3D X-Ray Nanotomography of Cells Grown on Electrospun Scaffolds

Bradley, Robert S.; Robinson, Ian; Yusuf, Mohammed

doi:10.1002/mabi.201600236

Cited by 24 publications

(31 citation statements)

References 44 publications

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“…Considering such complex and three-dimensional nanofibrous scaffolds, acquiring XCT images was challenging. In their work, Bradley et al (2017) were able to visualise human fibroblasts seeded on electrospun poly(lactide-co-glycolide) (PLGA) random microfibrous mats by using a laboratory XCT scanner thanks to the micrometric cross-section of the fibres and the different levels the X-rays attenuation between the PLGA and the cellular component. In the case of the PLLA/Coll nanofibres instead, it is difficult to obtain tomographic images fibres, due to the low absorption of the collagen of X-rays (Balint et al, 2016;Zidek et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

“…Recent studies have defined dedicated protocols to overcome such limitations even in case of submicron voxel sizes (Bosworth et al, 2014;Sensini et al, 2018b). Furthermore, Bradley et al (2017) have defined a procedure to document, by using a laboratory XCT, cell infiltration inside electrospun mats of random microfibres. However, to the best of our knowledge, no work has ever tried to investigate the cell growth and infiltration in complex three-dimensional electrospun nanofibrous scaffolds by combining XCT and other different imaging techniques.…”

Section: Introductionmentioning

confidence: 99%

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Hierarchical electrospun tendon‐ligament bioinspired scaffolds induce changes in fibroblasts morphology under static and dynamic conditions

Sensini

Cristofolini

Zucchelli

et al. 2019

Journal of Microscopy

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Summary The regeneration of injured tendons and ligaments is challenging because the scaffolds needs proper mechanical properties and a biomimetic morphology. In particular, the morphological arrangement of scaffolds is a key point to drive the cells growth to properly regenerate the collagen extracellular matrix. Electrospinning is a promising technique to produce hierarchically structured nanofibrous scaffolds able to guide cells in the regeneration of the injured tissue. Moreover, the dynamic stretching in bioreactors of electrospun scaffolds had demonstrated to speed up cell shape modifications in vitro. The aim of the present study was to combine different imaging techniques such as high‐resolution X‐ray tomography (XCT), scanning electron microscopy (SEM), fluorescence microscopy and histology to investigate if hierarchically structured poly (L‐lactic acid) and collagen electrospun scaffolds can induce morphological modifications in human fibroblasts, while cultured in static and dynamic conditions. After 7 days of parallel cultures, the results assessed that fibroblasts had proliferated on the external nanofibrous sheath of the static scaffolds, elongating themselves circumferentially. The dynamic cultures revealed a preferential axial orientation of fibroblasts growth on the external sheath. The aligned nanofibre bundles inside the hierarchical scaffolds instead, allowed a physiological distribution of the fibroblasts along the nanofibre direction. Inside the dynamic scaffolds, cells appeared thinner compared with the static counterpart. This study had demonstrated that hierarchically structured electrospun scaffolds can induce different fibroblasts morphological modifications during static and dynamic conditions, modifying their shape in the direction of the applied loads. Lay Description To enhance the regeneration of injured tendons and ligaments cells need to growth on dedicated structures (scaffolds) with mechanical properties and a fibrous morphology similar to the natural tissue. In particular, the morphological organisation of scaffolds is fundamental in leading cells to colonise them, regenerating the collagen extracellular matrix. Electrospinning is a promising technique to produce fibres with a similar to the human collagen fibres, suitable to design complex scaffolds able to guide cells in the reconstruction of the natural tissue. Moreover, it is well established that the cyclic stretching of these scaffolds inside dedicated systems called bioreactors, can speed up cells growth and their shape modification. The aim of the present study was to investigate how hierarchically structured electrospun scaffolds, made of resorbable material such as poly(L‐lactic acid) and collagen, could induce morphological changes in human fibroblasts, while cultured during static and dynamic conditions. These scaffolds were composed by an external electrospun membrane that grouped inside it a ring‐shaped bundle, made of axially aligned nanofibres, resembling the morphological arrangement of tendon and ligament tis...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Hierarchical electrospun tendon‐ligament bioinspired scaffolds induce changes in fibroblasts morphology under static and dynamic conditions

Sensini

Cristofolini

Zucchelli

et al. 2019

Journal of Microscopy

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“…Efi mov 1 возможности применения этого метода для иссле-дований биосовместимых матриксов и клеточно-инженерных конструкций [4,5]. Метод СЭМ/ФИП позволяет выполнять наномасштабную реконструк-цию структуры различных материалов.…”

Section: Three-dimensional Analysis Of Micro-and Nanostructure Of Biounclassified

Three-dimensional analysis of micro- and nanostructure of biomaterials and cells by method of scanning probe nanotomography

Ефимов

Agapova

Safonova

et al. 2018

RJTAO

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1 Лаборатория бионанотехнологий, ФГБУ «Национальный медицинский исследовательский центр трансплантологии и искусственных органов имени академика В.И. Шумакова» Минздрава России, Москва, Российская Федерация 2 Биологический факультет МГУ им. М.В. Ломоносова, Москва, Российская Федерация Цель. Провести трехмерный анализ микро-и наноструктуры и количественных морфологических па-раметров альгинатных сферических клеточных микроносителей и макроносителей на основе регенери-рованного шелка, модифицированных микрочастицами межклеточного матрикса децеллюляризованной печени крысы, а также клеток гепатокарциномы человека HepG2, адгезированных на микро-и макро-носители. Материалы и методы. Трехмерные пористые матриксы из регенерированного шелка, полу-ченные методом выщелачивания, и сферические альгинатные микроносители, полученные методом ин-капсуляции, были витализированы клетками гепатокарциномы человека HepG2. Изучение трехмерной структуры клеток и микро-и макроносителей производилось при температуре -120 °С методом ска-нирующей зондовой крионанотомографии при помощи экспериментальной установки, объединяющей криоультрамикротом и сканирующий зондовый микроскоп. Результаты. Получены трехмерные нано-томографические реконструкции клеток HepG2, адгезированных на стенку макропоры матрикса из ре-генерированного шелка и на сферический альгинатный микроноситель. Определены морфологические параметры поверхностей макро-и микроносителей и адгезированных клеток: средняя шероховатость, удельная эффективная площадь и длина автокорреляции. Установлено, что средняя шероховатость по-верхности альгинатных микроносителей составляет 76,4 ± 7,5 нм, а средняя шероховатость поверхности стенки макроносителя на основе регенерированного шелка -133,8 ± 16,2 нм, в то время как средняя шероховатость внешних поверхностей клеток, адгезированных на микро-и макроносители, составляет 118,5 ± 9,0 и 158,8 ± 21,6 нм соответственно. Получены трехмерные реконструкции внутриклеточных компартментов размером от 140 до 500 нм. Выводы. Полученные в результате исследования количест-венные характеристики морфологии поверхностей клеточных носителей и адгезированных на них клеток позволяют исследовать корреляцию морфологических параметров поверхностей клеточных носителей и их биологические свойства. Использование метода сканирующей зондовой крионанотомографии для трехмерного анализа структуры и характеристик биоматериалов, клеток и биоискусственных клеточных конструкций позволит повысить эффективность разработок по созданию новых клеточно-инженерных конструкций с заданными физико-химическими и биологическими характеристиками для задач тканевой инженерии и регенеративной медицины.Ключевые слова : альгинат, фиброин, биоматериалы, клетки HepG2, биосовместимые матриксы, сканирущая зондовая нанотомография.

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“…X-ray computed nanotomography (nCT) imaging is a technique enabling a 3D investigation of scaffold's structure and thus a quantification of cell's and scaffold's characteristics such as their size, 3D distribution, porosity, thickness etc. [4,5].In the present study, the collagen scaffold structure was observed using a nCT RIGAKU Nano3DX and a SEM Tescan MIRA3. These two machines have completely different geometrical arrangements.…”

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confidence: 93%

Correlation of X-ray Computed Nanotomography and Scanning Electron Microscopy Imaging of Collagen Scaffolds

et al. 2018

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Many tissue engineering approaches involve an application of 3D scaffolds. Numerous scaffolds produced from a variety of biomaterials (collagen, chitosan, elastin, etc.) are used in many fields to regenerate different tissues and organs such as bones [1] and for cartilage and cardiovascular repair [2]. As an example, the collagen is a popular extracellular matrix of molecules of connective tissues because of its abundance, biodegradability, and biocompatibility [3]. The morphology of porous scaffolds is usually observed using scanning electron microscopy (SEM). X-ray computed nanotomography (nCT) imaging is a technique enabling a 3D investigation of scaffold's structure and thus a quantification of cell's and scaffold's characteristics such as their size, 3D distribution, porosity, thickness etc. [4,5].In the present study, the collagen scaffold structure was observed using a nCT RIGAKU Nano3DX and a SEM Tescan MIRA3. These two machines have completely different geometrical arrangements. To be able to locate the same region of interest of the sample in both nCT and SEM measurements, a sample holder compatible with nCT and SEM had to be manufactured. Consequently, the sample didn't have to be removed from the holder during measurements on either machine. The sample holder was designed as a rounded bar split in two parts (Fig. 1). The upper part (12 mm height) allows fastening the sample to the MIRA3, whereas the entire sample holder allows fastening the sample to the Nano3DX (Fig. 2). For an easy dismantling, the upper part is connected by M2 set screw and can be demounted easily by bare hands. Both parts of the holder are made of stainless steel grade 1.4305 (AISI 303). This grade was chosen for its good temperature stability and excellent machinability.To locate precisely the same regions of interest, e.g. cells on scaffold, it is advantageous that the marker is placed on a sample and easily recognizable in both measurement methods. A test of several markers was performed to select the best one for both nCT and SEM. Tested materials were red and silver nail polish, carbon tape, carbon tape with pieces of aluminium foil, and handle tape. All of these were applied to the collagen scaffold and imaged with a light microscope (LM), SEM and nCT (Fig. 3). nCT data were processed in VGSTUDIO MAX 3.1 software and the markers were segmented and rendered in 3D (Fig. 3 bottom left). For the comparison of 3D nCT and 2D LM and SEM, the surface of the sample was rendered in the nCT data to obtain a similar view as in the SEM and LM.All markers containing metals (aluminium foil, handle tape) caused noticeable metal artefacts, which strongly decreased quality of nCT data (Fig. 3, nCT slice 2). Carbon tape does not have such a contrast at SEM as nail polish. Eventually, silver nail polish was chosen as the best marker, because of its easy detection and no artefacts in both nCT and SEM.The proper matching of SEM and nCT data by the use of a sample holder and a suitable marker allows to confirm the position of the cells within t...

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3D X-Ray Nanotomography of Cells Grown on Electrospun Scaffolds

Cited by 24 publications

References 44 publications

Hierarchical electrospun tendon‐ligament bioinspired scaffolds induce changes in fibroblasts morphology under static and dynamic conditions

Hierarchical electrospun tendon‐ligament bioinspired scaffolds induce changes in fibroblasts morphology under static and dynamic conditions

Three-dimensional analysis of micro- and nanostructure of biomaterials and cells by method of scanning probe nanotomography

Correlation of X-ray Computed Nanotomography and Scanning Electron Microscopy Imaging of Collagen Scaffolds

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