Regeneration of long-distance peripheral nerve defects after delayed reconstruction in healthy and diabetic rats is supported by immunomodulatory chitosan nerve guides

Background: Delayed reconstruction of transection or laceration injuries of peripheral nerves is inflicted by a reduced regeneration capacity. Diabetic conditions, more frequently encountered in clinical practice, are known to further impair regeneration in peripheral nerves. Chitosan nerve guides (CNGs) have recently been introduced as a new generation of medical devices for immediate peripheral nerve reconstruction. Here, CNGs were used for 45 days delayed reconstruction of critical length 15 mm rat sciatic nerve defects in either healthy Wistar rats or diabetic Goto-Kakizaki rats; the latter resembling type 2 diabetes. In short and long-term investigations, we comprehensively analyzed the performance of one-chambered hollow CNGs (hCNGs) and two-chambered CNGs (CFeCNGs) in which a chitosan film has been longitudinally introduced. Additionally, we investigated in vitro the immunomodulatory effect provided by the chitosan film. Results: Both types of nerve guides, i.e. hCNGs and CFeCNGs, enabled moderate morphological and functional nerve regeneration after reconstruction that was delayed for 45 days. These positive findings were detectable in generally healthy as well as in diabetic Goto-Kakizaki rats (for the latter only in short-term studies). The regenerative outcome did not reach the degree as recently demonstrated after immediate reconstruction using hCNGs and CFeCNGs. CFeCNG-treatment, however, enabled tissue regrowth in all animals (hCNGs: only in 80% of animals). CFeCNGs did further support with an increased vascularization of the regenerated tissue and an enhanced regrowth of motor axons. One mechanism by which the CFeCNGs potentially support successful regeneration is an immunomodulatory effect induced by the chitosan film itself. Our in vitro results suggest that the pro-regenerative effect of chitosan is related to the differentiation of chitosan-adherent monocytes into pro-healing M2 macrophages. Conclusions: No considerable differences appear for the delayed nerve regeneration process related to healthy and diabetic conditions. Currently available chitosan nerve grafts do not support delayed nerve regeneration to the same extent as they do after immediate nerve reconstruction. The immunomodulatory characteristics of the biomaterial may, however, be crucial for their regeneration supportive effects

Long-Term In Vivo Evaluation of Chitosan Nerve Guide Properties with respect to Two Different Sterilization Methods

Severe peripheral nerve injuries are reconstructed either with autologous nerve grafts (gold standard) or alternatively with clinically approved artificial nerve guides. The most common method used to sterilize these medical products is ethylene oxide gassing (EO). However, this method has several disadvantages. An alternative, which has been barely studied so far, represents beta irradiation (β). In previous studies, we developed an artificial nerve guide made of chitosan (chitosan nerve guide, CNG), a biomaterial that is known to potentially retain toxic residues upon EO sterilization. Therefore, we analyzed the long-term regeneration-supporting and mechanical properties of CNGs upon their sterilization with EO or β and their following application in unilateral repair of 12 mm gaps of the rat sciatic nerve. Over a period of 76 weeks, we serially evaluated the recovery of motor functions, the possible emergence of an inflammation in the surrounding connective tissue, the regrowth of axons into the distal nerve, and possible changes in the material properties. Our first long-term evaluation did not reveal significant differences between both sterilization methods. Thus, β is as appropriate as commonly used EO for sterilization of CNGs; however, it may slightly increase the stiffness of the biomaterial over time

[In Press] Tapping into the physiological responses to mistletoe infection during heat and drought stress

Mistletoes are important co-contributors to tree mortality globally, particularly during droughts. In Australia, mistletoe distributions are expanding in temperate woodlands, while their hosts experienced unprecedented heat and drought stress in recent years. We investigated whether the excessive water use of mistletoes increased the probability of xylem emboli in a mature woodland during the recent record drought that was compounded by multiple heatwaves. We continuously recorded transpiration (⁠TSLA⁠) of infected and uninfected branches from two eucalypt species over two summers, monitored stem and leaf water potentials (⁠Ψ⁠), and used hydraulic vulnerability curves to estimate percent loss in conductivity (PLC) for each species. Variations in weather (vapour pressure deficit, photosynthetic active radiation, soil water content), host species and % mistletoe foliage explained 78% of hourly TSLA⁠. While mistletoe acted as an uncontrollable sink for water in the host even during typical summer days, daily TSLA increased up to 4-fold in infected branches on hot days, highlighting the previously overlooked importance of temperature stress in amplifying water loss in mistletoes. The increased water use of mistletoes resulted in significantly decreased host Ψleaf and Ψtrunk⁠. It further translated to an estimated increase of up to 11% PLC for infected hosts, confirming greater hydraulic dysfunction of infected trees that place them at higher risk of hydraulic failure. However, uninfected branches of Eucalyptus fibrosa had much tighter controls on water loss than uninfected branches of Eucalyptus moluccana, which shifted the risk of hydraulic failure towards an increased risk of carbon starvation for E. fibrosa. The contrasting mechanistic responses to heat and drought stress between both co-occurring species demonstrates the complexity of host–parasite interactions and highlights the challenge in predicting species-specific responses to biotic agents in a warmer and drier climate