Injectable and Crosslinkable PLGA‐Based Microribbons as 3D Macroporous Stem Cell Niche

Poly(lactide‐co‐glycolide) (PLGA) has been widely used as a tissue engineering scaffold. However, conventional PLGA scaffolds are not injectable, and do not support direct cell encapsulation, leading to poor cell distribution in 3D. Here, a method for fabricating injectable and intercrosslinkable PLGA microribbon‐based macroporous scaffolds as 3D stem cell niche is reported. PLGA is first fabricated into microribbon‐shape building blocks with tunable width using microcontact printing, then coated with fibrinogen to enhance solubility and injectability using aqueous solution. Upon mixing with thrombin, firbornogen‐coated PLGA microribbons can intercrosslink into 3D scaffolds. When subject to cyclic compression, PLGA microribbon scaffolds exhibit great shock‐absorbing capacity and return to their original shape, while conventional PLGA scaffolds exhibit permanent deformation after one cycle. Using human mesenchymal stem cells (hMSCs) as a model cell type, it is demonstrated that PLGA μRB scaffolds support homogeneous cell encapsulation, and robust cell spreading and proliferation in 3D. After 28 days of culture in osteogenic medium, hMSC‐seeded PLGA μRB scaffolds exhibit an increase in compressive modulus and robust bone formation as shown by staining of alkaline phosphatase, mineralization, and collagen. Together, the results validate PLGA μRBs as a promising injectable, macroporous, non‐hydrogel‐based scaffold for cell delivery and tissue regeneration applications.     

3D Scaffolds to Study Basic Cell Biology

Mimicking the properties of the extracellular matrix is crucial for developing in vitro models of the physiological microenvironment of living cells. Among other techniques, 3D direct laser writing (DLW) has emerged as a promising technology for realizing tailored 3D scaffolds for cell biology studies. Here, results based on DLW addressing basic biological issues, e.g., cell‐force measurements and selective 3D cell spreading on functionalized structures are reviewed. Continuous future progress in DLW materials engineering and innovative approaches for scaffold fabrication will enable further applications of DLW in applied biomedical research and tissue engineering.     

Poly(l-lactide-co-glycolide) biodegradable microfibers and electrospun nanofibers for nerve tissue engineering: an in vitro study

For tissue engineering applications, the distribution and growth of cells on a scaffold are key requirements. The potential of biodegradable poly(l-lactide-co-glycolide) (PLGA) polymer with different microstructures, as scaffolds for nerve tissue engineering was investigated. In this study, an attempt was made to develop porous nanofibrous scaffolds by the electrospinning method. In this process, polymer fibers with diameters in the nanometer range are formed by subjecting a polymer fluid jet to a high electric field. Attempt was also made to develop microbraided and aligned microfiber scaffolds. A polymer film scaffold was made by solvent casting method. C17.2 nerve stem cells were seeded and cultured on all the four different types of scaffolds under static conditions for 3 days. Scanning electron micrographs showed that the nerve stem cells adhered and differentiated on all the scaffolds and supported neurite outgrowth. Interesting observation was seen in the aligned microfiber scaffolds, where the C17.2 nerve stem cells attached and differentiated along the direction of the fibers. The size and shape of the cell-polymer constructs remained intact. The present study suggests that PLGA is a potential scaffold for nerve tissue engineering and predicts the orientation and growth of nerve stem cells on the scaffold.     

Fabrication of hierarchical polycaprolactone/gel scaffolds via combined 3D bioprinting and electrospinning for tissue engineering

It is a severe challenge to construct 3D scaffolds which hold controllable pore structure and similar morphology of the natural extracellular matrix(ECM).In this study,a compound technology is proposed by combining the 3D bioprinting and electrospinning process to fabricate 3D scaffolds,which are composed by orthogonal array gel microfibers in a grid-like arrangement and intercalated by a nonwoven structure with randomly distributed polycaprolactone(PCL) nanofibers.Human adiposederived stem cells(hASCs) are seeded on the hierarchical scaffold and cultured 21 d for in vitro study.The results of cells culturing show that the microfibers structure with controlled pores can allow the easy entrance of cells and the efficient diffusion of nutrients,and the nanofiber webs layered in the scaffold can significantly improve initial cell attachment and proliferation.The present work demonstrates that the hierarchical PCL/gel scaffolds consisting of controllable 3D architecture with interconnected pores and biomimetic nanofiber structures resembling the ECM can be designed and fabricated by the combination of 3D bioprinting and electrospinning to improve biological performance in tissue engineering applications.     

Probing the pH Microenvironment of Mesenchymal Stromal Cell Cultures on Additive‐Manufactured Scaffolds

Despite numerous advances in the field of tissue engineering and regenerative medicine, monitoring the formation of tissue regeneration and its metabolic variations during culture is still a challenge and mostly limited to bulk volumetric assays. Here, a simple method of adding capsules‐based optical sensors in cell‐seeded 3D scaffolds is presented and the potential of these sensors to monitor the pH changes in space and time during cell growth is demonstrated. It is shown that the pH decreased over time in the 3D scaffolds, with a more prominent decrease at the edges of the scaffolds. Moreover, the pH change is higher in 3D scaffolds compared to monolayered 2D cell cultures. The results suggest that this system, composed by capsules‐based optical sensors and 3D scaffolds with predefined geometry and pore architecture network, can be a suitable platform for monitoring pH variations during 3D cell growth and tissue formation. This is particularly relevant for the investigation of 3D cellular microenvironment alterations occurring both during physiological processes, such as tissue regeneration, and pathological processes, such as cancer evolution.     

Multiphoton Direct Laser Writing and 3D Imaging of Polymeric Freestanding Architectures for Cell Colonization

The realization of 3D architectures for the study of cell growth, proliferation, and differentiation is a task of fundamental importance for both technological and biological communities involved in the development of biomimetic cell culture environments. Here we report the fabrication of 3D freestanding scaffolds, realized by multiphoton direct laser writing and seeded with neuroblastoma cells, and their multitechnique characterization using advanced 3D fluorescence imaging approaches. The high accuracy of the fabrication process (≈200 nm) allows a much finer control of the micro‐ and nanoscale features compared to other 3D printing technologies based on fused deposition modeling, inkjet printing, selective laser sintering, or polyjet technology. Scanning electron microscopy (SEM) provides detailed insights about the morphology of both cells and cellular interconnections around the 3D architecture. On the other hand, the nature of the seeding in the inner core of the 3D scaffold, inaccessible by conventional SEM imaging, is unveiled by light sheet fluorescence microscopy and multiphoton confocal imaging highlighting an optimal cell colonization both around and within the 3D scaffold as well as the formation of long neuritic extensions. The results open appealing scenarios for the use of the developed 3D fabrication/3D imaging protocols in several neuroscientific contexts.     

3D打印HA微球支架的制备与表征

生物材料表面微结构对于成骨具有重要的影响,该研究以不同粒径(<60μm)的羟基磷灰石(HA)微球状粉体为原料,通过3D打印技术制备了一系列(HA0、HA10、HA30、HA50)生物陶瓷支架。不同支架具有相似的理化性能,由于微球粒径不同形成了不同的微结构,对其生物学性能造成不同的影响。相比传统非微球颗粒打印的支架(HA0), HA微球构成的支架能够提供更多细胞粘附和生长位点, 24 h的粘附实验显示HA30支架能显著促进骨髓间充质干细胞的伪足伸长;培养5 d的细胞增殖实验显示,微球支架上的细胞数量与HA0支架出现显著性差异,表面微球结构与细胞尺度相当的HA30支架具有最好的促增殖效果。因此,3D打印技术在可控制备HA支架宏观结构的同时,还可以通过控制生物陶瓷粉体的颗粒形貌,调控3D打印支架的表面微结构,从而优化其生物学效应,在骨组织工程领域具有良好的应用前景。     

Performance of evacuated calcium phosphate microcarriers loaded with mesenchymal stem cells within a rat calvarium defect

Tissue engineering of stem cells in concert with 3-dimensional (3D) scaffolds is a promising approach for regeneration of bone tissues. Bioactive ceramic microspheres are considered effective 3D stem cell carriers for bone tissue engineering. Here we used evacuated calcium phosphate (CaP) microspheres as the carrier of mesenchymal stem cells (MSCs) derived from rat bone marrow. The performance of the CaP-MSCs construct in bone formation within a rat calvarium defect was evaluated. MSCs were first cultured in combination with the evacuated microcarriers for 7?days in an osteogenic medium, which was then implanted in the 6?mm-diameter calvarium defect for 12?weeks. For comparison purposes, a control defect and cell-free CaP microspheres were also evaluated. The osteogenic differentiation of MSCs cultivated in the evacuated CaP microcarriers was confirmed by alkaline phosphatase staining and real time polymerase chain reaction. The in vivo results confirmed the highest bone formation was attained in the CaP microcarriers combined with MSCs, based on microcomputed tomography and histological assays. The results suggest that evacuated CaP microspheres have the potential to be useful as stem cell carriers for bone tissue engineering.     

Effect of solid freeform fabrication-based polycaprolactone/poly(lactic-co-glycolic acid)/collagen scaffolds on cellular activities of human adipose-derived stem cells and rat primary hepatocytes

Highly biocompatible polycaprolactone (PCL)/poly(lactic-co-glycolic acid) (PLGA)/collagen scaffolds in which the PCL/PLGA collagen solution was selectively dispensed into every other space between the struts were fabricated using solid freeform fabrication (SFF) technology, as we described previously. The objective of this study was to evaluate and compare the PCL/PLGA/collagen scaffolds (group 3) with PCL/PLGA-only scaffolds (group 1) and PCL/PLGA scaffolds with collagen by the dip-coating method (group 2) using human adipose-derived stem cells (hASCs) and rat primary hepatocytes. The selectively dispensed collagen formed a three-dimensional (3D) network of nanofibers in group 3, as observed by scanning electron microscopy. The compressive strength and modulus of group 3 were approximately 140 and 510 times higher, respectively, than those of a sponge-type collagen scaffold whose weak mechanical properties were regarded as a critical drawback. Proliferation and osteogenic differentiation of hASCs were promoted significantly in group 3 compared to groups 1 and 2. In addition, we found that the viability and albumin secretion ability of rat primary hepatocytes were highly retained for 10 days in group 3 but not group 1. Interestingly, hepatocyte aggregation, which enhances hepatic function through cell–cell interactions, was observed particularly in group 3. In conclusion, group 3, in which the collagen was selectively dispensed in the 3D space of the porous PCL/PLGA framework, will be a promising 3D scaffold for culturing various cell types.     

Investigation of cancer cell behavior on nanofibrous scaffolds

Tissue engineering and the use of nanofibrous biomaterial scaffolds offer a unique perspective for studying cancer development in vitro. Current in vitro models of tumorigenesis are limited by the use of static, two-dimensional (2D) cell culture monolayers that lack the structural architecture necessary for cell-cell interaction and three-dimensional (3D) scaffolds that are too simplistic for studying basic pathological mechanisms. In this study, two nanofibrous biomaterials that mimic the structure of the extracellular matrix, bacterial cellulose and electrospun polycaprolactone (PCL)/collagen I, were investigated as potential 3D scaffolds for an in vitro cancer model. Multiple cancer cell lines were cultured on each scaffold material and monitored for cell viability, proliferation, adhesion, infiltration, and morphology. Both bacterial cellulose and electrospun PCL/collagen I, which have nano-scale structures on the order of 100-500 nm, have been used in many diverse tissue engineering applications. Cancer cell adhesion and growth were limited on bacterial cellulose, while all cellular processes were enhanced on the electrospun scaffolds. This initial analysis has demonstrated the potential of electrospun PCL/collagen I scaffolds toward the development of an improved 3D in vitro cancer model.     

Ultrafine fibrous gelatin scaffolds with deep cell infiltration mimicking 3D ECMs for soft tissue repair

In this research, ultrafine fibrous scaffolds with deep cell infiltration and sufficient water stability have been developed from gelatin, aiming to mimic the extracellular matrices (ECMs) as three dimensional (3D) stromas for soft tissue repair. The ultrafine fibrous scaffolds produced from the current technologies of electrospinning and phase separation are either lack of 3D oriented fibrous structure or too compact to be penetrated by cells. Whilst electrospun scaffolds are able to emulate two dimensional (2D) ECMs, they cannot mimic the 3D ECM stroma. In this work, ultralow concentration phase separation (ULCPS) has been developed to fabricate gelatin scaffolds with 3D randomly oriented ultrafine fibers and loose structures. Besides, a non-toxic citric acid crosslinking system has been established for the ULCPS method. This system could endow the scaffolds with sufficient water stability, while maintain the fibrous structures of scaffolds. Comparing with electrospun scaffolds, the ULCPS scaffolds showed improved cytocompatibility and more importantly, cell infiltration. This research has proved the possibility of using gelatin ULCPS scaffolds as the substitutes of 3D ECMs.     

Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF–Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF–Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.     

Towards ready-to-use 3-D scaffolds for regenerative medicine: adhesion-based cryopreservation of human mesenchymal stem cells attached and spread within alginate–gelatin cryogel scaffolds

Cultivation and proliferation of stem cells in three-dimensional (3-D) scaffolds is a promising strategy for regenerative medicine. Mesenchymal stem cells with their potential to differentiate in various cell types, cryopreserved adhesion-based in fabricated scaffolds of biocompatible materials can serve as ready-to-use transplantation units for tissue repair, where pores allow a direct contact of graft cells and recipient tissue without further preparation. A successful cryopreservation of adherent cells depends on attachment and spreading processes that start directly after cell seeding. Here, we analyzed different cultivation times (0.5, 2, 24 h) prior to adhesion-based cryopreservation of human mesenchymal stem cells within alginate–gelatin cryogel scaffolds and its influence on cell viability, recovery and functionality at recovery times (0, 24, 48 h) in comparison to non-frozen control. Analysis with confocal laser scanning microscopy and scanning electron microscopy indicated that 2 h cultivation time enhanced cryopreservation success: cell number, visual cell contacts, membrane integrity, motility, as well as spreading were comparable to control. In contrast, cell number by short cultivation time (0.5 h) reduced dramatically after thawing and expanded cultivation time (24 h) decreased cell viability. Our results provide necessary information to enhance the production and to store ready-to-use transplantation units for application in bone, cartilage or skin regenerative therapy.     

Stem cell homing-based tissue engineering using bioactive materials

Tissue engineering focuses on repairing tissue and restoring tissue functions by employing three elements: scaffolds, cells and biochemical signals. In tissue engineering, bioactive material scaffolds have been used to cure tissue and organ defects with stem cell-based therapies being one of the best documented approaches. In the review, different biomaterials which are used in several methods to fabricate tissue engineering scaffolds were explained and show good properties (biocompatibility, biodegradability, and mechanical properties etc.) for cell migration and infiltration. Stem cell homing is a recruitment process for inducing the migration of the systemically transplanted cells, or host cells, to defect sites. The mechanisms and modes of stem cell homing-based tissue engineering can be divided into two types depending on the source of the stem cells: endogenous and exogenous. Exogenous stem cell-based bioactive scaffolds have the challenge of long-term culturing in vitro and for endogenous stem cells the biochemical signal homing recruitment mechanism is not clear yet. Although the stem cell homing-based bioactive scaffolds are attractive candidates for tissue defect therapies, based on in vitro studies and animal tests, there is still a long way before clinical application.     

Electrospun nanofibers: Work for medicine?

Attempts have been made to fabricate nanofibrous scaffolds to mimic the chemical composition and structural properties of the extracellular matrix (ECM) for tissue/organ replacement. Nanofiber scaffolds with various patterns have been successfully produced from synthetic and natural polymers through a relatively simple technique of electrospinning. The resulting patterns can mimic some of the diverse tissue-specific orientation and three-dimensional (3D) fibrous structures. Studies on cell-nanofiber interactions, including studies on stem cells, have revealed the importance of nanotopography on cell adhesion, proliferation and differentiation. Furthermore, clinical application of electrospun nanofibers including wound healing, tissue regeneration, drug delivery and stem cell therapy are highly feasible due to the ease and flexibility of fabrication of making nanofiber with this cost-effective method using electrospinning. In this review, we have highlighted the current state of the art and provided future perspectives on electrospun nanofiber in medical applications.     

Fabrication and evaluation of biomimetic scaffolds by using collagen–alginate fibrillar gels for potential tissue engineering applications

Pore architecture and its stable functionality under cell culturing of three dimensional (3D) scaffolds are of great importance for tissue engineering purposes. In this study, alginate was incorporated with collagen to fabricate collagen–alginate composite scaffolds with different collagen/alginate ratios by lyophilizing the respective composite gels formed via collagen fibrillogenesis in vitro and then chemically crosslinking. The effects of alginate amount and crosslinking treatment on pore architecture, swelling behavior, enzymatic degradation and tensile property of composite scaffolds were systematically investigated. The relevant results indicated that the present strategy was simple but efficient to fabricate highly interconnected strong biomimetic 3D scaffolds with nanofibrous surface. NIH3T3 cells were used as a model cell to evaluate the cytocompatibility, attachment to the nanofibrous surface and porous architectural stability in terms of cell proliferation and infiltration within the crosslinked scaffolds. Compared with the mechanically weakest crosslinked collagen sponges, the cell-cultured composite scaffolds presented a good porous architecture, thus permitting cell proliferation on the top surface as well as infiltration into the inner part of 3D composite scaffolds. These composite scaffolds with pore size ranging from 150 to 300 μm, over 90% porosity, tuned biodegradability and water-uptake capability are promising for tissue engineering applications.     

Evaluation of synovium-derived mesenchymal stem cells and 3D printed nanocomposite scaffolds for tissue engineering

Abstract

Stem cells and scaffolds play a very important role in tissue engineering. Here, we isolated synovium-derived mesenchymal stem cells (SMSCs) from synovial membrane tissue and characterized stem-cell properties. Gelatin nanoparticles (NP) were prepared using a two-step desolvation method and then pre-mixed into different host matrix (silk fibroin (SF), gelatin (Gel), or SF–Gel mixture) to generate various 3D printed nanocomposite scaffolds (NP/SF, NP/SF–Gel, NP/Gel-1, and NP/Gel-2). The microstructure was examined by scanning electron microscopy. Biocompatibility assessment was performed through CCK-8 assay by coculturing with SMSCs at 1, 3, 7 and 14 days. According to the results, SMSCs are similar to other MSCs in their surface epitope expression, which are negative for CD45 and positive for CD44, CD90, and CD105. After incubation in lineage-specific medium, SMSCs could differentiate into chondrocytes, osteocytes and adipocytes. 3D printed nanocomposite scaffolds exhibited a good biocompatibility in the process of coculturing with SMSCs and had no negative effect on cell behavior. The study provides a strategy to obtain SMSCs and fabricate 3D printed nanocomposite scaffolds, the combination of which could be used for practical applications in tissue engineering.     

The Regulatory Functionality of Exosomes Derived from hUMSCs in 3D Culture for Alzheimer's Disease Therapy

Reducing amyloid‐β (Aβ) accumulation could be a potential therapeutic approach for Alzheimer's disease (AD). Particular functional biomolecules in exosomes vested by the microenvironment in which the original cells resided can be transferred to recipient cells to improve pathological conditions. However, there are few reports addressing whether exosomes derived from cells cultured on scaffolds with varying dimension can reduce Aβ deposition or ameliorate cognitive decline for AD therapy. Herein, both 3D graphene scaffold and 2D graphene film are used as the matrix for human umbilical cord mesenchymal stem cell culture, from which the supernatants are obtained to isolate exosomes. The levels of 195 kinds of miRNAs and proteins, including neprilysin, insulin‐degrading enzyme and heat shock protein 70, in 3D‐cultured exosomes (3D‐Exo) are dramatically different from those obtained from 2D culture. Hence, 3D‐Exo could up‐regulate the expression of α‐secretase and down‐regulate the β‐secretase to reduce Aβ production in both AD pathology cells and transgenic mice, through their special cargo. With rescuing Aβ pathology, 3D‐Exo exerts enhanced therapeutic effects on ameliorating the memory and cognitive deficits in AD mice. These findings provide a novel clinical application for scaffold materials and functional exosomes derived from stem cells.     

Novel porous scaffolds of poly(lactic acid) produced by phase-separation using room temperature ionic liquid and the assessments of biocompatibility

Here we prepared three-dimensional (3D) porous-structured biodegradable polymer scaffolds for tissue regeneration using room temperature ionic liquids (RTILs) as a novel porogen, and addressed their biological properties, including in vitro cell growth and differentiation and in vivo tissue compatibility. RTIL based on 1-butyl-3-methylimidazolium ([bmim]) bearing hydrophilic anion Cl was introduced within the polymer structure to provide a pore network. A mixture of poly(lactic acid) (PLA) with RTIL dissolved in an organic solvent formed a bi-continuous network during the drying process. Selective dissolution of the RTIL phase was facilitated in ethanol, which resulted in a porous network of the polymer phase with complete removal of the RTIL. The RTILs-assisted porous scaffolds showed a typical open-channeled network with pore sizes over 100 μm and porosities of about 86–94%. For the biocompatibility assessments of the scaffolds, mesenchymal stem cells (MSCs) derived from rat bone marrow were seeded onto the PLA scaffold, and the cell proliferation and osteoblastic differentiation behaviors were examined. Results showed a typical on-going increase in the cell population with a level comparable to that observed on the tissue culture plastic control, indicating good cell compatibility. When cultured in an osteogenic medium, the alkaline phosphatase (ALP) activity of the cells on the PLA scaffolds was stimulated to increase with time from 7 to 14 days, in a similar manner to that on the control. Moreover, the expression of genes related to osteoblasts, including collagen type I, osteocalcin and bone sialoprotein, was stimulated on the 3D PLA scaffold during culture for up to 14 days, with levels higher than those on the control, suggesting the developed scaffold provided a 3D matrix condition for osteogenesis. An in vivo pilot study conducted subcutaneously in rat for 4 weeks revealed good tissue compatibility of the scaffold, with the ingrowth of cells and formation of collageneous tissue around and deep within the pores of the scaffold and no significant inflammatory reaction. Taken together, this novel method of using RTILs as a pore generator is considered to be useful in the development of biocompatible porous polymer scaffolds for tissue regeneration.     

A study of the cellular response to orientated fibronectin material in healing extensor rat tendon

3D orientated fibronectin (Fn) mats have been used as biocompatible and biodegradeable scaffolds to provide orientated cues using contact guidance for cell migration/adhesion and deposition of extracellular matrix. We have implanted Fn scaffolds in an established rat tendon(partial tenotomy) injury model to test its efficacy and monitor the early cellular and inflammatory response. Tendons were harvested at 0, 6 h, 1, 3, 5, 7 and 14 days for H&E, immunohistochemistry and TEM. Total cell counts within the window increased progressively with time with no significant differences between the Fn scaffolds and controls. CD45 (pan leukocyte) positive cell numbers peaked at 6 h and when expressed as a percentage of total cell counts as determined by H&E staining constituted 20% of the total cell number at 6 h but decreased to 5% of total number by 72 h. There were no significant differences in the inflammatory response between the control and implanted groups. Few CD44 (mesenchymal stem cell) positive cells identified had a surface location. A novel cell with long exaggerated cytoplasmic processes was identified by TEM. Our results show that the Fn scaffold did not degrade or elicit any untoward inflammatory response at the time points tested and has potential use in guiding the repair process.