Cell transplantation is one of the most promising strategies for the minimally invasive treatment of a raft of injuries and diseases. However, a standing challenge to its efficacy is poor cell ...survival due to a lack of mechanical protection during administration and an unsupportive milieu thereafter. In response, a shear‐injectable nanoscaffold vector is engineered considering the three equal requirements of protection, support, and survival. Here, the programmed peptide assembly of tissue‐specific epitopes presents a safe sanctuary microenvironment for the transplantation of cells. For the first time, a mechanistic understanding of the multifactorial role of the nanoscaffold in promoting cell survival is presented, where initial cell survival is dependent on the fluid mechanic process of droplet formation rather than on shear rate. However, provided is the first report of the most critical component of a transplantation vector, distinguishing feigned biological support from mechanical properties from true ongoing biological support post transplantation. This is achieved via the presentation of amino acid constituents that significantly improve the efficacy of the vector compared to a biocompatible, yet inert analogue. Together, the peptide‐programmed hydrogels enable fundamental rules for the engineering of advanced treatment strategies with wide reaching implications for tissue repair and biofabrication.
Cell transplantation is one of the most promising minimally invasive treatments to repair damaged tissue. However, unacceptably high numbers of cells die during the procedure before they can have a regenerative effect due to a lack of support. In response, a mechanistic study of a programmed hydrogel to support the cells before, during, and after transplantation is presented here.
Abstract With the brain's limited capacity for repair there is a need for new and innovative therapies to promote regeneration. Stem/progenitor cell transplantation has received increasing attention, ...and whilst clinical trials demonstrating functional integration exist, inherent variability between patients has hindered development of this therapy. Variable outcomes have largely been attributed to poor survival and insufficient reinnervation of target tissues due in part to the suboptimal host environment. Here we examined whether improving the physical properties of the host milieu, by way of bioengineered scaffolds, may enhance engraftment. We developed a composite scaffold, incorporating electrospun poly( l -lactic acid) short nanofibers embedded within a thermo-responsive xyloglucan hydrogel, which could be easily injected into the injured brain. Furthermore, to improve the trophic properties of the host brain, glial derived neurotrophic factor (GDNF), a protein known to promote cell survival and axonal growth, was blended into and/or covalently attached onto the composite scaffolds to provide controlled delivery. In vitro we confirmed the ability of the scaffolds to support ventral midbrain (VM) dopamine progenitors, and provide sustained delivery of GDNF – capable of eliciting effects on cell survival and dopaminergic axon growth. In Parkinsonian mice, we show that these composite scaffolds, whilst having no deleterious impact on the host immune response, enhanced the survival of VM grafts and reinnervation of the striatum, an effect that was augmented through the scaffold delivery of GDNF. Taken together, these functionalized composite scaffolds provide a means to significantly improve the milieu of the injured brain, enabling enhanced survival and integration of grafted neurons.
Stem cell transplants offer significant hope for brain repair following ischemic damage. Pre-clinical work suggests that therapeutic mechanisms may be multi-faceted, incorporating bone-fide circuit ...reconstruction by transplanted neurons, but also protection/regeneration of host circuitry. Here, we engineered hydrogel scaffolds to form “bio-bridges” within the necrotic lesion cavity, providing physical and trophic support to transplanted human embryonic stem cell-derived cortical progenitors, as well as residual host neurons. Scaffolds were fabricated by the self-assembly of peptides for a laminin-derived epitope (IKVAV), thereby mimicking the brain’s major extracellular protein. Following focal ischemia in rats, scaffold-supported cell transplants induced progressive motor improvements over 9 months, compared to cell- or scaffold-only implants. These grafts were larger, exhibited greater neuronal differentiation, and showed enhanced electrophysiological properties reflective of mature, integrated neurons. Varying graft timing post-injury enabled us to attribute repair to both neuroprotection and circuit replacement. These findings highlight strategies to improve the efficiency of stem cell grafts for brain repair.
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•Peptide scaffolds structurally and functionally support neural grafts in a stroke model•Scaffolds promote human stem cell graft differentiation and integration•Cell + scaffold implants reduce host tissue atrophy•Cell + scaffold implants promote prolonged recovery of motor function
Somaa et al. examine the capacity of peptide-based scaffolds to structurally and functionally support human pluripotent stem cell-derived neural transplants in the stroke-injured brain. Scaffolds promoted graft maturation and integration and reduced host tissue atrophy, resulting in improved motor function over a period of 9 months.
Tissue-specific self-assembling peptide (SAP) hydrogels designed based on biologically relevant peptide sequences have great potential in regenerative medicine. These materials spontaneously form 3D ...networks of physically assembled nanofibres utilising non-covalent interactions. The nanofibrous structure of SAPs is often compared to that of electrospun scaffolds. These electrospun nanofibers are produced as sheets that can be engineered from a variety of polymers that can be chemically modified to incorporate many molecules including drugs and growth factors. However, their macroscale morphology limits them to wrapping and bandaging applications. Here, for the first time, we combine the benefits of these systems to describe a two-component composite scaffold from these biomaterials, with the design goal of providing a hydrogel scaffold that presents 3D structures, and also has temporal control over drug delivery. Short fibres, cut from electrospun scaffolds, were mixed with our tissue-specific SAP hydrogel to provide a range of nanofibre sizes found in the extracellular matrix (10-300 nm in diameter). The composite material maintained the shear-thinning and void-filling properties of SAP hydrogels that have previously been shown to be effective for minimally invasive material injection, cell delivery and subsequent in vivo integration. Both scaffold components were separately loaded with growth factors, important signaling molecules in tissue regeneration whose rapid degradation limits their clinical efficacy. The two biomaterials provided sequential growth factor delivery profiles: the SAP hydrogel provided a burst release, with the release rate decreasing over 12 hours, while the electrospun nanofibres provided a more constant, sustained delivery. Importantly, this second release commenced 6 days later. The design rules established here to provide temporally distinct release profiles can enable researchers to target specific stages in regeneration, such as the acute immune response versus sustained protection and survival of cells following injury. In summary, this novel composite material combines the physical advantages of SAP hydrogels and electrospun nanofibres, while additionally providing a superior vehicle for the stabilisation and controlled delivery of growth factors necessary for optimal tissue repair.
Gene delivery has been extensively investigated for introducing foreign genetic material into cells to promote expression of therapeutic proteins or to silence relevant genes. This approach can ...regulate genetic or epigenetic disorders, offering an attractive alternative to pharmacological therapy or invasive protein delivery options. However, the exciting potential of viral gene therapy has yet to be fully realized, with a number of clinical trials failing to deliver optimal therapeutic outcomes. Reasons for this include difficulty in achieving localized delivery, and subsequently lower efficacy at the target site, as well as poor or inconsistent transduction efficiency. Thus, ongoing efforts are focused on improving local viral delivery and enhancing its efficiency. Recently, biomaterials have been exploited as an option for more controlled, targeted and programmable gene delivery. There is a growing body of literature demonstrating the efficacy of biomaterials and their potential advantages over other delivery strategies. This review explores current limitations of gene delivery and the progress of biomaterial-mediated gene delivery. The combination of biomaterials and gene vectors holds the potential to surmount major challenges, including the uncontrolled release of viral vectors with random delivery duration, poorly localized viral delivery with associated off-target effects, limited viral tropism, and immune safety concerns.
The brain is a complex 3-dimensional structure, the organization of which provides a local environment that directly influences the survival, proliferation, differentiation, migration, and plasticity ...of neurons. To probe the effects of damage and disease on these cells, a synthetic environment is needed. Three-dimensional culturing of stem cells, neural progenitors, and neurons within fabricated biomaterials has demonstrated superior biomimetic properties over conventional 2-dimensional cultureware, offering direct recapitulation of both cell-cell and cell-extracellular matrix interactions. Within this review we address the benefits of deploying biomaterials as advanced cell culture tools capable of influencing neuronal fate and as in vitro models of the native in vivo microenvironment. We highlight recent and promising biomaterials approaches toward understanding neural network and their function relevant to neurodevelopment and provide our perspective on how these materials can be engineered and programmed to study both the healthy and diseased nervous system.
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Biomaterials; Cellular Neuroscience; Materials Science; Neuroscience
Various engineering applications have been utilized to deliver molecules and compounds in both innate and biological settings. In the context of biological applications, the timely delivery of ...molecules can be critical for cellular and organ function. As such, previous studies have demonstrated the superiority of long-term protein delivery, by way of protein tethering onto bioengineered scaffolds, compared with conventional delivery of soluble protein in vitro and in vivo. Despite such benefits little knowledge exists regarding the stability, release kinetics, longevity, activation of intracellular pathway, and functionality of these proteins over time. By way of example, here we examined the stability, degradation and functionality of a protein, glial-derived neurotrophic factor (GDNF), which is known to influence neuronal survival, differentiation, and neurite morphogenesis. Enzyme-linked immunosorbent assays (ELISA) revealed that GDNF, covalently tethered onto polycaprolactone (PCL) electrospun nanofibrous scaffolds, remained present on the scaffold surface for 120 days, with no evidence of protein leaching or degradation. The tethered GDNF protein remained functional and capable of activating downstream signaling cascades, as revealed by its capacity to phosphorylate intracellular Erk in a neural cell line. Furthermore, immobilization of GDNF protein promoted cell survival and differentiation in culture at both 3 and 7 days, further validating prolonged functionality of the protein, well beyond the minutes to hours timeframe observed for soluble proteins under the same culture conditions. This study provides important evidence of the stability and functionality kinetics of tethered molecules.
Protein growth factors have demonstrated great potential for tissue repair, but their inherent instability and large size prevents meaningful presentation to biologically protected nervous tissue. ...Here, we create a nanofibrous network from a self-assembling peptide (SAP) hydrogel to carry and stabilize the growth factors. We significantly reduced growth factor degradation to increase their lifespan by over 40 times. To control the temporal release profile we covalently attached polysaccharide chitosan molecules to the growth factor to increase its interactions with the hydrogel nanofibers and achieved a 4 h delay, demonstrating the potential of this method to provide temporally controlled growth factor delivery. We also describe release rate based analysis to examine the growth factor delivery in more detail than standard cumulative release profiles allow and show that the chitosan attachment method provided a more consistent release profile with a 60% reduction in fluctuations. To prove the potential of this system as a complex growth factor delivery platform we demonstrate for the first time temporally distinct release of multiple growth factors from a single tissue specific SAP hydrogel: a significant goal in regenerative medicine.
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The nanofibrillar structures that underpin self-assembling peptide (SAP) hydrogels offer great potential for the development of finely tuned cellular microenvironments suitable for ...tissue engineering. However, biofunctionalisation without disruption of the assembly remains a key issue. SAPS present the peptide sequence within their structure, and studies to date have typically focused on including a single biological motif, resulting in chemically and biologically homogenous scaffolds. This limits the utility of these systems, as they cannot effectively mimic the complexity of the multicomponent extracellular matrix (ECM). In this work, we demonstrate the first successful co-assembly of two biologically active SAPs to form a coassembled scaffold of distinct two-component nanofibrils, and demonstrate that this approach is more bioactive than either of the individual systems alone. Here, we use two bioinspired SAPs from two key ECM proteins: Fmoc-FRGDF containing the RGD sequence from fibronectin and Fmoc-DIKVAV containing the IKVAV sequence from laminin. Our results demonstrate that these SAPs are able to co-assemble to form stable hybrid nanofibres containing dual epitopes. Comparison of the co-assembled SAP system to the individual SAP hydrogels and to a mixed system (composed of the two hydrogels mixed together post-assembly) demonstrates its superior stable, transparent, shear-thinning hydrogels at biological pH, ideal characteristics for tissue engineering applications. Importantly, we show that only the coassembled hydrogel is able to induce in vitro multinucleate myotube formation with C2C12 cells. This work illustrates the importance of tissue engineering scaffold functionalisation and the need to develop increasingly advanced multicomponent systems for effective ECM mimicry.
Successful control of stem cell fate in tissue engineering applications requires the use of sophisticated scaffolds that deliver biological signals to guide growth and differentiation. The complexity of such processes necessitates the presentation of multiple signals in order to effectively mimic the native extracellular matrix (ECM). Here, we establish the use of two biofunctional, minimalist self-assembling peptides (SAPs) to construct the first co-assembled SAP scaffold. Our work characterises this construct, demonstrating that the physical, chemical, and biological properties of the peptides are maintained during the co-assembly process. Importantly, the coassembled system demonstrates superior biological performance relative to the individual SAPs, highlighting the importance of complex ECM mimicry. This work has important implications for future tissue engineering studies.
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