Multimodal additive manufacturing of biomimetic tympanic membrane replacements with near tissue-like acousto-mechanical and biological properties.

The three additive manufacturing techniques fused deposition modeling, gel plotting and melt electrowriting were combined to develop a mimicry of the tympanic membrane (TM) to tackle large TM perforations caused by chronic otitis media. The mimicry of the collagen fiber orientation of the TM was accompanied by a study of multiple funnel-shaped mimics of the TM morphology, resulting in mechanical and acoustic properties similar to those of the eardrum. For the different 3D printing techniques used, the process parameters were optimized to allow reasonable microfiber arrangements within the melt electrowriting setup. Interestingly, the fiber pattern was less important for the acousto-mechanical properties than the overall morphology. Furthermore, the behavior of keratinocytes and fibroblasts is crucial for the repair of the TM, and an in vitro study showed a high biocompatibility of both primary cell types while mimicking the respective cell layers of the TM. A simulation of the in vivo ingrowth of both cell types resulted in a cell growth orientation similar to the original collagen fiber orientation of the TM. Overall, the combined approach showed all the necessary parameters to support the growth of a neo-epithelial layer with a similar structure and morphology to the original membrane. It therefore offers a suitable alternative to autologous materials for the treatment of chronic otitis media. STATEMENT OF SIGNIFICANCE: Millions of people worldwide suffer from chronic middle ear infections. Although the tympanic membrane (TM) can be reconstructed with autologous materials, the grafts used for this purpose require extensive manual preparation during surgery. This affects not only the hearing ability but also the stability of the reconstructed TM, especially in the case of full TM reconstruction. The synthetic alternative presented here mimicked not only the fibrous structure of the TM but also its morphology, resulting in similar acousto-mechanical properties. Furthermore, its high biocompatibility supported the migration of keratinocytes and fibroblasts to form a neo-epithelial layer. Overall, this completely new TM replacement was achieved by combining three different additive manufacturing processes.

Application of neurotrophic and proangiogenic factors as therapy after peripheral nervous system injury.

The intrinsic ability of peripheral nerves to regenerate after injury is extremely limited, especially in case of severe injury. This often leads to poor motor function and permanent disability. Existing approaches for the treatment of injured nerves do not provide appropriate conditions to support survival and growth of nerve cells. This drawback can be compensated by the use of gene therapy and cell therapy-based drugs that locally provide an increase in the key regulators of nerve growth, including neurotrophic factors and extracellular matrix proteins. Each growth factor plays its own specific angiotrophic or neurotrophic role. Currently, growth factors are widely studied as accelerators of nerve regeneration. Particularly noteworthy is synergy between various growth factors, that is essential for both angiogenesis and neurogenesis. Fibroblast growth factor 2 and vascular endothelial growth factor are widely known for their proangiogenic effects. At the same time, fibroblast growth factor 2 and vascular endothelial growth factor stimulate neural cell growth and play an important role in neurodegenerative diseases of the peripheral nervous system. Taken together, their neurotrophic and angiogenic properties have positive effect on the regeneration process. In this review we provide an in-depth overview of the role of fibroblast growth factor 2 and vascular endothelial growth factor in the regeneration of peripheral nerves, thus demonstrating their neurotherapeutic efficacy in improving neuron survival in the peripheral nervous system.

Angiogenesis and nerve regeneration induced by local administration of plasmid pBud-coVEGF165-coFGF2 into the intact rat sciatic nerve.

Vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) are well-known growth factors involved in the regeneration of various tissues and organs, including peripheral nerve system. In the present study, we elucidated the local and systemic effects of plasmid construct рBud-coVEGF165-coFGF2 injected into the epineurium of intact rat sciatic nerve. Results of histological examination of sciatic nerve and multiplex immunoassays of serum showed the absence of immunogenicity and biosafety of plasmid рBud-coVEGF165-coFGF2. Moreover, local administration of plasmid DNA construct resulted in significantly decreased levels of pro-inflammatory cytokines in the peripheral blood, including tumor necrosis factor α (TNFα) and interleukin-12, and significantly increased levels of cytokines and chemokines including Regulated upon Activation, Normal T Cell Expressed and Presumably Secrete (RANTES), epidermal growth factor, interleukin-2, and monocyte chemoattractant protein 1. These changes in the peripheral blood on day 7 after injection of plasmid construct рBud-coVEGF165-coFGF2 show that the plasmid construct has systemic effects and may modulate immune response. At the same time, reverse transcription-polymerase chain reaction revealed transient expression of coFGF2, coVEGF165, ratFGF2 and ratVEGFA with direct transport of transcripts from distal part to proximal part of the sciatic nerve. Immunohistochemical staining revealed prolonged presence of VEGFA in sciatic nerve till 14 days post-injection. These findings suggest that local administration of plasmid construct рBud-coVEGF165-coFGF2 at a concentration of 30 ng/µL results in the formation of pro-angiogenic stimuli and, and the plasmid construct, used as a drug for gene therapy, might potentially facilitate regeneration of the sciatic nerve. The study was approved by the Animal Ethics Committee of Kazan Federal University, procedures were approved by the Local Ethics Committee (approval No. 5) on May 27, 2014.

Adipose-Derived Mesenchymal Stem Cells Applied in Fibrin Glue Stimulate Peripheral Nerve Regeneration.

Mesenchymal stem cells (MSCs) hold a great promise for cell therapy. To date, they represent one of the best choices for the treatment of post-traumatic injuries of the peripheral nervous system. Although autologous can be easily transplanted in the injured area, clinical advances in this filed have been impaired by lack of preservation of graft cells into the injury area after transplantation. Indeed, cell viability is not retained after injection into the blood stream, and cells injected directly into the area of injury either are washed off or inhibit regeneration through scar formation and neuroma development. This study proposes a new way of MSCs delivery to the area of traumatic injury by using fibrin glue, which not only fixes cells at the site of application but also provides extracellular matrix support. Using a sciatic nerve injury model, MSC derived from adipose tissue embedded in fibrin glue were able to enter the nerve and migrate mainly retrogradely after transplantation. They also demonstrated a neuroprotective effect on DRG L5 sensory neurons and stimulated axon growth and myelination. Post-traumatic changes of the sensory neuron phenotype were also improved. Importantly, MSCs stimulated nerve angiogenesis and motor function recovery. Therefore, our data suggest that MSC therapy using fibrin glue is a safe and efficient method of cell transplantation in cases of sciatic nerve injury, and that this method of delivery of regeneration stimulants could be beneficial for the successful treatment of other central and peripheral nervous system conditions.