Traumatic spinal cord injury (SCI) is a damage to the spinal cord that results in loss or impaired motor and/or sensory function. SCI is a sudden and unexpected event characterized by high morbidity and mortality rate during both acute and chronic stages, and it can be devastating in human, social and economical terms. Despite significant progresses in the clinical management of SCI, there remain no effective treatments to improve neurological outcomes. Among experimental strategies, bioengineered scaffolds have the potential to support and guide injured axons contributing to neural repair. The major aim of this study was to investigate a novel composite type I collagen scaffold with micropatterned porosity in a rodent model of severe spinal cord injury. After segment resection of the thoracic spinal cord we implanted the scaffold in female Sprague-Dawley rats. Controls were injured without receiving implantation. Behavioral analysis of the locomotor performance was monitored up to 55 days postinjury. Two months after injury histopathological analysis were performed to evaluate the extent of scar and demyelination, the presence of connective tissue and axonal regrowth through the scaffold and to evaluate inflammatory cell infiltration at the injured site. We provided evidence that the new collagen scaffold was well integrated with the host tissue, slightly ameliorated locomotor function, and limited the robust recruitment of the inflammatory cells at the injury site during both the acute and chronic stage in spinal cord injured rats. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016.

Crosslinking and denaturation were two variables that deeply affected the performance of collagen-based scaffolds designed for tissue regeneration. If crosslinking enhances the mechanical properties and the enzymatic resistance of collagen, while masking or reducing the available cell binding sites, denaturation has very opposite effects, as it impairs the mechanical and the enzymatic stability of collagen, but increases the number of exposed cell adhesive domains. The quantification of both crosslinking and denaturation was thus fundamental to the design of collagen-based scaffolds for selected applications. The aim of this work was to investigate the extents of crosslinking and denaturation of collagen-based films upon dehydrothermal (DHT) treatment, that is, one of the most commonly employed methods for zero-length crosslinking that shows the unique ability to induce partial denaturation. Swelling measurements, differential scanning calorimetry, Fourier transform infrared spectroscopy, colorimetric assays for the quantification of primary amines, and mechanical tests were performed to analyze the effect of the DHT temperature on crosslinking and denaturation. In particular, chemically effective and elastically effective crosslink densities were evaluated. Both crosslinking and denaturation were found to increase with the DHT temperature, although according to different trends. The results also showed that DHT treatments performed at temperatures up to 120°C maintained the extent of denaturation under 25%. Coupling a mild DHT treatment with further crosslinking may thus be very useful not only to modulate the crosslink density, but also to induce a limited amount of denaturation, which shows potential to partially compensate the loss of cell binding sites caused by crosslinking. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 186-194, 2016.

Autologous nerve grafting is the current gold standard treatment for peripheral nerve injury, in cases where direct suturing of nerve ends is not possible. Even though the functional restoration achieved by the autograft is not optimal, autologous nerve tissues still show higher regenerative capability than several synthetic conduits available in the clinical setting, the latter used only for gaps that do not exceed 3 cm in length. The aim of this chapter is to highlight how bio-mimicry, inspired by nerve development, structure and spontaneous regeneration following mild nerve injury, can help in the design of synthetic templates with optimized bioactivity for nerve regeneration.

A porous collagen-based hydrogel scaffold was prepared in the presence of iron oxide nanoparticles (NPs) and was characterized by means of infrared spectroscopy and scanning electron microscopy. The hybrid scaffold was then loaded with fluorescein sodium salt as a model compound. The release of the hydrosoluble species was triggered and accurately controlled by the application of an external magnetic field, as monitored by fluorescence spectroscopy. The biocompatibility of the proposed matrix was also tested by the MTT assay performed on 3T3 cells. Cell viability was only slightly reduced when the cells were incubated in the presence of the collagen-NP hydrogel, compared to controls. The economicity of the chemical protocol used to obtain the paramagnetic scaffolds as well as their biocompatibility and the safety of the external trigger needed to induce the drug release suggest the proposed collagen paramagnetic matrices for a number of applications including tissue engeneering and drug delivery.

The microstructural, mechanical, compositional, and degradative properties of a nerve conduit are known to strongly affect the regenerative process of the injured peripheral nerve. Starting from the fabrication of micropatterned collagen-based nerve guides, according to a spin-casting process reported in the literature, this study further investigates the possibility to modulate the degradation rate of the scaffolds over a wide time frame, in an attempt to match different rates of nerve regeneration that might be encountered in vivo. To this aim, three different crosslinking methods, that is, dehydrothermal (DHT), carbodiimide-based (EDAC), and glutaraldehyde-based (GTA) crosslinking, were selected. The elastically effective degree of crosslinking, attained by each method and evaluated according to the classical rubber elasticity theory, was found to significantly tune the in vitro half-life (t1/2) of the matrices, with an exponential dependence of the latter on the crosslink density. The high crosslinking efficacy of EDAC and GTA treatments, respectively threefold and fourfold when compared to the one attained by DHT, led to a sharp increase of the corresponding in vitro half-lives (ca., 10, 172, and 690 h, for DHT, EDAC, and GTA treated matrices, respectively). As shown by cell viability assays, the cytocompatibility of both DHT and EDAC treatments, as opposed to the toxicity of GTA, suggests that such methods are suitable to crosslink collagen-based scaffolds conceived for clinical use. In particular, nerve guides with expected high residence times in vivo might be produced by finely controlling the biocompatible reaction(s) adopted for crosslinking.

Collagen is one of the most used materials in scaffolding production; this is due to its peculiar characteristics that make the polymer highly biocompatible and efficient in regeneration induction and growth cone guidance. We aimed to investigate whether collagen could per se induce Schwann cell differentiation/proliferation and how it would do so. Results obtained in immortalized rat Schwann cells showed differential effects on several proliferation and differentiation markers depending on the type of collagen used to produce the scaffolds.

Dehydrothermal (DHT) crosslinking is routinely performed to increase the stiffness and the enzymatic resistance of collagen-based devices. Amide and ester bonds are formed among the collagen macromolecules, as a result of the high temperatures and high vacuum involved in the process. The extent of crosslinking is known to increase with the DHT temperature and duration, but simultaneous collagen denaturation might be induced. The aim of this work was to investigate the extent of crosslinking and denaturation of DHT-treated collagen-based films, by means of thermal and physicochemical analyses. With the ultimate goal of optimizing the DHT process, five different temperatures (110, 120, 140, 160 and 180°C) were used, while the DHT duration was kept constant (24 hours). Differential scanning calorimetry (DSC) was carried out to measure the denaturation temperature (Td) and enthalpy (ΔHd) of the collagen films. The reaction of 2,4,6-trinitrobenzenesulfonic acid (TNBS) with primary amines (-NH2) allowed determining the number of free -NH2 in the collagen films, whereas Fourier transform infrared spectroscopy (FTIR) was used to investigate the chemical modifications occurring upon DHT treatment. Higher degrees of crosslinking were attained for increasing DHT temperatures, as demonstrated by reduced number of free -NH2, lower absorbance of amide II band (1545 cm-1) and higher Td values. However, the sharp reduction of ΔHd detected for samples treated at 140, 160 and 180°C indicated a significant denaturation associated to crosslinking. The analysis of the absorbance band at 1236 cm-1 confirmed that collagen denaturation was particularly pronounced for DHT temperatures higher than 120°C, suggesting that, at those temperatures, denaturation might predominate over crosslinking. Further stress relaxation tensile tests and dynamic mechanical analysis (DMA) are currently being performed to measure the stiffness of DHT-treated samples and to estimate the elastically effective crosslink density, according to the rubber elasticity theory.

The aim of this work was to investigate the structural features of type I collagen isoforms and collagen-based films at atomic and molecular scales, in order to evaluate whether and to what extent different protocols of slurry synthesis may change the protein structure and the final properties of the developed scaffolds. Wide Angle X-ray Scattering data on raw materials demonstrated the preferential orientation of collagen molecules in equine tendon-derived collagens, while randomly oriented molecules were found in bovine skin collagens, together with a lower crystalline degree, analyzed by the assessment of FWHM (Full Width at Half Maximum), and a certain degree of salt contamination. WAXS and FT-IR (Fourier Transform Infrared) analyses on bovine collagen-based films, showed that mechanical homogenization of slurry in acidic solution was the treatment ensuring a high content of super-organization of collagen into triple helices and a high crystalline domain into the material. In vitro tests on rat Schwannoma cells showed that Schwann cell differentiation into myelinating cells was dependent on the specific collagen film being used, and was found to be stimulated in case of homogenization-treated samples. Finally DHT/EDC crosslinking treatment was shown to affect mechanical stiffness of films depending on collagen source and processing conditions.

The development of biocompatible collagen substrates able to conduct electric current along specific pathways represent an appealing issue in tissue engineering, since it is well known that electrical stimuli significantly affects important cell behaviour, such as proliferation, differentiation, directional migration, and, therefore, tissue regeneration. In this work, a cheap and easy approach was proposed to produce collagen-based films exhibiting enhanced electrical conductivity, through the simple manipulation of a weak external magnetic trigger. Paramagnetic iron oxide nanoparticles (NPs) capped by a biocompatible polyethylene-glycol coating were synthetized by a co-precipitation and solvothermic method and sprayed onto a collagen suspension. The system was then subjected to a static external magnetic field in order to conveniently tune NPs organization. Under the action of the external stimulus, NPs were induced to orient along the magnetic field lines, forming long-range aligned micropatterns within the collagen matrix. Drying of the substrate following water evaporation permanently blocked the magnetic architecture produced, thereby preserving NPs organization even after magnetic field removal. Electrical conductivity measurements clearly showed that the presence of such a magnetic framework endowed collagen with marked conductive properties in specific directions. The biocompatibility of the paramagnetic collagen films was also demonstrated by MTT cell cytotoxicity test.

The development of bio-devices for complete regeneration of ligament and tendon tissues is presently one of the biggest challenges in tissue engineering. Such device must simultaneously possess optimal mechanical performance, suitable porous structure, and biocompatible microenvironment. This study proposes a novel collagen-BDDGE-elastin (CBE)-based device for tendon tissue engineering, by the combination of two different modules: (i) a load-bearing, non-porous, "core scaffold" developed by braiding CBE membranes fabricated via an evaporative process and (ii) a hollow, highly porous, "shell scaffold" obtained by uniaxial freezing followed by freeze-drying of CBE suspension, designed to function as a physical guide and reservoir of cells to promote the regenerative process. Both core and shell materials demonstrated good cytocompatibility in vitro, and notably, the porous shell architecture directed cell alignment and population within the sample. Finally, a prototype of the core module was implanted in a rat tendon lesion model, and histological analysis demonstrated its safety, biocompatibility, and ability to induce tendon regeneration. Overall, our results indicate that such device may have the potential to support and induce in situ tendon regeneration.

Poly(ethylene glycol) diacrylate (PEGDA) cryogels, particularly useful for biotechnological applications, are currently fabricated exploiting crosslinking systems that require long freezing/crosslinking times (20 h or longer). The aim of this work was to assess whether fast UV irradiation (up to 60 s) of frozen PEGDA solutions could be an advantageous alternative for cryogel production. By using different polymer concentrations and UV times, cryogels with highly interconnected macropores (about 50–90 μm) were produced. A gelation yield in the range 60–80% was recorded, with higher values obtained for low PEGDA concentrations (5 and 10% w/v). Interestingly, while decreasing the swelling and increasing the stiffness of the cryogels, a higher polymer concentration was also found to reduce the pore size. Furthermore, increasing the UV time resulted in significantly higher swelling and larger pores for 10% PEGDA samples, while having negligible effect on other cryogel types and/or features. Although deserving further exploration, fast UV irradiation is an effective method to produce PEGDA cryogels with tunable properties.

In tissue engineering field, the production of a porous resorbable matrix, termed scaffold, allows to host cells and guide them towards the synthesis of physiological tissue. Porous scaffolds provide mechanical stability and an initial framework for migrating cells and vascular infiltration. Sustained delivery of bioactive molecules at the defect site may be also particularly important for tissue regeneration. In this context, the goal of this work was the fabrication of highly porous collagen-based scaffolds incorporating uniformly dispersed poly(lactide-co-glycolide) (PLGA) microparticles as depots for the sustained and localized delivery of bioactive molecules. Collagen scaffolds loaded with different amounts of PLGA-microparticles were prepared by freeze-drying and crosslinking. The scaffolds microstructure was assessed to evaluate the spatial distribution of microparticles and the achieved pore size. The impact of the microparticles on the scaffolds stiffness was investigated through compression tests. Preliminarily, the cell-microparticles interactions were also evaluated by imaging of cell morphology in vitro, adopting a human derived epithelial cell model. The experimental findings showed that collagen scaffolds with different amounts of uniformly dispersed PLGA-microparticles were successfully produced. The microparticles did not negatively affect the scaffold porous structure, while acting as a mechanical reinforcement. Additionally, microparticles show high permissiveness to cell adhesion, and the interactions between microparticles and epithelial cell membranes did not interfere with the correct cells morphological differentiation. Such promising results suggest the potential of the developed scaffolds for tissue engineering applications.

INTRODUCTION Peripheral nerve injuries often result in painful neuropathies owing to reduction in motor function and sensory perception. When large nerve gaps exist (20mm or longer in humans), sensory nerve autografts are conventionally used to treat neural defects. The main issues related to autografts are shortage of donor nerves, a mismatch of donor nerve size with the recipient site, and occurrences of neuroma formation. Recent advances in nanotechnology and tissue engineering have been found to cover a broad range of applications in regenerative medicine and offer the most effective strategy to repair neural defects. Prior work in this area has shown the utility of collagen-based scaffolds for the regeneration of nerve tissue. This work focuses on the fabrication of collagen scaffolds with two different pore sizes, with the aim of evaluating the effects of pore size on the migration of Schwann cell lines. EXPERIMENTAL METHODS Scaffold fabrication and crosslinking Porous cylindrical scaffolds (diameter=2mm, length=10mm) with aligned channels were fabricated by freeze-drying a 2wt% collagen suspension along a one-dimensional temperature gradient (along the length of the cylindrical scaffold). Scaffolds with two different pore sizes were fabricated by freezing the collagen suspension at two different final freezing temperatures (-20°C and -60°C). The scaffolds were then subjected to dehydrothermal (DHT) cross-linking, followed by a carbodiimide based chemical crosslinking. Qualitative characterization of the pore structure was performed by means of scanning electron microscopy (SEM). Cell culture and cytocompatibility A rat Schwann cell line, RSC96, was expanded in monolayer culture in a 96-well plate. The plate was then incubated at 37°C and 5% CO2 for 24 hours. After 24 hours, sterilized scaffolds were placed vertically to the wells of the 96-well plate and incubated again at 37°C and 5% CO2. At 1, 3, 7, and 10 days, the cell-seeded scaffolds were fixed in 10% formalin and processed for paraffin embedding. Schwann cells were quantified by embedding the cell-seeded scaffolds in paraffin blocks, sectioning, staining them with hematoxylin & eosin stain (H&E stain) and visualizing under a microscope. MTT assay was also performed at 1, 3, 7 and 10 days to evaluate the cell viability. RESULTS AND DISCUSSION SEM demonstrated that both freezing temperature and rate of freezing affect significantly the pore size. As shown in Fig.1, lower temperatures (-60°C) resulted in smaller pore sizes (~85µm), while higher temperatures (-20°C) resulted in much larger pores (~120µm). The longitudinal sections of the samples showed that the pores were in axial orientation disregard of the freezing temperature. MTT assay revealed that cell viability on the two different types of scaffolds increased gradually from first to the tenth day after seeding. Although there was not much difference between the two porous scaffolds on day 1, 3 and 7, on day 10 there was a slight increase in the cell number in the scaffolds with a larger pore size (-20°C). In spite of the different pore dimensions under investigation, the cell migration studies revealed that Schwann cells could migrate through the entire length of both types of scaffolds, by day 7. CONCLUSION Both types of scaffolds were found to support Schwann cell growth and migration, which is the key factor required for the regeneration of nerve tissue. Further studies are proposed regarding the addition of laminin and the evaluation of its effects on the cell growth and migration. REFERENCES 1. W. Daly et al., J. R. Soc. Interface 9:202-221, 2012.

In this chapter, we aim at providing an up-to-date review on nerve tissue engineering, focusing on both the peripheral and the central nervous systems (PNS and CNS, respectively). After introducing the pathophysiology of nerves and the social impact of nerve injuries, we overview the therapeutic approaches oriented toward inducing nerve regeneration, involving cellular, molecular, and scaffold-based strategies. A section is dedicated specifically to the PNS, with a critical focus on the actual therapeutic potential of experimental devices for the development of tissue-engineered medical products. A case study regarding the implementation of micropatterned collagen-based conduits in a clinical trial on PNS regeneration is also presented. Another section is dedicated to the ongoing research investigating the regenerative mechanisms of the CNS. In this context, spinal cord injury is assumed as a model lesion, for which complex tissue-engineered devices are being developed, at least in animal studies. With such a structure, this chapter is intended to provide a comprehensive, though not exhaustive, overview of nerve tissue engineering, which might be useful to students, researchers, clinicians, and biomedical entrepreneurs.

The specific design of a collagen scaffold containing iron oxide nanostructures capped by a TiO2 (anatase) layer is reported. The TiO2 shell is proposed with a dual role: as an innovative and biocompatible cross-linker agent, providing binding sites to the protein moiety, through the well-known TiO2 chemical affinity towards carboxyl groups, and as a protective surface layer from oxidation for the paramagnetic core. Simultaneously, the presence of the nanostructures confers to the collagen gel the sensitivity to an external stimulus, i.e. the application of a magnetic field. The hybrid biomaterial was demonstrated to be healthy and was proposed as a smart scaffold for the on demand release of bioactive compounds. The tunable release upon magnetic field application of a model protein, i.e. myoglobin, was investigated. Myoglobin was loaded in the microporous material and the discharging was induced by consecutive magnet applications, obtaining the release of the protein with a high spatio-temporal and dosage control.

Several bioengineering approaches have been proposed for peripheral nervous system repair, with limited results and still open questions about the underlying molecular mechanisms. We assessed the biological processes that occur after the implantation of collagen scaffold with a peculiar porous microstructure of the wall in a rat sciatic nerve transection model compared to commercial collagen conduits and nerve crush injury using functional, histological and genome wide analyses. We demonstrated that within 60 days, our conduit had been completely substituted by a normal nerve. Gene expression analysis documented a precise sequential regulation of known genes involved in angiogenesis, Schwann cells/axons interactions and myelination, together with a selective modulation of key biological pathways for nerve morphogenesis induced by porous matrices. These data suggest that the scaffold's microstructure profoundly influences cell behaviors and creates an instructive micro-environment to enhance nerve morphogenesis that can be exploited to improve recovery and understand the molecular differences between repair and regeneration.

The aim of this work was the superficial activation, by means of plasma treatments, of crosslinked collagen-based scaffolds for nerve regeneration, in order to immobilize anionic and cationic microcapsules (MCPs) for drug delivery. Matrices with axially oriented pores have the potential to improve the regeneration of peripheral nerves and spinal cord by physically supporting and guiding the growth of neural structures across the site of injury. To improve mechanical resistance and stability in water solutions, it is necessary to crosslink collagenous fibres by formation of amide bonds with consequent reduction of free amino and carboxylic groups useful for immobilization approach of drug delivery systems like MCPs. Plasma chemical processes represent a successful approach because allow polar groups to be grafted on the surface, without modifying the massive properties of the bulk. Plasma surface modification was performed in a capacitively-coupled rf (13.56 MHz) glass reactor fed with different precursors like N2, H2O, C2H4 to study the effect of plasma parameters on the chemical properties of the resulting material and its ability to improve the immobilization of polyelectrolyte MCPs. Cylindrical scaffolds were synthesized by freeze-drying technique and dehydrothermally crosslinked. Polyelectrolyte capsules were obtained by LbL method. Scaffolds were characterized by means of WCA and XPS. Fluorescence microscopy was used to verify MCPs immobilization. After treatments, scaffolds became hydrophilic and able to absorb water. The success of grafting, on the external surface and within the scaffold core, was clearly revealed. The obtained results demonstrate that plasma processing of cross-linked collagen allows to enhance MCPs immobilization and that, by changing the typology of functional groups on the plasma treated surfaces, a different attitude to immobilize negatively or positively charged MCPs is observed.

In this study we investigated the impact of three different sterilization methods, dry heat (DHS), ethylene oxide (EtO) and electron beam radiation (β), on the properties of cylindrical collagen scaffolds with longitudinally oriented pore channels, specifically designed for peripheral nerve regeneration. Scanning electron microscopy, mechanical testing, quantification of primary amines, differential scanning calorimetry and enzymatic degradation were performed to analyze possible structural and chemical changes induced by the sterilization. Moreover, in vitro proliferation and infiltration of the rat Schwann cell line RSC96 within the scaffolds was evaluated, up to 10 days of culture. No major differences in morphology and compressive stiffness were observed among scaffolds sterilized by the different methods, as all samples showed approximately the same structure and stiffness as the unsterilized control. Proliferation, infiltration, distribution and morphology of RSC96 cells within the scaffolds were also comparable throughout the duration of the cell culture study, regardless of the sterilization treatment. However, we found a slight increase of chemical crosslinking upon sterilization (EtO < DHS < β), together with an enhanced resistance to denaturation of the EtO treated scaffolds and a significantly accelerated enzymatic degradation of the β sterilized scaffolds. The results demonstrated that β irradiation impaired the scaffold properties to a greater extent, whereas EtO exposure appeared as the most suitable method for the sterilization of the proposed scaffolds.

Le lesioni del midollo spinale nell’uomo sono molto eterogenee. L’incapacità di rigenerazione del midollo lesionato è attribuita all’instaurarsi di un ambiente inibitorio e alla formazione di una cicatrice gliale che funge da barriera chimica e meccanica alla rigenerazione assonale. L’impianto di scaffold microporosi rappresenta una valida strategia per guidare la rigenerazione, nel tentativo di ripristinare i collegamenti con i target di innervazione e promuovere il recupero funzionale. Porosità, distribuzione delle dimensioni dei pori, area superficiale specifica, interconnettività ed orientazione dei pori sono parametri cruciali che influenzano la bioattività dello scaffold. Lo scopo del presente lavoro è quello di modulare e caratterizzare la struttura microporosa di scaffold cilindrici in collagene, con porosità orientata in direzione longitudinale o assiale, destinati ad uno studio sulla rigenerazione del midollo spinale. Gli scaffold (3mm diametro, 3 cm lunghezza) sono stati realizzati mediante freezing unidirezionale di sospensioni di collagene di tipo I da derma bovino (a diverse concentrazioni), liofilizzazione e reticolazione termica e chimica. La porosità degli scaffold è stata quindi analizzata qualitativamente e quantitativamente mediante microscopia elettronica a scansione e ottica, al fine di determinare morfologia, omogeneità, diametro medio e grado di orientazione dei pori. L’analisi delle sezioni trasversali e longitudinali degli scaffold ha mostrato rispettivamente una distribuzione pressoché omogenea e una buona orientazione uniassiale dei pori per tutte le tipologie di campioni analizzate, evidenziando una leggera diminuzione della dimensione media all’aumentare della concentrazione di collagene utilizzata in fase di sintesi. Tuttavia, si è anche osservato un gradiente crescente del diametro medio dei pori lungo l’asse longitudinale degli scaffold, legato al gradiente di temperatura che si instaura durante il processo di freezing uniassiale. Inoltre, i trattamenti di reticolazione investigati sembrano non influenzare significativamente la microstruttura. Studi futuri saranno rivolti a comprendere l’effetto della microstruttura sul comportamento di cellule neuronali, immortalizzate e primarie, in vitro.

In this work, we synthesized porous nanohydroxyapatite/collagen composite scaffold (nHA-COL), which resemble extracellular matrices in bone and cartilage tissues. Nano hydroxyapatite (nHA) was successfully nucleated in to the collagen matrix using hen eggshell as calcium biogenic source. Porosity was evaluated by apparent and theoretical density measurement. Porosity of all scaffolds was in the range of 95–98%. XRD and TEM analyses show the purity and size of nucleated HA around 10 nm and selected area electron diffraction (SAED) analysis reveals the polycrystalline nature of nucleated HA. SEM analysis reveals (i) all the scaffolds have interconnected pores with an average pore diameter of 130 micron and (ii) aggregates of hydroxyapatite were strongly embedded in the collagen matrix for both composite scaffolds compared with pure collagen scaffold. EDS analysis shows the Ca/P stoichiometric ratio around 1.67 and FTIR reveals the chemical interaction between the collagen molecule and HA particles. The testing of mechanical properties evidenced that incorporation of HA resulted in up to a two-fold increase in compressive modulus with high reinforcement level (∼ 7 kPa for 50HA–50COL) compared to pure collagen scaffold.

Porosity is a key parameter in the design of tissue engineering scaffolds, as bioactivity can be controlled and tailored to the synthesis of the target tissue by finely tuning the porous structure of the scaffolding biomaterial. This chapter discusses the effect of structural parameters, such as pore volume fraction, pore size and distribution, pore shape, pore interconnectivity and pore orientation, on the performance of sponge-like scaffolds, with a special focus on those directed to nerve regeneration.

This study evaluated a tendon substitute model. Tenocytes were isolated from pig Achilles tendon, seeded onto scaffolds (Opocrin 2%, Typeone 3% and Symatese 2%) and studied by histology, immunofluorescence for collagen type 1 and 3 and biochemical analysis to assess cellularity. The permeability of these compounds was evaluated in the presence or absence of fibrin glue. Opocrin 2% was the best choice for cellular distribution within the scaffolds, which were then cultured for T0, T4, T7 and T10 days. Fibrin glue has been strongly supportive for the survival of cells with a significant increase in DNA content at T10 (P<0.05). Moreover, the synthetic activity of fibrin-free scaffolds was always negative. Lastly, a progressive increase in collagen 1 and 3 with fibrin-glue was observed. However, static culture is not sufficient to support long-term cellular activities and at T10 there is still a lack of organized matrix similar to the native tissue.