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The control of stem cell morphology and differentiation using three-dimensional printed scaffold architecture.

ABSTRACT: In this work, we investigated the interactions of human mesenchymal stem cells (hMSCs) with three-dimensional (3D) printed scaffolds displaying different scaffold architectures. Pressure-assisted microsyringe system was used to fabricate scaffolds with square (SQR), hexagonal (HEX), and octagonal (OCT) architectures defined by various degrees of curvatures. OCT represents the highest degree of curvature followed by HEX, and SQR is composed of linear struts without curvature. Scaffolds were fabricated from poly(L-lactic acid) and poly(tyrosol carbonate). We found that hMSCs attached and spread by taking the shape of the individual struts, exhibiting high aspect ratios (ARs) and mean cell area when cultured on OCT scaffolds as compared with those cultured on HEX and SQR scaffolds. In contrast, cells appeared bulkier with low AR on SQR scaffolds. These significant changes in cell morphology directly correlate with the stem cell lineage commitment, such that 80 ± 1% of the hMSCs grown on OCT scaffolds differentiated into osteogenic lineage, compared with 70 ± 4% and 62 ± 2% of those grown on HEX and SQR scaffolds, respectively. Cells on OCT scaffolds also showed 2.5 times more alkaline phosphatase activity compared with cells on SQR scaffolds. This study demonstrates the importance of scaffold design to direct stem cell differentiation, and aids in the development of novel 3D scaffolds for bone regeneration.

SUBMITTER: Guvendiren M

PROVIDER: S-EPMC6561507 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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The control of stem cell morphology and differentiation using three-dimensional printed scaffold architecture.

Guvendiren Murat M Fung Stephanie S Kohn Joachim J De Maria Carmelo C Montemurro Francesca F Vozzi Giovanni G

MRS communications 20170829 3

In this work, we investigated the interactions of human mesenchymal stem cells (hMSCs) with three-dimensional (3D) printed scaffolds displaying different scaffold architectures. Pressure-assisted microsyringe system was used to fabricate scaffolds with square (SQR), hexagonal (HEX), and octagonal (OCT) architectures defined by various degrees of curvatures. OCT represents the highest degree of curvature followed by HEX, and SQR is composed of linear struts without curvature. Scaffolds were fabri ...[more]

PMID: 31192033

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Project description:Background/objectiveA novel porous scaffold poly (lactide-co-glycolide) and tricalcium phosphate (PLGA/TCP) was developed by three-dimensional printing technology for bone defect repair. As a Class 2 solvent with less severe toxicity, content of residual 1,4-dioxane in this newly developed scaffold should be rigorously controlled when it is translated to clinical use. In this study, a headspace gas chromatography-mass spectrometric (HS-GC-MS) method and related testing protocol were developed for quantitative determination of 1,4-dioxane in the PLGA/TCP composite scaffolds.MethodsMatrix effect analysis was used to optimise the pretreatment method of the scaffolds. Then, the procedure for testing 1,4-dioxane using HS-GC-MS was set up. The accuracy, precision, and robustness of this newly developed quantitative method were also validated before quantification of 1,4-dioxane in the scaffolds with different drying procedures.ResultsDimethyl formamide (DMF) was the optimal solvent for dissolving scaffolds for GC-MS with proper sensitivity and without matrix effect. Then, the optimised procedure was determined as: the scaffolds were dissolved in DMF and kept at 90°C for 40 minutes, separated on a HP-5MS column, and detected by mass spectroscopy. Recovery experiments gave 97.9-100.7% recovery for 1,4-dioxane. The linear range for 1,4-dioxane was determined as 1-40 ppm with linear correlation coefficient ≥ 0.9999. Intraday and interday precision was determined as being within relative standard deviation of below 0.68%. The passable drying procedure was related to lyophilising (-50°C, 50 Pa) the scaffolds for 2 days and drying in vacuum (50 Pa) for 7 days.ConclusionThis is the first quantitative method established to test 1,4-dixoane in a novel scaffold. This method was validated with good accuracy and reproducibility, and met the methodological requirements of the Guideline 9101 documented in the Chinese Pharmacopoeia 2015 Edition.The translational potential of this articleThis quantitative method for determination of residual 1,4-dioxane in the novel scaffolds is a key technical method during its translation into clinical use because this method is an important and indispensable file in the enterprise standard when the porous scaffold is registered as a Class III implanted medical device for bone defect repair, which is used to guarantee the safety of the scaffolds. It is also applied to optimise the drying process of scaffolds and to monitor the quality of scaffolds in the industrialisation process. Further, this method provides references for other solvents quantitative determination in porous scaffolds or materials.

Project description:ObjectiveApplications of biological scaffolds for regenerative medicine are increasing. Such scaffolds improve cell attachment, migration, proliferation and differentiation. In the current study decellularised mouse whole testis was used as a natural 3 dimensional (3D) scaffold for culturing spermatogonial stem cells.Materials and methodsIn this experimental study, adult mouse whole testes were decellularised using sodium dodecyl sulfate (SDS) and Triton X-100. The efficiency of decellularisation was determined by histology and DNA quantification. Masson's trichrome staining, alcian blue staining, and immunohistochemistry (IHC) were done for validation of extracellular matrix (ECM) proteins. These scaffolds were recellularised through injection of mouse spermatogonial stem cells in to rete testis. Then, they were cultured for eight weeks. Recellularised scaffolds were assessed by histology, real-time polymerase chain reaction (PCR) and IHC.ResultsHaematoxylin-eosin (H and E) staining showed that the cells were successfully removed by SDS and Triton X-100. DNA content analysis indicated that 98% of the DNA was removed from the testis. This conﬁrmed that our decellularisation protocol was efﬁcient. Masson's trichrome and alcian blue staining respectively showed that glycosaminoglycans (GAGs) and collagen are preserved in the scaffolds. IHC analysis confirmed the preservation of fibronectin, collagen IV, and laminin. MTT assay indicated that the scaffolds were cell-compatible. Histological evaluation of recellularised scaffolds showed that injected cells were settled on the basement membrane of the seminiferous tubule. Analyses of gene expression using real-time PCR indicated that expression of the Plzf gene was unchanged over the time while expression of Sycp3 gene was increased significantly (P=0.003) after eight weeks in culture, suggesting that the spermatogonial stem cells started meiosis. IHC confirmed that PLZF-positive cells (spermatogonial stem cells) and SYCP3-positive cells (spermatocytes) were present in seminiferous tubules.ConclusionSpermatogonial stem cells could proliferate and differentiated in to spermatocytes after being injected in the decellularised testicular scaffolds.

Project description:BACKGROUND:The role of bone tissue engineering in the field of regenerative medicine has been a main research topic over the past few years. There has been much interest in the use of three-dimensional (3D) engineered scaffolds (PLA) complexed with human gingival mesenchymal stem cells (hGMSCs) as a new therapeutic strategy to improve bone tissue regeneration. These devices can mimic a more favorable endogenous microenvironment for cells in vivo by providing 3D substrates which are able to support cell survival, proliferation and differentiation. The present study evaluated the in vitro and in vivo capability of bone defect regeneration of 3D PLA, hGMSCs, extracellular vesicles (EVs), or polyethyleneimine (PEI)-engineered EVs (PEI-EVs) in the following experimental groups: 3D-PLA, 3D-PLA?+?hGMSCs, 3D-PLA?+?EVs, 3D-PLA?+?EVs?+?hGMSCs, 3D-PLA?+?PEI-EVs, 3D-PLA?+?PEI-EVs?+?hGMSCs. METHODS:The structural parameters of the scaffold were evaluated using both scanning electron microscopy and nondestructive microcomputed tomography. Nanotopographic surface features were investigated by means of atomic force microscopy. Scaffolds showed a statistically significant mass loss along the 112-day evaluation. RESULTS:Our in vitro results revealed that both 3D-PLA?+?EVs?+?hGMSCs and 3D-PLA?+?PEI-EVs?+?hGMSCs showed no cytotoxicity. However, 3D-PLA?+?PEI-EVs?+?hGMSCs exhibited greater osteogenic inductivity as revealed by morphological evaluation and transcriptomic analysis performed by next-generation sequencing (NGS). In addition, in vivo results showed that 3D-PLA?+?PEI-EVs?+?hGMSCs and 3D-PLA?+?PEI-EVs scaffolds implanted in rats subjected to cortical calvaria bone tissue damage were able to improve bone healing by showing better osteogenic properties. These results were supported also by computed tomography evaluation that revealed the repair of bone calvaria damage. CONCLUSION:The re-establishing of the integrity of the bone lesions could be a promising strategy in the treatment of accidental or surgery trauma, especially for cranial bones.

Project description:BackgroundCritical bone defects remain challenges for clinicians, which cannot heal spontaneously and require medical intervention. Following the development of three-dimensional (3D) printing technology is widely used in bone tissue engineering for its outstanding customizability. The 3D printed scaffolds were usually accompanied with growth factors, such as bone morphometric protein 2 (BMP-2), whose effects have been widely investigated on bone regeneration. We previously fabricated and investigated the effect of a polylactic acid (PLA) cage/Biogel scaffold as a carrier of BMP-2. In this study, we furtherly investigated the effect of another shape of PLA cage/Biogel scaffold as a carrier of BMP-2 in a rat calvaria defect model and an ectopic ossification (EO) model.MethodThe PLA scaffold was printed with a basic commercial 3D printer, and the PLA scaffold was combined with gelatin and alginate-based Biogel and BMP-2 to induce bone regeneration. The experimental groups were divided into PLA scaffold, PLA scaffold with Biogel, PLA scaffold filled with BMP-2, and PLA scaffold with Biogel and BMP-2 and were tested both in vitro and in vivo. One-way ANOVA with Bonferroni post-hoc analysis was used to determine whether statistically significant difference exists between groups.ResultThe in vitro results showed the cage/Biogel scaffold released BMP-2 with an initial burst release and followed by a sustained slow-release pattern. The released BMP-2 maintained its osteoinductivity for at least 14 days. The in vivo results showed the cage/Biogel/BMP-2 group had the highest bone regeneration in the rat calvarial defect model and EO model. Especially, the bone regenerated more regularly in the EO model at the implanted sites, which indicated the cage/Biogel had an outstanding ability to control the shape of regenerated bone.ConclusionIn conclusion, the 3D printed PLA cage/Biogel scaffold system was proved to be a proper carrier for BMP-2 that induced significant bone regeneration and induced bone formation following the designed shape.

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The control of stem cell morphology and differentiation using three-dimensional printed scaffold architecture.

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The control of stem cell morphology and differentiation using three-dimensional printed scaffold architecture.

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