Synthetic scaffolds for 3D cell cultures and organoids: applications in regenerative medicine.

Three-dimensional (3D) cell cultures offer an unparalleled platform to recreate spatial arrangements of cells in vitro. 3D cell culture systems have gained increasing interest due to their evident advantages in providing more physiologically relevant information and more predictive data compared to their two-dimensional (2D) counterpart. Design and well-established fabrication of organoids (a particular type of 3D cell culture system) are nowadays considered a pivotal achievement for their ability to replicate in vitro cytoarchitecture and the functionalities of an organ. In this condition, pluripotent stem cells self-organize into 3D structures mimicking the in vivo microenvironments, architectures and multi-lineage differentiation. Scaffolds are key supporting elements in these 3D constructs, and Matrigel is the most commonly used matrix despite its relevant translation limitations including animal-derived sources, non-defined composition, batch-to-batch variability and poorly tailorable properties. Alternatively, 3D synthetic scaffolds, including self-assembling peptides, show promising biocompatibility and biomimetic properties, and can be tailored on specific target tissue/cells. In this review, we discuss the recent advances on 3D cell culture systems and organoids, promising tools for tissue engineering and regenerative medicine applications. For this purpose, we will describe the current state-of-the-art on 3D cell culture systems and organoids based on currently available synthetic-based biomaterials (including tailored self-assembling peptides) either tested in in vivo animal models or developed in vitro with potential application in the field of tissue engineering, with the aim to inspire researchers to take on such promising platforms for clinical applications in the near future.

Multifunctionalized hydrogels foster hNSC maturation in 3D cultures and neural regeneration in spinal cord injuries.

Three-dimensional cell cultures are leading the way to the fabrication of tissue-like constructs useful to developmental biology and pharmaceutical screenings. However, their reproducibility and translational potential have been limited by biomaterial and culture media compositions, as well as cellular sources. We developed a construct comprising synthetic multifunctionalized hydrogels, serum-free media, and densely seeded good manufacturing practice protocol-grade human neural stem cells (hNSC). We tracked hNSC proliferation, differentiation, and maturation into GABAergic, glutamatergic, and cholinergic neurons, showing entangled electrically active neural networks. The neuroregenerative potential of the "engineered tissue" was assessed in spinal cord injuries, where hNSC-derived progenitors and predifferentiated hNSC progeny, embedded in multifunctionalized hydrogels, were implanted. All implants decreased astrogliosis and lowered the immune response, but scaffolds with predifferentiated hNSCs showed higher percentages of neuronal markers, better hNSC engraftment, and improved behavioral recovery. Our hNSC-construct enables the formation of 3D functional neuronal networks in vitro, allowing novel strategies for hNSC therapies in vivo.

Age-associated differences in motor output variability and coordination during the simultaneous dorsiflexion of both feet.

Older adults are more variable than young adults on tasks that demand the simultaneous control of more than one effector, and the difference between age groups may be related to their different capacity of coordinating the force output of the involved effectors. The goal of this study was to determine whether age-associated differences in motor output variability during tasks involving the simultaneous dorsiflexion of two feet can be partially explained by differences in coordination and possibly attenuated by physical training. Ten young and 22 old adults (10 trained and 12 untrained old adults) volunteered to participate in the study. Trained older adults had experience in a high-intensity mixed modality training (MMT) regime for a minimum of 1 year. Volunteers performed successive trials of a constant force task and a goal-directed task, with and without visual feedback. Within- and between-trial variability were calculated and coordination was quantified using the uncontrolled manifold (UCM) approach (i.e., co-variation of the force outputs of both feet were used to quantify a motor synergy index). Older adults exhibited greater variability and lower synergy (p < .05), independently of physical training status, than young adults. Removal of visual feedback caused greater variability and lower synergy for all groups (p < .05). Our results suggest that older adults exhibit greater motor output variability in tasks involving the simultaneous dorsiflexion of both feet possibly due to a lack of coordination between the feet.

In Situ Transglutaminase Cross-Linking Improves Mechanical Properties of Self-Assembling Peptides for Biomedical Applications.

The development of three-dimensional (3D) biomaterials that mimic natural tissues is required for efficiently restoring physiological functions of injured tissues and organs. In the field of soft hydrogels, self-assembled peptides (SAPs) stand out as distinctive biomimetic scaffolds, offering tunable properties. They have garnered significant attention in nanomedicine due to their innate ability to self-assemble, resulting in the creation of fibrous nanostructures that closely mimic the microenvironment of the extracellular matrix (ECM). This unique feature ensures their biocompatibility and bioactivity, making them a compelling area of study over the past few decades. As they are soft hydrogels, approaches are necessary to enhance the stiffness and resilience of the SAP materials. This work shows an enzymatic strategy to selectively increase the stiffness and resiliency of functionalized SAPs using transglutaminase (TGase) type 2, an enzyme capable of triggering the formation of isopeptide bonds. To this aim, we synthesized a set of SAP sequences and characterized their cross-linking via rheological experiments, atomic force microscopy (AFM), thioflavin-T binding assay, and infrared spectroscopy (ATR-FTIR) tests. The results showed an improvement of the storage modulus of cross-linked SAPs at no cost of the maximum stress-at-failure. Further, in in vitro tests, we examined and validated the TGase capability to cross-link SAPs without hampering seeded neural stem cells (hNSCs) viability and differentiation, potentially leaving the door open for safe in situ cross-linking reactions in vivo.

Low-power microwaves: a cell-compatible physical treatment to enhance the mechanical properties of self-assembling peptides.

Biomaterials designed for tissue engineering applications should, among other requirements, mimic the native extracellular matrix (ECM) of the tissues to be regenerated, both in terms of biomimetic and mechanical properties. Ideally, the scaffold stiffness and stress resistance should be tuned for each specific implantation therapy. Self-assembling peptides (SAPs) are promising synthetic bionanomaterials prone to easy multi-functionalization, bestowing biomimetic properties. However, they usually yield soft and fragile hydrogels unsuited for the regeneration of medium-to-hard tissues. For this purpose, chemical cross-linking of SAPs is an option, but it often requires a moderately toxic and expensive chemical compound and/or the presence of specific residues/reactive sites, posing issues for its feasibility and translational potential. In this work, we introduced, characterized by rheology, atomic force microscopy (AFM), Thioflavin-T assay (ThT), and Fourier transform infrared (FT-IR) tests, and optimized (by tuning the power, temperature and treatment time) a novel fast, green and affordable methodology using mild microwave (MW) irradiation to increase the mechanical properties of diverse classes of SAPs. Low-power MWs increase stiffness, resilience, and ß-structuration, while high-power MW treatments partially denature the tested SAPs. Our pure-physical methodology does not alter the SAP biomimetic properties (verified via in vitro tests of viability and differentiation of human neural stem cells), is compatible with already seeded cells, and is also synergic with genipin-based cross-linking of SAPs; therefore, it may become the next standard for SAP preparation in tissue engineering applications at hand of all research labs and in clinics.

Biomimetic Electrospun Self-Assembling Peptide Scaffolds for Neural Stem Cell Transplantation in Neural Tissue Engineering.

Spinal cord regeneration using stem cell transplantation is a promising strategy for regenerative therapy. Stem cells transplanted onto scaffolds that can mimic natural extracellular matrix (ECM) have the potential to significantly improve outcomes. In this study, we strived to develop a cell carrier by culturing neural stem cells (NSCs) onto electrospun 2D and 3D constructs made up of specific crosslinked functionalized self-assembling peptides (SAPs) featuring enhanced biomimetic and biomechanical properties. Morphology, architecture, and secondary structures of electrospun scaffolds in the solid-state and electrospinning solution were studied step by step. Morphological studies showed the benefit of mixed peptides and surfactants as additives to form thinner, uniform, and defect-free fibers. It has been observed that ß-sheet conformation as evidence of self-assembling has been predominant throughout the process except for the electrospinning solution. In vitro NSCs seeded on electrospun SAP scaffolds in 2D and 3D conditions displayed desirable proliferation, viability, and differentiation in comparison to the gold standard. In vivo biocompatibility assay confirmed the permissibility of implanted fibrous channels by foreign body reaction. The results of this study demonstrated that fibrous 2D/3D electrospun SAP scaffolds, when shaped as micro-channels, can be suitable to support NSC transplantation for regeneration following spinal cord injury.

Long-term cultures of human pancreatic islets in self-assembling peptides hydrogels.

Human pancreatic islets transplantation is an experimental therapeutic treatment for Type I Diabetes. Limited islets lifespan in culture remains the main drawback, due to the absence of native extracellular matrix as mechanical support after their enzymatic and mechanical isolation procedure. Extending the limited islets lifespan by creating a long-term in vitro culture remains a challenge. In this study, three biomimetic self-assembling peptides were proposed as potential candidates to recreate in vitro a pancreatic extracellular matrix, with the aim to mechanically and biologically support human pancreatic islets, by creating a three-dimensional culture system. The embedded human islets were analyzed for morphology and functionality in long-term cultures (14-and 28-days), by evaluating ß-cells content, endocrine component, and extracellular matrix constituents. The three-dimensional support provided by HYDROSAP scaffold, and cultured into MIAMI medium, displayed a preserved islets functionality, a maintained rounded islets morphology and an invariable islets diameter up to 4 weeks, with results analogues to freshly-isolated islets. In vivo efficacy studies of the in vitro 3D cell culture system are ongoing; however, preliminary data suggest that human pancreatic islets pre-cultured for 2 weeks in HYDROSAP hydrogels and transplanted under subrenal capsule may restore normoglycemia in diabetic mice. Therefore, engineered self-assembling peptide scaffolds may provide a useful platform for long-term maintenance and preservation of functional human pancreatic islets in vitro.

Multi-Functionalized Self-Assembling Peptides as Reproducible 3D Cell Culture Systems Enabling Differentiation and Survival of Various Human Neural Stem Cell Lines.

Neural stem cells-based therapies have shown great potential for central nervous system regeneration, with three-dimensional (3D) culture systems representing a key technique for tissue engineering applications, as well as disease modeling and drug screenings. Self-assembling peptides (SAPs), providing biomimetic synthetic micro-environments regulating cellular functionality and tissue repair, constitute a suitable tool for the production of complex tissue-like structures in vitro. However, one of the most important drawbacks in 3D cultures, obtained via animal-derived substrates and serum-rich media, is the reproducibility and tunability of a standardized methodology capable to coax neural differentiation of different human cell lines. In this work we cultured four distinct human neural stem cell (hNSC) lines in 3D synthetic multifunctionalized hydrogel (named HYDROSAP) for up to 6 weeks. Three-dimensional cultures of differentiating hNSCs exhibited a progressive differentiation and maturation over time. All hNSCs-derived neurons in 3D culture system exhibited randomly organized entangled networks with increasing expression of GABAergic and glutamatergic phenotypes and presence of cholinergic ones. Oligodendrocytes formed insulating myelin sheaths positive for myelin basic protein (MBP). In summary, results demonstrated a successfully standardized and reproducible 3D cell culture system for hNSC differentiation and maturation in serum-free conditions useful for future therapies.

Mixed Modal Training to Help Older Adults Maintain Postural Balance.

OBJECTIVE: Older adults have poorer balance compared with younger adults, but exercise may slow this age-related loss. Although the best type of exercise to optimize balance gains remains unclear, it is likely that a training regimen incorporating several different types of exercise, termed mixed modality training (MMT) (popularized by CrossFit), would be effective. Accordingly, this study aims to assess whether regular MMT leads to improved balance in older adults. METHODS: Ten trained young (28 ± 4 years, minimum of 1 year MMT) and 22 older (67 ± 6 years) adults participated in this study. Older adults were divided into 2 groups: trained (minimum of 1 year MMT) and untrained. An electronic baropodometer was used to assess baseline postural balance using the postural sway (both open and closed eyes) test. RESULTS: Compared with untrained older adults, those who trained performed similarly to young trained adults in the postural sway test. In addition, with eyes closed, trained older adults demonstrated better center of pressure total displacement area than untrained older adults. CONCLUSION: These data suggest that regular MMT can lead to a level of postural control in older adults similar to that observed in young adults. The favorable effects of MMT on postural control in older adults may be attributable to improvements in both muscle strength and proprioception.

Branched peptides integrate into self-assembled nanostructures and enhance biomechanics of peptidic hydrogels.

Self-assembling peptides (SAP) have drawn an increasing interest in the tissue engineering community. They display unquestionable biomimetic properties, tailorability and promising biocompatibility. However their use has been hampered by poor mechanical properties making them fragile soft scaffolds. To increase SAP hydrogel stiffness we introduced a novel strategy based on multiple ramifications of (LDLK)3, a well-known linear SAP, connected with one or multiple "lysine knots". Differently branched SAPs were tested by increasing the number of (LDLK)3-like branches and by adding the neuro-regenerative functional motif BMHP1 as a single branch. While pure branched peptides did not have appealing self-assembling propensity, when mixed with the corresponding linear SAP they increased the stiffness of the overall hydrogel of multiple times. Notably, optimal results (or peak) were obtained 1) at similar molar ratio (between linear and branched peptides) for all tested sequences and 2) for the branched SAPs featuring the highest number of branches made of (LDLK)3. The functional motif BMHP1, as expected, seemed not to contribute to the increase of the storage modulus as efficiently as (LDLK)3. Interestingly, branched SAPs improved the ß-sheet self-arrangement of (LDLK)3 and allowed for the formation of assembled nanofibers. Indeed in coarse-grained molecular dynamics we showed they readily integrate in the assembled aggregates providing "molecular connections" among otherwise weakly paired ß-structures. Lastly, branched SAPs did not affect the usual response of human neural stem cells cultured on (LDLK)3-like scaffolds in vitro. Hence, branched SAPs may be a valuable new tool to enhance mechanical properties of self-assembling peptide biomaterials harmlessly; as neither chemical nor enzymatic cross-linking reactions are involved. As a consequence, branched SAPs may enlarge the field of application of SAPs in tissue engineering and beyond. STATEMENT OF SIGNIFICANCE: Self-assembling peptides stand at the forefront of regenerative medicine because they feature biomimetic nano-architectures that mimic the complexity of natural peptide-based extracellular matrices of living tissues. Their superior biocompatibility and ease of scale-up production are hampered by weak mechanical properties due to transient non-covalent interactions among and within the self-assembled peptide chains, thus limiting their potential applications. We introduced new branched self-assembling peptides to be used as "molecular connectors" among self-assembled nanostructures made of linear SAPs. Branched SAPs could be mixed with linear SAPs before self-assembling in order to have them intermingled with different ß-sheets of linear SAPs after gelation. This strategy caused a manifold increase of the stiffness of the assembled hydrogels (proportional to the number of self-assembling branches), did not affect SAP propensity to form ß-sheet but, instead, further stimulated their secondary structure rearrangements. It is now possible to modularly improve SAP scaffold mechanical properties without using harmful chemical reactions. Therefore, branched SAPs represent an additional tool to be adopted for efficient and harmless SAP scaffold customization in tissue engineering.