Three‐Dimensional Scaffolds of Carbonized Polyacrylonitrile for Bone Tissue Regeneration

Carbon‐based materials have been extensively studied for stem cell culture. However, difficulties associated with engineering pure carbon materials into 3D scaffolds have hampered applications in tissue engineering and regenerative medicine. Carbonized polyacrylonitrile (cPAN) could be a promising alternative, as cPAN is a highly ordered carbon isomorph that resembles the graphitic structure and can be easily processed into 3D scaffolds. Despite the notable features of cPAN, application of cPAN in tissue engineering and regenerative medicine have not been explored. This study, for the first time, demonstrates the fabrication of microporous 3D scaffolds of cPAN and excellent osteoinductivity of cPAN, suggesting utility of 3D cPAN scaffolds as synthetic bone graft materials. The combination of excellent processability and unique bioactive properties of cPAN may lead to future applications in orthopedic regenerative medicine.     

Self‐Assembled Peptide‐Based Hydrogels as Scaffolds for Proliferation and Multi‐Differentiation of Mesenchymal Stem Cells

Fluorenyl‐9‐methoxycarbonyl (Fmoc)‐diphenylalanine (Fmoc‐FF) and Fmoc‐arginine‐glycine‐­aspartate (Fmoc‐RGD) peptides self‐assemble to form a 3D network of supramolecular hydrogel (Fmoc‐FF/Fmoc‐RGD), which provides a nanofibrous network that uniquely presents bioactive ligands at the fiber surface for cell attachment. In the present study, mesenchymal stem cells (MSCs) in Fmoc‐FF/Fmoc‐RGD hydrogel increase in proliferation and survival compared to those in Fmoc‐FF/Fmoc‐RGE hydrogel. Moreover, MSCs encapsulated in Fmoc‐FF/Fmoc‐RGD hydrogel and induced in each defined induction medium undergo in vitro osteogenic, adipogenic, and chondrogenic differentiation. For in vivo differentiation, MSCs encapsulated in hydrogel are induced in each defined medium for one week, followed by injection into gelatin sponges and transplantation into immunodeficient mice for four weeks. MSCs in Fmoc‐FF/Fmoc‐RGD hydrogel increase in differentiation into osteogenic, adipogenic, and chondrogenic differentiation, compared to those in Fmoc‐FF/Fmoc‐RGE hydrogel. This study concludes that nanofibers formed by the self‐assembly of Fmoc‐FF and Fmoc‐RGD are suitable for the attachment, proliferation, and multi‐differentiation of MSCs, and can be applied in musculoskeletal tissue engineering.

     

Human Elastin‐Based Recombinant Biopolymers Improve Mesenchymal Stem Cell Differentiation

Elastin‐based polypeptides are a class of smart biopolymers representing an important model in the design of biomaterials. The combination of biomimetic materials with cells that have great plasticity provides a promising strategy for the realization of highly engineered cell‐based constructs for regenerative medicine and tissue repair applications. Two recombinant biopolymers inspired by human elastin are assessed as coating agents to prepare biomimetic surfaces for cell culture. These substrates are assayed for hBM MSC culture. The coated surfaces are also characterized with AFM to evaluate the topographical features of the deposited biopolymers. The results suggest that the elastin‐derived biomimetic surfaces play a stimulatory role on osteogenic differentiation of MSCs.

     

Human Mesenchymal Stem‐Cell Behaviour On Direct Laser Micropatterned Electrospun Scaffolds with Hierarchical Structures

Direct laser machining and electrospinning are utilized to obtain a bi‐layered hybrid scaffold with hierarchical topographical features to mimic extracellular matrix‐like microenvironment of cells. Adult bone marrow derived human mesenchymal stem cells (hMSCs) are cultured in vitro in these hybrid scaffolds, and cell orientation, proliferation, viability, and differentiation are evaluated. The results show that this novel hybrid scaffold not only supports cell growth like traditional scaffolds, but also elicits positive responses from the cells, like lineage commitment and alignment, which are essential features of future scaffolds.

     

Controlling the Poly(ε‐caprolactone) Degradation to Maintain the Stemness and Function of Adipose‐Derived Mesenchymal Stem Cells in Vascular Regeneration Application

Biodegradable poly(ε‐caprolactone) (PCL) scaffolds with adipose‐derived mesenchymal stem cells (ADSCs) have been used in vascular regeneration studies. An evaluation method of the effect of PCL degradation products (DP) on the viability, stemness, and differentiation capacities of ADSCs is established. ADSCs are cultured in medium containing different concentrations of PCL DP before evaluating the effect of PCL DP on the cell apoptosis and proliferation, cell surface antigens, adipogenic and osteogenic differentiation capacities, and capacities to differentiate into endothelial cells and smooth muscle cells. The results demonstrate that PCL DP exceed 0.05 mg mL^?1 may change the stemness and differentiation capacities of ADSCs. Therefore, to control the proper concentration of PCL DP is essential for ADSCs in vascular regeneration application.     

Modifying the Pores of an Inverse Opal Scaffold With Chitosan Microstructures for Truly Three‐Dimensional Cell Culture

Inverse opal scaffolds have recently emerged as a novel class of scaffolds with uniform and controllable pore sizes for tissue engineering to provide better nutrient transport, a uniform cell distribution, and an adjustable microenvironment for cell differentiation. However, when the pore size of the scaffold is much larger than the dimension of a cell, the cell actually encounters a local 2D environment and the void space associated with the pore can not be efficiently utilized. Here, we demonstrate that a truly 3D microenvironment can be created inside a pore by further functionalizing the as‐prepared inverse opal scaffold with a second polymer by freeze‐drying. The resultant inverse opal scaffold with hierarchically structured pores can enhance both cell proliferation and tissue infiltration.     

Viability of Human Mesenchymal Stem Cells Seeded on Crosslinked Entropy‐Elastic Gelatin‐Based Hydrogels

Biomimetic polymer network systems with tailorable properties based on biopolymers represent a class of degradable hydrogels that provides sequences for protein adsorption and cell adhesion. Such materials show potential for in vitro MSC proliferation as well as in vivo applications and were obtained by crosslinking different concentrations of gelatin using varying amounts of ethyl lysine diisocyanate in the presence of a surfactant in pH 7.4 PBS solution. Material extracts, which were tested for cytotoxic effects using L929 mouse fibroblasts, were non‐toxic. The hydrogels were seeded with human bone marrow‐derived MSCs and supported viable MSCs for the incubation time of 9 d. Preadsorption of fibronectin on materials improved this biofunctionality.

     

Stabilizer‐Free Poly(lactide‐co‐glycolide) Nanoparticles Conjugated with Quantum Dots as a Potential Carrier Applied in Human Mesenchymal Stem Cells

We report that human mesenchymal stem cells (hMSCs) were successfully labeled with poly(lactide‐co‐glycolide) nanoparticles (PLGA NPs) surface‐conjugated quantum dots (QDs) (PLGA‐QD NPs) via endocytosis pathway. These NPs were not toxicity even treated with PLGA‐QD NPs at high concentrations for at least four weeks. Besides, PLGA‐QD NPs‐labeled hMSCs did not change their proliferation and differentiation capability toward the cell fates of adipocytes, osteocytes, and chrondrocytes. It's known that PLGA has been widely employed to act as delivery carrier which encapsulates drugs and releases them under a controlled way. Currently, we have also demonstrated that FITC‐loaded PLGA‐QD NPs degraded in hMSCs to achieve intracellular release of FITC. The aim of this research is to investigate viability, proliferation and differentiation capability and the potential for gene delivery of MSCs labeled with PLGA‐QD NPs. In addition to PLGA‐QD NPs, QDs alone were used to serve as a control set for comparison.     

Evaluation of 3D Printed Gelatin‐Based Scaffolds with Varying Pore Size for MSC‐Based Adipose Tissue Engineering

Adipose tissue engineering aims to provide solutions to patients who require tissue reconstruction following mastectomies or other soft tissue trauma. Mesenchymal stromal cells (MSCs) robustly differentiate into the adipogenic lineage and are attractive candidates for adipose tissue engineering. This work investigates whether pore size modulates adipogenic differentiation of MSCs toward identifying optimal scaffold pore size and whether pore size modulates spatial infiltration of adipogenically differentiated cells. To assess this, extrusion‐based 3D printing is used to fabricate photo‐crosslinkable gelatin‐based scaffolds with pore sizes in the range of 200–600 µm. The adipogenic differentiation of MSCs seeded onto these scaffolds is evaluated and robust lipid droplet formation is observed across all scaffold groups as early as after day 6 of culture. Expression of adipogenic genes on scaffolds increases significantly over time, compared to TCP controls. Furthermore, it is found that the spatial distribution of cells is dependent on the scaffold pore size, with larger pores leading to a more uniform spatial distribution of adipogenically differentiated cells. Overall, these data provide first insights into the role of scaffold pore size on MSC‐based adipogenic differentiation and contribute toward the rational design of biomaterials for adipose tissue engineering in 3D volumetric spaces.     

Two‐Photon Polymerization as a Tool for Studying 3D Printed Topography‐Induced Stem Cell Fate

Geometric topographies are known to influence cellular differentiation toward specific phenotypes, but to date the range of features and type of substrates that can be easily fabricated to study these interactions is somewhat limited. In this study, an emerging technology, two‐photon polymerization, is used to print topological patterns with varying feature‐size and thereby study their effect on cellular differentiation. This technique offers rapid manufacturing of topographical surfaces with good feature resolution for shapes smaller than 3 µm. Human‐induced pluripotent stem cells, when attached to these substrates or a non‐patterned control for 1 week, express an array of genetic markers that suggest their differentiation toward a heterogeneous population of multipotent progenitors from all three germ layers. Compared to the topographically smooth control, small features (1.6 µm) encourage differentiation toward ectoderm while large features (8 µm) inhibit self‐renewal. This study demonstrates the potential of using two‐photon polymerization to study and control stem cell fate as a function of substrate interactions. The ability to tailor and strategically design biomaterials in this way can enable more precise and efficient generation or maintenance of desired phenotypes in vitro and in vivo.     

Tuning the Mechanical Properties of Poly(Ethylene Glycol) Microgel‐Based Scaffolds to Increase 3D Schwann Cell Proliferation

2D in vitro studies have demonstrated that Schwann cells prefer scaffolds with mechanical modulus approximately 10× higher than the modulus preferred by nerves, limiting the ability of many scaffolds to promote both neuron extension and Schwann cell proliferation. Therefore, the goals of this work are to develop and characterize microgel‐based scaffolds that are tuned over the stiffness range relevant to neural tissue engineering and investigate Schwann cell morphology, viability, and proliferation within 3D scaffolds. Using thiol‐ene reaction, microgels with surface thiols are produced and crosslinked into hydrogels using a multiarm vinylsulfone (VS). By varying the concentration of VS, scaffold stiffness ranges from 0.13 to 0.76 kPa. Cell morphology in all groups demonstrates that cells are able to spread and interact with the scaffold through day 5. Although the viability in all groups is high, proliferation of Schwann cells within the scaffold of G* = 0.53 kPa is significantly higher than other groups. This result is ≈5× lower than previously reported optimal stiffnesses on 2D surfaces, demonstrating the need for correlation of 3D cell response to mechanical modulus. As proliferation is the first step in Schwann cell integration into peripheral nerve conduits, these scaffolds demonstrate that the stiffness is a critical parameter to optimizing the regenerative process.

     

Temperature Responsive Shape‐Memory Scaffolds with Circumferentially Aligned Nanofibers for Guiding Smooth Muscle Cell Behavior

Structural simulation of the smooth muscle layer plays an important role in tissue engineering of blood vessels for the replacement of damaged arteries. However, it is difficult to construct small‐diameter tubular scaffolds to homogenously locate and align smooth muscle cells (SMCs). In this work, novel temperature responsive shape‐memory scaffolds are designed for SMC culturing. The scaffolds are composed of an outer layer of poly(lactide–glycolide–trimethylene carbonate) (PLGATMC) for programming the deformation from planar to small‐diameter tubular shape and an inner layer of aligned nanofibrous membrane of poly(lactide–glycolide)/chitosan (PLGA/CS) to regulate cell adhesion, proliferation, and morphology. The SMC behaviors and functions are dependent on the PLGA/CS ratios of membranes, and the scaffold with PLGA/CS 7:3 membrane exhibits the most suitable ability to regulate SMC behavior. The PLGA/CS@PLGATMC scaffold can be deformed into a temporary planar at 20 °C for convenient seeding and attachment of SMCs and then immediately self‐rolled into 3D tube at 37 °C. The proposed strategy offers a practical approach for the development of small‐diameter vascular scaffolds from 2D planar into 3D tubular shape by self‐rolling.     

Mastoid Obliteration Using Three‐Dimensional Composite Scaffolds Consisting of Polycaprolactone/β‐Tricalcium Phosphate/Collagen Nanofibers: An In Vitro and In Vivo Study

Two different composite scaffolds, solid‐freeform‐fabricated PCL/β‐TCP supplemented with and without collagen nanofibers are fabricated. These scaffolds are evaluated whether a combination of collagen nanofibers with PCL/β‐TCP can promote osteogenesis in a mastoid obliteration. To assess the effects of the cellular activities of osteoblast‐like‐cells (MG63), SEM images and MTT assays are conducted. Experimental mastoid obliteration is performed using guinea pigs that are divided group A (PCL/β‐TCP/collagen‐nanofiber scaffold) and group B (PCL/β‐TCP scaffold). The results reveal that PCL/β‐TCP/collagen scaffold provide much broader cell attachment sites than PCL/β‐TCP scaffold. The µ‐CT and fluorescent microscopy results reveal that the acceleration of early new bone formation within the pores and scaffold itself at week 4 post‐operation is more effective in group A. In addition, based on the results of the histological and µ‐CT at 12 weeks post‐surgery, the effective regeneration of bone in the PCL/β‐TCP/collagen scaffold is appeared.

     

3D Microfibrous Scaffolds Selectively Promotes Proliferation and Glial Differentiation of Adult Neural Stem Cells: A Platform to Tune Cellular Behavior in Neural Tissue Engineering

Biomaterials are essential for the development of innovative biomedical and therapeutic applications. Biomaterials‐based scaffolds can influence directed cell differentiation to improve cell‐based strategies. Using a novel microfluidics approach, poly (ε‐caprolactone) (PCL), is used to fabricate microfibers with varying diameters (3–40 µm) and topographies (straight and wavy). Multipotent adult rat hippocampal stem/progenitor cells (AHPCs) are cultured on 3D aligned PCL microfibrous scaffolds to investigate their ability to differentiate into neurons, astrocytes, and oligodendrocytes. The results indicate that the PCL microfibers significantly enhance proliferation of the AHPCs compared to control, 2D planar substrates. While the AHPCs maintained their multipotent differentiation capacity when cultured on the PCL scaffolds, there is a significant and dramatic increase in immunolabeling for astrocyte and oligodendrocyte differentiation when compared with growth on planar surfaces. Our results show a 3.5‐fold increase in proliferation and 23.4‐fold increase in astrocyte differentiation for cells on microfibers. Transplantation of neural stem/progenitor cells within a PCL microfiber scaffold may provide important biological and topographic cues that facilitate the survival, selective differentiation, and integration of transplanted cells to improve therapeutic strategies.     

Poly‐l‐Lactic Acid Nanofiber–Polyamidoamine Hydrogel Composites: Preparation,Properties, and Preliminary Evaluation as Scaffolds for Human Pluripotent Stem Cell Culturing

Electrospun poly‐l ‐lactic acid (PLLA) nanofiber mats carrying surface amine groups, previously introduced by nitrogen atmospheric pressure nonequilibrium plasma, are embedded into aqueous solutions of oligomeric acrylamide‐end capped AGMA1, a biocompatible polyamidoamine with arg‐gly‐asp (RGD)‐reminiscent repeating units. The resultant mixture is finally cured giving PLLA‐AGMA1 hydrogel composites that absorb large amounts of water and, in the swollen state, are translucent, soft, and pliable, yet as strong as the parent PLLA mat. They do not split apart from each other when swollen in water and remain highly flexible and resistant, since the hydrogel portion is covalently grafted onto the PLLA nanofibers via the addition reaction of the surface amine groups to a part of the terminal acrylic double bonds of AGMA1 oligomers. Preliminary tested as scaffolds, the composites prove capable of maintaining short‐term undifferentiated cultures of human pluripotent stem cells in feeder‐free conditions.

     

3D and Porous RGDC‐Functionalized Polyester‐Based Scaffolds as a Niche to Induce Osteogenic Differentiation of Human Bone Marrow Stem Cells

Polyester‐based scaffolds covalently functionalized with arginine‐glycine‐aspartic acid‐cysteine (RGDC) peptide sequences support the proliferation and osteogenic differentiation of stem cells. The aim is to create an optimized 3D niche to sustain human bone marrow stem cell (hBMSC) viability and osteogenic commitment, without reliance on differentiation media. Scaffolds consisting of poly(lactide‐co‐trimethylene carbonate), poly(LA‐co‐TMC), and functionalized poly(lactide) copolymers with pendant thiol groups are prepared by salt‐leaching technique. The availability of functional groups on scaffold surfaces allows for an easy and straightforward method to covalently attach RGDC peptide motifs without affecting the polymerization degree. The strategy enables the chemical binding of bioactive motifs on the surfaces of 3D scaffolds and avoids conventional methods that require harsh conditions. Gene and protein levels and mineral deposition indicate the osteogenic commitment of hBMSC cultured on the RGDC functionalized surfaces. The osteogenic commitment of hBMSC is enhanced on functionalized surfaces compared with nonfunctionalized surfaces and without supplementing media with osteogenic factors. Poly(LA‐co‐TMC) scaffolds have potential as scaffolds for osteoblast culture and bone grafts. Furthermore, these results contribute to the development of biomimetic materials and allow a deeper comprehension of the importance of RGD peptides on stem cell transition toward osteoblastic lineage.