفرغ منخفض. ظهرت هذه النتائج لتحقيق الاستقرار في جميع أنحاء السادس إلى اليوم السابع 14،17، وبالتالي، وربما كان توقيت مثالي لتقييم cystometric. ومع ذلك، فإن معظم التقارير في الأدب أداء قياس المثانة في غضون أول 3 أيام من قسطرة 18، والحسابات هذا لتباين واسع في المعلمات أعلاه بالنسبة للوقت. ترك قسطرة فوق العانة لمدة أطول من 3 أيام تحمل معها حالة مرضية مختلفة مثل خطر من الحجارة، والخلع، والعدوى، بيلة دموية وانسداد القسطرة مع الحطام. &lt;وقد وصفت ف الطبقة = &quot;jove_content&quot;&gt; معدلات ضخ مختلفة خلال قياس المثانة من 1-3mL/hr بالنسبة للفئران و15،16 10-11mL/hr للفئران 13،19،20. يمكن أن معدلات ضخ Supraphysiologic يسبب ضغوطا مرتفعة زورا 14. نحن نستخدم معدل ضخ 12.5 ميكرولتر / دقيقة (0.75 مل / ساعة) ويمكن أيضا للفئران و 100 ميكروليتر / دقيقة (6 مل / ساعة) بالنسبة للفئران في الإعداد لدينا، ولكن انخفاض معدلات استخدامها. وينبغي أن درجة حرارة المياه المالحة الفسيولوجية تكون على الأقل درجة حرارة الغرفة، على الرغم من دافئ (37 درجة) المالحة هو أكثر المثلى لتجنب فرط نشاط المثانة وأثار مع غرس حل الباردة. في قياس المثانة مستيقظا، من الأهمية بمكان للسماح لتحقيق الاستقرار في نمط يفرغ كما الحيوان تعديلها ليصبح القفص، والتي في تجربتنا يتطلب فترة من 10-20 دقيقة ~. بعد هذا، يمكن تسجيل الدورات العادية للتبول 45-120 دقيقة أو في الحد الأدنى دورات يفرغ 3-4. ينبغي مراعاة هذا الحيوان في الوقت الحقيقي منذ الحيوان موفن بحريةويمكن ز والمضاعفات مثل التواء أو مجعد القسطرة تغيير تحليل cystometric. الحد من الضوضاء البيئية خلال قياس المثانة هو المطلوب لتقليل حركة الحيوانات والأعمال الفنية اللاحقة. قياس المثانة فاقد الوعي لا يملك المشاكل المصاحبة مثل قياس المثانة مستيقظا، ولكن ثبت التخدير متعددة لمنع تقلصات المثانة عفوية. هذا تثبيط يقابل مباشرة إلى المدة المتوقعة لعمل أدوية التخدير، أي عندما تخف حدة تأثير مخدر، تقلصات عفوية استئناف 14. وعلاوة على ذلك، كانت الضغوط تقاس عندما فاضت المثانة، وزيادة إحصائيا في الفئران تخدير، وكلاهما على قيد الحياة وبعد الوفاة، مما يشير إلى تأثير على خصائص الإذعان السلبي للجدار المثانة. ويظهر هذا التأثير مع 21 بنتوباربيتال، الكيتامين، و. كلورالوز IM / IP، بالإضافة إلى هالوثان المستنشق وداخل القراب nesacaine 14 وأجرت دراسة واسعة من مختلف confir التخديرم هذه النتيجة مع قمع منعكس التبول لمواد التخدير على حد سواء الاستنشاق (isoflurane وmethoxyflurane)، والباربيتورات (بنتوباربيتال وthiobutabarbital) تحت التخدير مستويات معتدلة 17. وقد لوحظ هذا التأثير مع ضوء المستويات حتى المسكنات أو التخدير مع أدوية مثل الفنتانيل-دروبيريدول والكيتامين الديازيبام، وكما في الدراسة السابقة، حيث أن تأثير التخدير هدأت، وكذلك فعل تثبيط 17. لهذا الإجراء، يمكن استخدام الحقن داخل الصفاق يوريتان منذ تم التدليل على أن يتم الاحتفاظ تبول لا ارادي في الوقت الذي تسمح أيضا للتخدير كاف 17،22. وعلاوة على ذلك، يلاحظ وجود أي تأثير فيما يتعلق الضغوط التبول 23. يوصف وضع القسطرة فوق العانة لقياس المثانة هنا، لأنه تبين القسطرة داخل الاحليل لديها أعلى منحنيات ضغط المثانة وانخفاض معدلات تدفق تتفق مع النسبية المثانة انسداد مخرج 24.وعلاوة على ذلك، قسطرة داخل الاحليل هو ممكن فقط في الحيوانات تخدير، وحتى ذلك الحين، قسطرة يمكن أن يكون صعبا، وخصوصا في القوارض والفئران الذكور. في الختام، واختيار النموذج الذي ترغب في استخدامه لتكبير المثانة و / أو تحليل cystometric يعتمد على أهداف دراسة محددة. من الناحية التقنية في نموذج الفئران يحمل بوضوح ميزة للأسباب التي ذكرناها آنفا. ومع ذلك، يمكن استخدام نموذج الفأر في دراسات تقييم أدوار محددة المنتجات النهائية الجينات المرمزة في أمراض المسالك البولية، وذلك بسبب قابليتها للتلاعب الجيني. هذا ليس ممكنا عادة في الفئران. قياس المثانة مستيقظا معظم بدقة يحاكي الحالة الطبيعية الفسيولوجية في هذه الحيوانات التي تخضع لدورات التبول بهم، وغير ذلك، من المرجح أن يعطي عزم أكثر موثوقية الفسيولوجية في وظيفة المثانة. وعلاوة على ذلك، فإن المتغير التباس من الآثار المباشرة لليتم تجنب nesthetics على وظيفة المثانة.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> المواد: </td><td> وصف / الاستخدام: </td></tr><tr><td> حلق المقصات </td><td> إعداد جرذ / الماوس لاجراء عملية جراحية </td></tr><tr><td> معقم الستائر، betadine، والإيثانول بنسبة 70٪، شاش معقم </td><td> إعداد حقل الجراحية المعقمة </td></tr><tr><td></td><td></td><td></td><td colspan="2"> الأدوات: </td></tr><tr><td> مشرط شفرة </td><td> شق الجلد </td></tr><tr><td> ملقط مع أسنان </td><td> التلاعب في الجلد </td></tr><tr><td> غرامة ملقط </td><td> ارضحي (لا أسنان)، لا التسنين أو التسنين مع غرامة للتلاعب </td></tr><tr><td> إبرة صغيرة سائق </td><td> تشريح الأنسجة حاد </td></tr><tr><td> Metzenbaum مقص </td><td> Bldder شق </td></tr><tr><td> بضع الوتر مقص </td><td> لخياطة الجروح تراجع ووضع تحت الجلد تيunnel (قسطرة فغر المثانة) </td></tr><tr><td> منحني المشابك الصغيرة </td><td> نفق تحت الجلد (القسطرة فغر المثانة) </td></tr><tr><td></td><td></td><td></td><td colspan="2"> الخيوط الجراحية: </td></tr><tr><td> خيوط البولي بروبلين 6-0 </td><td> خيوط تحقيق المثانة وخياطة pursestring </td></tr><tr><td> 7-0 polyglactin خاط </td><td> مفاغرة من سقالة على المثانة </td></tr><tr><td> 4-0 polyglactin خاط </td><td> إغلاق عضلة / الجلد </td></tr><tr><td> 3-0 أو 4-0 خياطة الحرير </td><td> تأمين طرف القسطرة على الجلد </td></tr><tr><td></td><td></td><td></td><td colspan="2"> الإبر والحقن: </td></tr><tr><td> قياس 18 إبرة </td><td> ثقب في المثانة لقسطرة فغر المثانة </td></tr><tr><td> الإبر قياس 25 و 30 </td><td> المثانة اختبار للتسرب </td></tr><tr><td> 1 مل و ملحيilled محقنة </td><td></td></tr><tr><td> 22 نصيحة فظة قياس إبرة </td><td></td></tr><tr><td></td><td></td><td></td><td colspan="2"> فغر المثانة قسطرة: </td></tr><tr><td> PE-50 أنابيب </td><td></td></tr><tr><td> ولاعة </td><td> حرق PE-50 أنابيب </td></tr><tr><td> منحني المشبك صغير </td><td> النامي تحت الجلد نفق </td></tr><tr><td></td><td></td><td></td><td colspan="2"> قياس المثانة: </td></tr><tr><td> MLT844 التقاط البيانات والبرمجيات ADInstruments LabChart </td><td> ضغط والحصول على البيانات </td></tr><tr><td> هارفارد مضخة محقنة 22 (هارفارد جهاز، Holliston، ماجستير) </td><td> السائل مضخة التسريب </td></tr><tr><td></td><td></td><td></td><td colspan="2"> التخدير (قياس المثانة اللاوعي): </td></tr><tr><td> أناsoflurane </td><td> الاستقراء / صيانة التخدير العام </td></tr><tr><td> يوريتان </td><td> Unconconscious قياس المثانة </td></tr><tr><td> Bupivicaine أو ما يعادلها </td><td> التخدير الموضعي </td></tr><tr><td> Meloxicam </td><td> بعد العمليات الجراحية للوعي </td></tr><tr><td> البوبرينورفين </td><td> بعد العمليات الجراحية للوعي </td></tr></tbody></table>

evaluation of biomaterials for bladder augmentation using cystometric analyses in various rodent models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Watch this Scientific Journal Video about Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models at JoVE.com

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

وصفت مراحل العمليات الجراحية في زيادة المثانة باستخدام 3-D السقالات في نماذج الفئران والجرذان. لاختبار فعالية من تكوينات مادة بيولوجية لاستخدامها في عمليات تكبير المثانة، ويتم تقديم تقنيات لقياس المثانة كلا مستيقظا وتخدير.

تقييم المواد الحيوية لتكبير المثانة باستخدام تحليلات Cystometric في نماذج القوارض مختلفة

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

evaluation-biomaterials-for-bladder-augmentation-using-cystometric

Research

JoVE Journal

Bioengineering

19.1K Views.  Harvard Medical School. وصفت مراحل العمليات الجراحية في زيادة المثانة باستخدام 3-D السقالات في نماذج الفئران والجرذان. لاختبار فعالية من تكوينات مادة بيولوجية لاستخدامها في عمليات تكبير المثانة، ويتم تقديم تقنيات لقياس المثانة كلا مستيقظا وتخدير.

Video: Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

تصنيع الأنسجة الصغيرة باستخدام وحدات من الخلايا التي تحتوي على الكولاجين جل

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

[ميكروأرس] البوليمر لاكتشاف إنتاجية عالية من المواد الحيوية

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

جلد كامل عيب نموذج في تقييم الأوعية الدموية من الحيوية<em&gt; في فيفو</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microenvironments-شطيرة مثل لتسخير خلية / المواد التفاعلات

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

توليف للأنابيب النانوية الكربونية متعددة الجدران تعديل مع جسيمات فضة نانوية وتقييم الخصائص السامة للخلايا والأنشطة المضادة للبكتيريا

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

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.

الشخصية التوافق البيولوجي في الحيوية لتجديد العظام

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

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

تقييم السلوكيات التركيبية الضوئية من خلال القياسات المتزامنة لانعكاس الأوراق وتحليلات الفلورة الكلوروفيل

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...

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.

نموذج كالفاري لتكبير العظام في الأرنب لتقييم نمو العظام والأوعية الدموية الجديدة في مواد استبدال العظام

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 ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

التوضيح الأولي لسائل زراعة الخلايا المقطوعة CHO باستخدام فاصل صوتي

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

التقييمات الهيكلية في الجسم الحي لأمراض العين في نماذج القوارض باستخدام التصوير المقطعي للتماسك البصري

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...