In vivo studies of silk based gold nano-composite conduits for functional peripheral nerve regeneration

We report a novel silk-gold nanocomposite based nerve conduit successfully tested in a neurotmesis grade sciatic nerve injury model in rats over a period of eighteen months. The conduit was fabricated by adsorbing gold nanoparticles onto silk fibres and transforming them into a nanocomposite sheet by electrospinning which is finally given a tubular structure by rolling on a stainless steel mandrel of chosen diameter. The conduits were found to promote adhesion and proliferation of Schwann cells in vitro and did not elicit any toxic or immunogenic responses in vivo. We also report for the first time, the monitoring of muscular regeneration post nerve conduit implantation by recording motor unit potentials (MUPs) through needle electromyogram. Pre-seeding the conduits with Schwann cells enhanced myelination of the regenerated tissue. Histo-morphometric and electrophysiological studies proved that the nanocomposite based conduits pre-seeded with Schwann cells performed best in terms of structural and functional regeneration of severed sciatic nerves. The near normal values of nerve conduction velocity (50 m/sec), compound muscle action potential (29.7 mV) and motor unit potential (133 μV) exhibited by the animals implanted with Schwann cell loaded nerve conduits in the present study are superior to those observed in previous reports with synthetic materials as well as collagen based nerve conduits. Animals in this group were also able to perform complex locomotory activities like stretching and jumping with excellent sciatic function index (SFI) and led a normal life.     

A comparison of the performance of mono- and bi-component electrospun conduits in a rat sciatic model

Synthetic nerve conduits represent a promising strategy to enhance functional recovery in peripheral nerve injury repair. However, the efficiency of synthetic nerve conduits is often compromised by the lack of molecular factors to create an enriched microenvironment for nerve regeneration. Here, we investigate the in vivo response of mono (MC) and bi-component (BC) fibrous conduits obtained by processing via electrospinning poly(ε-caprolactone) (PCL) and gelatin solutions. In vitro studies demonstrate that the inclusion of gelatin leads to uniform electrospun fiber size and positively influences the response of Dorsal Root Ganglia (DRGs) neurons as confirmed by the preferential extensions of neurites from DRG bodies. This behavior can be attributed to gelatin as a bioactive cue for the cultured DRG and to the reduced fibers size. However, in vivo studies in rat sciatic nerve defect model show an opposite response: MC conduits stimulate superior nerve regeneration than gelatin containing PCL conduits as confirmed by electrophysiology, muscle weight and histology. The G-ratio, 0.71 ± 0.07 for MC and 0.66 ± 0.05 for autograft, is close to 0.6, the value measured in healthy nerves. In contrast, BC implants elicited a strong host response and infiltrating tissue occluded the conduits preventing the formation of myelinated axons.     

Different types of extrafusal muscle fibres in snake costocutaneous muscles

1. Tonic and twitch muscle fibres were identified physiologically in m. costocutanei superiores and inferiores of garter snakes and grass snakes.

2. Tonic fibres were multiterminally innervated and showed s.j.p.s in response to nerve stimulation. They did not show propagated A.P.s. They were innervated by motor axons with lower conduction velocities than those to twitch fibres, and often gave a contraction and developed tension in response to a single shock to the nerve. Intracellular square pulse analysis showed that C_m = 1 μF/cm² and R_m = 40,000 Ω cm².

3. Twitch fibres showed a conducted action potential in response to nerve stimulation, and focal, as opposed to diffuse, innervation. They showed a variety of isometric twitch contraction times (times-to-peak of about 30-65 msec). Groups of similar motor units contained fibres of approximately similar contraction times. Slow twitch (and tonic) fibres often appeared silvery under dark field illumination, while faster twitch fibres appeared clear. No difference in C_m, R_m or λ was found between faster and slow twitch fibres. Values were approximately 3-4 μF/cm², 3000-4000 Ω cm² and 2 mm respectively.

     

The effect of urine-derived stem cells expressing VEGF loaded in collagen hydrogels on myogenesis and innervation following after subcutaneous implantation in nude mice

Impairment of sphincter muscles or their neural and vascular support leads to stress urinary incontinence. The aim of this study was to determine the role of urine-derived stem cells (USCs) over-expressing vascular endothelial growth factor (VEGF) in collagen-I gel on angiogenesis, cell survival, cell growth, myogenic phenotype differentiation of the implanted cells and innervations following implantation in vivo. USCs were infected with adenovirus containing the human VEGF₁₆₅ and green fluorescent protein genes. A total of 5 × 10⁶ cells, USCs alone, or plus endothelial cells or human skeletal myoblasts (as control) suspended in collagen-I gel were subcutaneously implanted into nude mice. Extensive vascularization and more implanted cells was noted in VEGF-expressing USCs groups compared to the non-VEGF groups in vivo. Numbers of the cells displaying endothelial markers (CD 31 and von Willebrand's factor) and myogenic markers (myf-5, MyoD and desmin), and regenerated nerve fibers displaying neural markers (S-100, GFAP and neurofilament) significantly increased in the grafts of VEGF-expressing USCs. Improved angiogenesis by VEGF-expressing USCs enhanced grafted cell survival, recruited the resident cells and promoted myogenic phenotype differentiation of USCs and innervation. This approach has important clinical implications for the development of cell therapies for the correction of stress urinary incontinence.     

Nerve guides manufactured from photocurable polymers to aid peripheral nerve repair

The peripheral nervous system has a limited innate capacity for self-repair following injury, and surgical intervention is often required. For injuries greater than a few millimeters autografting is standard practice although it is associated with donor site morbidity and is limited in its availability. Because of this, nerve guidance conduits (NGCs) can be viewed as an advantageous alternative, but currently have limited efficacy for short and large injury gaps in comparison to autograft. Current commercially available NGC designs rely on existing regulatory approved materials and traditional production methods, limiting improvement of their design. The aim of this study was to establish a novel method for NGC manufacture using a custom built laser-based microstereolithography (μSL) setup that incorporated a 405 nm laser source to produce 3D constructs with ∼50 μm resolution from a photocurable poly(ethylene glycol) resin. These were evaluated by SEM, in vitro neuronal, Schwann and dorsal root ganglion culture and in vivo using a thy-1-YFP-H mouse common fibular nerve injury model. NGCs with dimensions of 1 mm internal diameter × 5 mm length with a wall thickness of 250 μm were fabricated and capable of supporting re-innervation across a 3 mm injury gap after 21 days, with results close to that of an autograft control. The study provides a technology platform for the rapid microfabrication of biocompatible materials, a novel method for in vivo evaluation, and a benchmark for future development in more advanced NGC designs, biodegradable and larger device sizes, and longer-term implantation studies.     

Orthotopic replacement of the aortic valve with decellularized allograft in a sheep model

Tissue engineered (TE) allografts have been successfully applied in pulmonary circulation. The behavior of TE valves based on decellularized scaffolds in systemic circulation remains unexplored. We investigated the function, histological changes, potential of in-vivo re-endothelialization of decellularized aortic valve allografts in orthotopic position in sheep. Ovine aortic valve conduits (n = 12) were decellularized with detergents and implanted as an aortic root in lambs (35–45 kg). For controls, fresh native ovine aortic valve conduits (n = 6) were implanted. The valves were explanted at 3 and 9 months. In the experimental group, the valves exhibited trivial regurgitation and normal morphology with no signs of graft dilatation, degeneration or rejection. In some animals (n = 2), we documented minimal calcification in the area of arterial anastomosis and in one, microthrombi formation on the leaflet surface. The luminal sides of the grafts were partially covered with an endothelial cell monolayer, neovasculogenesis was observed at the adventitial side. The valves in the control group appeared thickened, shrunken with marked calcification/degeneration signs, and advanced valve insufficiency. Detergent decellularized aortic valve allografts satisfy the higher requirements of the systemic circulation in sheep. As valve conduits become repopulated by endothelial and interstitial cells, they may re-gain the potential for growth.     

Bladder tissue regeneration using acellular bi-layer silk scaffolds in a large animal model of augmentation cystoplasty

Acellular scaffolds derived from Bombyx mori silk fibroin were investigated for their ability to support functional tissue regeneration in a porcine model of augmentation cystoplasty. Two bi-layer matrix configurations were fabricated by solvent-casting/salt leaching either alone (Group 1) or in combination with silk film casting (Group 2) to yield porous foams buttressed by heterogeneous surface pore occlusions or homogenous silk films, respectively. Bladder augmentation was performed with each scaffold group (6 × 6 cm²) in juvenile Yorkshire swine for 3 m of implantation. Augmented animals exhibited high rates of survival (Group 1: 5/6, 83%; Group 2: 4/4, 100%) and voluntary voiding over the course of the study period. Urodynamic evaluations demonstrated mean increases in bladder capacity over pre-operative levels (Group 1: 277%; Group 2: 153%) which exceeded nonsurgical control gains (144%) encountered due to animal growth. Similarly, elevations in bladder compliance were substantially higher in augmented animals from baseline (Group 1: 357%; Group 2: 147%) in comparison to controls (41%). Gross tissue evaluations revealed that both matrix configurations supported extensive de novo tissue formation throughout the entire original implantation site which exhibited ultimate tensile strength similar to nonsurgical counterparts. Histological and immunohistochemical analyses showed that both implant groups promoted comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within defect sites similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent cytokeratin, uroplakin, and p63 protein expression in both matrix groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by synaptophysin-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. Ex vivo organ bath studies demonstrated that regenerated tissues supported by both silk matrices displayed contractile responses to carbachol, α,β-methylene-ATP, KCl, and electrical field stimulation similar to controls. Our data detail the ability of acellular silk scaffolds to support regeneration of innervated, vascularized smooth muscle and urothelial tissues within 3 m with structural, mechanical, and functional properties comparable to native tissue in a porcine model of bladder repair.     

Multiplexed in vivo fluorescence optical imaging of the therapeutic efficacy of photodynamic therapy

In our study we wanted to elucidate a time frame for in vivo optical imaging of the therapeutic efficacy of photodynamic therapy (PDT) by using a multiplexed imaging approach for detecting apoptosis and vascularization. The internalization of the photosensitizer Foslip^® into tongue-squamous epithelium carcinoma cells (CAL-27) was examined in vitro and in vivo. For detecting apoptosis, annexin V was covalently coupled to the near-infrared dye DY-734 and the spectroscopic properties and binding affinity to apoptotic CAL-27 cells were elucidated. CAL-27 tumor bearing mice were treated with PDT and injected 2 days and 2 weeks thereafter with DY-734-annexin V. PDT-induced changes in tumor vascularization were detected with the contrast agent IRDye^® 800CW RGD up to 3 weeks after PDT. A perinuclear enrichment of Foslip^® could be seen in vitro which was reflected in an accumulation in CAL-27 tumors in vivo. The DY-734-annexin V (coupling efficiency 30–50%) revealed a high binding affinity to apoptotic compared to non-apoptotic cells (17.2% vs. 1.2%) with a K_D-value of 20 nm. After PDT-treatment, the probe showed a significantly higher (p <0.05) contrast in tumors at 2 days compared to 2 weeks after therapy (2–8 h post injection). A reduction of the vascularization could be detected after PDT especially in the central tumor areas. To detect the therapeutic efficacy of PDT, a multiplexed imaging approach is necessary. A detection of apoptotic cells is possible just shortly after therapy, whereas at later time points the efficacy can be verified by investigating the vascularization.     

Silk fibroin rods for sustained delivery of breast cancer therapeutics

A silk-protein based reservoir rod was developed for zero-order and long-term sustained drug delivery applications. Silk reservoir rod formulations were processed in three steps. First, a regenerated silk fibroin solution, rich in random-coil content was transformed into a tubular silk film with controllable dimensions, uniform film morphology and a structure rich in silk II, β-sheet content via “film-spinning.” Second, the drug powder was loaded into swollen silk tubes followed by tube end clamping. Last, clamped silk tube ends were sealed completely via dip coating. Anastrozole, an FDA approved active ingredient for the treatment of breast cancer, was used as a model drug to investigate viability of the silk reservoir rod technology for sustained drug delivery. The in vitro and in vivo pharmacokinetic data (in a female Sprague–Dawley rat model) analyzed via liquid chromatography-tandem mass spectroscopy indicated zero-order release for 91 days. Both in vitro and in vivo anastrozole release rates could be controlled simply by varying silk rod dimensions. The swelling behavior of silk films and zero-order anastrozole release kinetics indicated practically immediate film hydration and formation of a linear anastrozole concentration gradient along the silk film thickness. The dependence of anastrozole release rate on the overall silk rod dimensions was in good agreement with an essentially diffusion-controlled sustained release from a reservoir cylindrical geometry. In vivo results highlighted a strong in vitro-in vivo pharmacokinetic correlation and a desirable biocompatibility profile of silk reservoir rods. During a 6-month implantation in rats, the apparent silk molecular weight values decreased gradually, while rod dry mass and β-sheet crystal content values remained essentially constant, providing a suitable timeframe for controlled, long-term sustained delivery applications. Overall, the silk reservoir rod may be a viable candidate for sustained delivery of breast cancer therapeutics.     

Mechano growth factor (MGF) and transforming growth factor (TGF)-β3 functionalized silk scaffolds enhance articular hyaline cartilage regeneration in rabbit model

Damaged cartilage has poor self-healing ability and usually progresses to scar or fibrocartilaginous tissue, and finally degenerates to osteoarthritis (OA). Here we demonstrated that one of alternative isoforms of IGF-1, mechano growth factor (MGF) acted synergistically with transforming growth factor β3 (TGF-β3) embedded in silk fibroin scaffolds to induce chemotactic homing and chondrogenic differentiation of mesenchymal stem cells (MSCs). Combination of MGF and TGF-β3 significantly increased cell recruitment up to 1.8 times and 2 times higher than TGF-β3 did in vitro and in vivo. Moreover, MGF increased Collagen II and aggrecan secretion of TGF-β3 induced hMSCs chondrogenesis, but decreased Collagen I in vitro. Silk fibroin (SF) scaffolds have been widely used for tissue engineering, and we showed that methanol treated pured SF scaffolds were porous, similar to compressive module of native cartilage, slow degradation rate and excellent drug released curves. At 7days after subcutaneous implantation, TGF-β3 and MGF functionalized silk fibroin scaffolds (STM) recruited more CD29+/CD44 + cells (P < 0.05). Similarly, more cartilage-like extracellular matrix and less fibrillar collagen were detected in STM scaffolds than that in TGF-β3 modified scaffolds (ST) at 2 months after subcutaneous implantation. When implanted into articular joints in a rabbit osteochondral defect model, STM scaffolds showed the best integration into host tissues, similar architecture and collagen organization to native hyaline cartilage, as evidenced by immunostaining of aggrecan, collagen II and collagen I, as well as Safranin O and Masson's trichrome staining, and histological evalution based on the modified O'Driscoll histological scoring system (P < 0.05), indicating that MGF and TGF-β3 might be a better candidate for cartilage regeneration. This study demonstrated that TGF-β3 and MGF functionalized silk fibroin scaffolds enhanced endogenous stem cell recruitment and facilitated in situ articular cartilage regeneration, thus providing a novel strategy for cartilage repair.     

Allometry of left ventricular myocardial innervation

Body mass (BM) of terrestrial mammalian species ranges from a few grams in the case of the Etruscan shrew to a few tonnes for an elephant. The mass‐specific metabolic rate, as well as heart rate, decrease with increasing BM, whereas heart mass is proportional to BM. In the present study, we investigated the scaling behaviour of several compartments of the left ventricular myocardium, notably its innervation, capillaries and cardiomyocytes. Myocardial samples were taken from 10 mammalian species with BM between approximately 2 g and 900 kg. Samples were analysed by design‐based stereology and electron microscopy and the resulting data were subjected to linear regression and correlation analyses. The total length of nerve fibres (axons) in the left ventricle increased from 0.017 km (0.020 km) in the shrew to 7237 km (13 938 km) in the horse. The innervation density was similar among species but the mean number of axons per nerve fibre profile increased with rising BM. The total length of capillaries increased from 0.119 km (shrew) to 10 897 km (horse). The volume of cardiomyocytes was 0.017 cm³ in the shrew and 1818 cm³ in the horse. Scaling of the data against BM indicated a higher degree of complexity of the axon tree in larger animals and an allometric relationship between total length of nerve fibres/axons and BM. In contrast, the density of nerve fibres is independent of BM. It seems that the structural components of the autonomic nervous system in the heart are related to BM and heart mass rather than to functional parameters such as metabolic rate.     

Label-free imaging of gelatin-containing hydrogel scaffolds

Composite hyaluronic acid (HA) hydrogels containing gelatin are used in regenerative medicine as tissue-mimicking scaffolds for improving stem cell survival. Once implanted, it is assumed that these biomaterials disintegrate over time, but at present there is no non-invasive imaging technique available with which such degradation can be directly monitored in vivo. We show here the potential of chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) as a label-free non-invasive imaging technique to monitor dynamic changes in scaffold composition in vivo. The CEST properties of the three individual hydrogel components (HA, GelinS, and polyethylene glycol diacrylate) were first measured in vitro. The complete hydrogel was then injected into the brain of immunodeficient rag2^−/− mice and CEST MR images were obtained at day 1 and 7 post-transplantation. In vitro, GelinS gave the strongest CEST signal at 3.6 ppm offset from the water peak, originating from the amide protons present in gelatin. In vivo, a significant decrease in CEST signal was observed at 1 week post-implantation. These results were consistent with the biodegradation of the GelinS component, as validated by fluorescent microscopy of implanted hydrogels containing Alexa Fluor 488-labeled GelinS. Our label-free imaging approach should be useful for further development of hydrogel formulations with improved composition and stability.     

Conductive PPY/PDLLA conduit for peripheral nerve regeneration

The significant drawbacks and lack of success associated with current methods to treat critically sized nerve defects have led to increased interest in neural tissue engineering. Conducting polymers show great promise due to their electrical properties, and in the case of polypyrrole (PPY), its cell compatibility as well. Thus, the goal of this study is to synthesize a conducting composite nerve conduit with PPY and poly(d, l-lactic acid) (PDLLA), assess its ability to support the differentiation of rat pheochromocytoma 12 (PC12) cells in vitro, and determine its ability to promote nerve regeneration in vivo. Different amounts of PPY (5%, 10%, and 15%) are used to synthesize the conduits resulting in different conductivities (5.65, 10.40, and 15.56 ms/cm, respectively). When PC12 cells are seeded on these conduits and stimulated with 100 mV for 2 h, there is a marked increase in both the percentage of neurite-bearing cells and the median neurite length as the content of PPY increased. More importantly, when the PPY/PDLLA nerve conduit was used to repair a rat sciatic nerve defect it performed similarly to the gold standard autologous graft. These promising results illustrate the potential that this PPY/PDLLA conducting composite conduit has for neural tissue engineering.     

Visualization of the tentorial innervation of human dura mater

Posterior projections of the ophthalmic division of the trigeminal nerve (the ophthalmic nerve) are distributed in the tentorium cerebelli as recurrent meningeal branches. We investigated the morphological tentorial distribution of the ophthalmic nerve. Fifty‐two sides of the tentorium cerebelli and adjacent dura mater obtained from 29 human specimens were stained using Sihler's method to examine the nerve fibres in the dural sheets. The innervation patterns of the tentorium cerebelli were classified into the following four types according to their distributions: Type 1, where nerve fibres projected to both the straight and transverse sinuses; Type 2, where nerve fibres projected only to the transverse sinus and lateral convexity; Type 3, where nerve fibres projected medially only to the straight sinus and the posterior part of the falx cerebri; and Type 4, where the nerve fibres terminated within the tentorium cerebelli. Images of the tentorium cerebelli were superimposed to identify areas of dense innervation. The incidence rates of Types 1–4 were 71.2% (n = 37), 21.2% (n = 11), 3.8% (n = 2) and 3.8% (n = 2), respectively. More branches of nerve fibres traversed towards the transverse sinus posterolaterally than towards the straight sinus medially. The space between the anterior half of the straight sinus and the medial tentorial notch can be considered a safe surgical area where innervation is scarce. The posterior part of the falx cerebri was innervated by the ophthalmic nerve that traversed to the straight sinus. The parietal branches of the middle meningeal artery in the lateral convexity that were projected orthogonally by the ophthalmic nerve traversed the transverse sinus, implicating their vulnerability and possible sensitivity under physiological or neurosurgical conditions. This study has revealed the macroscopic tentorial innervation of the dura mater in humans, which could be useful information for both neurosurgeons and neurologists.     

Quantitative examination of calcitonin gene-related peptide immunoreactive nerve fibres in the cat knee joint capsule

 The knee joint of the cat has been used extensively to study the morphology and function of primary afferents in a deep somatic tissue. A proportion of these neurones synthesizes various neuropeptides, with calcitonin gene-related peptide being the most prominent. In the present study we examined the distribution and density of nerve fibres immunoreactive for calcitonin gene-related peptide within the medial articular capsule. The fibres were predominantly located in the superficial layer of the capsule. They formed a dense innervation pattern, mainly accompanying blood vessels. Electron microscopy showed that most fibres were in close proximity to small arteries. The highest innervation density was found in parts of the capsule that were located over the epicondyle of the femur with 21±12 fibres per mm² (mean±SD). In the tissue over the joint cleft this density was lower, with 11±6 fibres per mm². In conclusion, the high innervation density of the knee joint capsule by nerve fibres containing calcitonin gene-related peptide supports the hypothesis of an important regulatory function of this peptide in normal tissue. Accepted: 24 January 1997     

In vivo assessment of guided neural stem cell differentiation in growth factor immobilized chitosan-based hydrogel scaffolds

In this study, we demonstrate that a unique growth factor-biomaterial system can offer spatial control of growth factors with sustained signaling to guide the specific lineage commitment of neural stem/progenitor cells (NSPCs) in vivo. First, recombinant fusion proteins incorporating an N-terminal biotin tag and interferon-γ (IFN-γ), platelet derived growth factor-AA (PDGF-AA), or bone morphogenic protein-2 (BMP-2) were immobilized to a methacrylamide chitosan (MAC) based biopolymer via a streptavidin linker to specify NSPC differentiation into neurons, oligodendrocytes, or astrocytes, respectively. MAC was mixed with growth factors (immobilized or adsorbed), acrylated laminin, NSPCs, and crosslinked within chitosan conduits. This system mimics regenerative aspects of the central nervous system ECM, which is largely composed of a crosslinked polysaccharide matrix with cell-adhesive regions, and adds the new functionality of protein sequestration. We demonstrated that these growth factors are maintained at functionally significant levels for 28 d in vitro. In the main study, immobilized treatments were compared to absorbed and control treatments after 28 d in vivo (rat subcutaneous). Masson's Trichrome staining revealed that small collagen capsules formed around the chitosan conduits with an average acceptable thickness of 153.07 ± 6.02 μm for all groups. ED-1 staining showed mild macrophage clustering around the outside of chitosan conduits in all treatments with no macrophage invasion into hydrogel portions. Importantly, NSPC differentiation staining demonstrated that immobilized growth factors induced the majority of cells to differentiate into the desired cell types as compared with adsorbed growth factor treatments and controls by day 28. Interestingly, immobilized IFN-γ resulted in neural rosette-like arrangements and even structures resembling neural tubes, suggesting this treatment can lead to guided dedifferentiation and subsequent neurulation.     

Silk protein fibroin from Antheraea mylitta for cardiac tissue engineering

The human heart cannot regenerate after an injury. Lost cardiomyocytes are replaced by scar tissue resulting in reduced cardiac function causing high morbidity and mortality. One possible solution to this problem is cardiac tissue engineering. Here, we have investigated the suitability of non-mulberry silk protein fibroin from Indian tropical tasar Antheraea mylitta as a scaffold for engineering a cardiac patch in vitro. We have tested cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication and contractility of 3-days postnatal rat cardiomyocytes on silk fibroin. Our data demonstrate that A. mylitta silk fibroin exhibits similar properties as fibronectin, a component of the natural matrix for cardiomyocytes. Comparison to mulberry Bombyx mori silk protein fibroin shows that A. mylitta silk fibroin is superior probably due to its RGD domains. 3D scaffolds can efficiently be loaded with cardiomyocytes resulting in contractile patches. In conclusion, our findings demonstrate that A. mylitta silk fibroin 3D scaffolds are suitable for the engineering of cardiac patches.     

Specificity of innervation of iris musculature by sympathetic nerve fibres in tissue culture

Summary Irides from 3–5 day old rats have been grown 1–3 mm from superior cervical or lumbar paravertebral sympathetic ganglia in modified Rose chambers.The two muscles of the iris received distinctly different innervation patternsin vitro, and these were similar to those seenin vivo. Varicose, adrenergic fibres were consistently associated with the dilator pupillae rather than with the sphincter pupillae while excitatory, cholinergic junctions developed between the nerve fibres and the muscle cells of the sphincter but not the dilator.There was a lack of specificity shown by the sympathetic neurons during this innervation. Fibres from lumbar ganglia formed plexuses within the dilator similar to those formed by superior cervical fibres, and sympathetic, cholinergic fibres were able to substitute for the normal parasympathetic, cholinergic fibres in the sphincter.     

Improved implant osseointegration of a nanostructured titanium surface via mediation of macrophage polarization

The use of endosseous implanted materials is often limited by undesirable effects that may be due to macrophage-related inflammation. The purpose of this study was to fabricate a nanostructured surface on a titanium implant to regulate the macrophage inflammatory response and improve the performance of the implant. Anodization at 5 and 20 V as well as UV irradiation were used to generate hydrophilic, nanostructured TiO₂ surfaces (denoted as NT5 and NT20, respectively). Their surface characteristics and in vivo osseointegration as well as the inflammatory response they elicit were analyzed. In addition, the behavior of macrophages in vitro was evaluated. Although the in vitro osteogenic activity on the two surfaces was similar, the NT5 surface was associated with more bone formation, less inflammation, and a reduced CD68⁺ macrophage distribution in vivo compared to the NT20 and polished Ti surfaces. Consistently, further experiments revealed that the NT5 surface induced healing-associated M2 polarization in vitro and in vivo. By contrast, the NT20 surface promoted the pro-inflammatory M1 polarization, which could further impair bone regeneration. The results demonstrate the dominant role of macrophage-related inflammation in bone healing around implants and that surface nanotopography can be designed to have an immune-regulating effect in support of the success of implants.     

Three-dimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration

Acellular scaffolds from complex whole organs such as lung are being increasingly studied for ex vivo organ generation and for in vitro studies of cell–extracellular matrix interactions. We have established effective methods for efficient de and recellularization of large animal and human lungs including techniques which allow multiple small segments (∼1–3 cm³) to be excised that retain 3-dimensional lung structure. Coupled with the use of a synthetic pleural coating, cells can be selectively physiologically inoculated via preserved vascular and airway conduits. Inoculated segments can be further sliced for high throughput studies. Further, we demonstrate thermography as a powerful noninvasive technique for monitoring perfusion decellularization and for evaluating preservation of vascular and airway networks following human and porcine lung decellularization. Collectively, these techniques are a significant step forward as they allow high throughput in vitro studies from a single lung or lobe in a more biologically relevant, three-dimensional acellular scaffold.