Srinivas Madduri

Trophically and topographically functionalized silk fibroin nerve conduits for guided peripheral nerve regeneration

Artificial nerve conduits (NC) can be used as an alternative to autologous nerve grafts to enhance the repair of small nerve gaps. Current NC lack adequate molecular and structural functionalities. Thus, we developed silk fibroin NC (SF NC) that were loaded with glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) and topographically functionalized with aligned and non-aligned SF nanofibers. The SF NC were produced from fully functionalized SF membranes on which initial experiments were performed. DRG (dorsal root ganglions) sensory neurons and spinal cord (SpC) motor neurons, both from chicken embryos, exhibited an augmented length and rate of axonal outgrowth parallel to the aligned nanofibers. In addition, glial cells from DRG proliferated and migrated in close association and even slightly ahead of the outgrowing axons. On the contrary, axonal and glial growth was slower and randomly oriented on non-aligned nanofibers. The DRG and SpC explants were also inserted into the lumen of the finished SF NC. The unidirectional orientation of axo-glial outgrowth from the explants evidenced the preservation of the trophic and topographical functionalities in the SF NC. Bioactive GDNF and NGF were released in vitro from SF NC over 4 weeks. Thus, the developed functionalized SF NC hold promise to enhance functional recovery of injured peripheral nerves.

Schwann cell delivery of neurotrophic factors for peripheral nerve regeneration

Current treatments of injured peripheral nerves often fail to mediate satisfactory functional recovery. For axonal regeneration, neurotrophic factors (NTFs) play a crucial role. Multiple NTFs and other growth‐promoting factors are secreted, amongst others, by Schwann cells (SCs), which also provide cellular guidance for regenerating axons. Therefore, delivery of NTFs and transplantation of autologous or genetically modified SCs with therapeutic protein expression have been proposed. This article reviews polymer‐based and cellular approaches for NTF delivery, with a focus on SCs and strategies to modulate SC gene expression. Polymer‐based NTF delivery has mostly resided on nerve conduits (NC). While NC have generally provided prolonged NTF release, their therapeutic effect has remained significantly below that achieved with autologous nerve grafts. Several studies demonstrated enhanced nerve regeneration using NC seeded with SCs. The SCs have sometimes been modified genetically using non‐viral or viral vectors. Whereas non‐viral vectors produced poor transgene delivery, adenoviral vectors mediated high transgene transduction efficiency of SCs. Further improvements of safety and transgene expression of adenoviral vector may lead to rapid translation of pre‐clinical research to clinical trials.

Synergistic effect of GDNF and NGF on axonal branching and elongation in vitro

There is a clinical need to enhance functional recovery of injured peripheral nerves. Local administration of neurotrophic factors (NTFs) after surgical repair has been proposed for this purpose. Little is known, however, on the optimal local dose and dosing frequency of NTFs in a peripheral nerve defect. For increasing our knowledge on biologically relevant local NTFs concentrations and for making available an in vitro assay for assessing the bioactivity of NTFs in connection with implantable localized delivery systems, we developed in this study a bioassay for NTFs, which is based on dorsal root ganglion (DRG) explants from E9 (9 days old) chicken embryos. Axonal elongation and extent of axonal branching was analyzed microscopically after addition of glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF), each alone and in combination. GDNF significantly promoted axonal elongation, but only little axonal branching, whereas NGF induced extensive axonal branching with modest axonal elongation. The combination of GDNF and NGF exerted a synergistic effect on the axonal elongation, axonal branching and growth kinetics. GDNF and NGF also enhanced the expression of their respective functional receptors Ret and TrkA on the DRG neurons. This information should be relevant for the development of implants containing NTFs and on drug therapy of damaged peripheral nerves.

Collagen nerve conduits releasing the neurotrophic factors GDNF and NGF

Artificial nerve conduits (NC) can clinically be instrumental for facilitating the surgery of damaged peripheral nerves. To improve axonal regeneration of injured peripheral nerves, we have developed collagen nerve conduits (NC) releasing glial cell line-derived neurotrophic factor (GDNF) alone or in combination with nerve growth factor (NGF), which exert synergistic action on axonal growth. Degradation of the NC and their mechanical and drug release properties were controlled by two means: (i) cross-linking the collagen tubes by physical means, through a dehydro-thermal treatment (DHT), before loading with the neurotrophic factors (NTFs) GDNF or GDNF/NGF; and (ii) coating the drug-loaded collagen tubes with layers of poly(lactide-co-glycolide) (PLGA). Non-cross-linked collagen NC (C-NC) released high amounts of NTFs during the initial 2-3 days of incubation, whereas the DHT-treated collagen NC (C(dht)-NC) did not show a prominent burst effect. The release kinetics was similar for GDNF alone and GDNF co-delivered with NGF. Within 30 days, the C-NC released 78% and 83% of the total doses of GDNF and NGF, respectively, whereas the C(dht)-NC released only 68% of GDNF and 56% of NGF. The bioactivity of the NTFs released up to 30 days was confirmed by an in vitro bioassay using chicken embryonic dorsal root ganglion (DRG) explants. The C(dht)-NC also possessed adequate mechanical resistance against radial compression, the pull-out of a suture thread, and loss of patency upon bending. Modulus and pull-out strength increased in the order of C-NC, C(dht)-NC approximately Neuragen, and Neurolac, with the latter two products being commercially available collagen and polyester NC, respectively. In vitro degradation time upon incubation with collagenase increased in the same order for the collagen-based NC. In conclusion, co-delivery of synergistically acting GDNF and NGF from structurally improved NC may be a promising tool for the successful repair of peripheral nerve defects.

Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats

Present interventions to repair severed peripheral nerves provide slow and poor early axonal regeneration, which may cause unsatisfactory functional reinnervation. To improve early axonal regeneration in a 10 mm rat sciatic nerve gap model, we developed collagen nerve conduits loaded with the synergistically acting glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF). For controlling the concomitant GDNF and NGF release, the collagen tubes were cross-linked by a dehydro-thermal treatment (110 degrees C; 20 mbar; 5 days) prior to impregnating the tubes with GDNF and NGF and by coating drug-loaded tubes with layers of poly(lactide-co-glycolide). The conduits made of cross-linked collagen released low initial amounts of GDNF and NGF (2% of both during first 3 days) and enhanced significantly the early (2 weeks) nerve regeneration in terms of axonal outgrowth and Schwann cell migration in a 10 mm rat sciatic nerve gap model, as compared to the conduits made of non-cross-linked collagen releasing higher initial amounts of GDNF and NGF (12-16% within 3 days), or those releasing GDNF alone. The enhancement of early axonal regeneration using controlled co-delivery of multiple synergistic neurotrophic factors is an important requisite for eventually establishing functional connections with the target organ.

Engineering functional bladder tissues

Purpose: End stage bladder disease can seriously affect patient quality of life and often requires surgical reconstruction with bowel tissue, which is associated with numerous complications. Bioengineering of functional bladder tissue using tissue‐engineering techniques could provide new functional tissues for reconstruction. In this review, we discuss the current state of this field and address different approaches to enable physiologic voiding in engineered bladder tissues in the near future. Materials and Methods: In a collaborative effort, we gathered researchers from four institutions to discuss the current state of functional bladder engineering. A MEDLINE® and PubMed® search was conducted for articles related to tissue engineering of the bladder, with special focus on the cells and biomaterials employed as well as the microenvironment, vascularisation and innervation strategies used. Results: Over the last decade, advances in tissue engineering technology have laid the groundwork for the development of a biological substitute for bladder tissue that can support storage of urine and restore physiologic voiding. Although many researchers have been able to demonstrate the formation of engineered tissue with a structure similar to that of native bladder tissue, restoration of physiologic voiding using these constructs has never been demonstrated. The main issues hindering the development of larger contractile tissues that allow physiologic voiding include the development of correct muscle alignment, proper innervation and vascularization. Conclusion: Tissue engineering of a construct that will support the contractile properties that allow physiologic voiding is a complex process. The combination of smart scaffolds with controlled topography, the ability to deliver multiple trophic factors and an optimal cell source will allow for the engineering of functional bladder tissues in the near future. Copyright

Growth factor delivery systems and repair strategies for damaged peripheral nerves

Artificial nerve conduits (NCs) are, in certain cases, instrumental for repairing damaged peripheral nerves, although therapeutic efficacy remains often suboptimal. Considerable efforts have been made to improve the therapeutic performance of NCs. This article reviews recent developments in NC-technology for peripheral nerve regeneration with a main focus on growth factors delivery systems and repair strategies. Commonly used materials for NC fabrication include collagen, silk fibroin, and biodegradable aliphatic polyesters. The basic NC structure, i.e., a hollow tube, can be manufactured by diverse methods: spinning mandrel technology, sheet rolling, injection-molding, freeze-drying, and electro-spinning. Polymeric and cellular delivery systems for growth factors can be integrated in the NC wall or within luminal structures such as gels, fibers, or biological materials providing binding affinity for the bioactive compounds. NCs with sustained growth factor delivery generally improve significantly the axonal outgrowth in nerve defect models, although restoration of sensory and motor functions remains inferior to that achieved with autologous nerve grafts. To improve therapeutic outcomes, further biofunctionalization of NCs will be needed, i.e., adjusting degradation kinetics of NC scaffolding to be compatible with axonal regeneration; delivering multiple growth factors at individually optimized kinetics; incorporating modality specific glial cells and furnishing NCs with guiding nanostructures.

Nerve conduit scaffolds for discrete delivery of two neurotrophic factors.

Axonal repair and regeneration remain critical due to lack of appropriate delivery systems for efficient release of neurotrophic factors (NTFs). Recently, we have demonstrated the synergistic activity of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) on axonal regeneration. Combined delivery of GDNF and NGF with individually controlled release kinetics may be crucial for exploiting their synergistic action on axonal elongation in animals. For engineering discrete NTF release kinetics, we have developed several nerve conduits (NCs) using collagen (Col) and silk fibroin (SF); the NC were made of Col or SF alone, or of Col and SF layers, or of Col/SF blends, all loaded with GDNF and NGF. All NC types provided sustained combined release of NGF and GDNF over 28 days. NC made of combinations of Col and SF showed reduced burst and more sustained dual release of GDNF and NGF. SF/Col-based NC scaffolds provide an adaptable delivery system for growth factors and hold potential for nerve regeneration and possibly for other tissue engineering applications.

Increased porosity of electrospun hybrid scaffolds improved bladder tissue regeneration

The object of this study was to investigate the role of scaffold porosity on tissue ingrowth using hybrid scaffolds consisting of bladder acellular matrix and electrospun poly (lactide-co-glycolide) (PLGA) microfibers that mimic the morphological characteristics of the bladder wall in vitro and in vivo. We compared single-spun (SS) PLGA scaffolds with more porous cospun (CS) scaffolds (PLGA and polyethylene glycol). Scaffolds were characterized by scanning electron microscopy. Bladder smooth muscle cells (SMCs) were seeded, and proliferation and histological assays were performed. Sixteen rats were subjected to augmentation cystoplasty with seeded SS or CS scaffolds, morphological, and histological studies were performed 2 and 4 weeks after implantation. The porosities of SS and CS scaffolds were 73.1 ± 2.9% and 80.9 ± 1.5%, respectively. The in vitro evaluation revealed significantly deeper cell migration into CS scaffolds. The in vivo evaluation showed significant shrinkage of SS scaffolds (p = 0.019). The histological analysis revealed a bladder wall-like structure with urothelial lining and SMC infiltration in both groups. The microvessel density was significantly increased in the CS scaffolds (p < 0.001). Increasing the porosity of electrospun hybrid scaffolds is an effective strategy to enhance cell proliferation and distribution in vitro and tissue ingrowth in vivo.

Scaffold Characteristics for Functional Hollow Organ Regeneration

Many medical conditions require surgical reconstruction of hollow organs. Tissue engineering of organs and tissues is a promising new technique without harvest site morbidity. An ideal biomaterial should be biocompatible, support tissue formation and provide adequate structural support. It should degrade gradually and provide an environment allowing for cell-cell interaction, adhesion, proliferation, migration, and differentiation. Although tissue formation is feasible, functionality has never been demonstrated. Mainly the lack of proper innervation and vascularisation are hindering contractility and normal function. In this chapter we critically review the current state of engineering hollow organs with a special focus on innervation and vascularisation.