Histological assessment of new bone formation with biomimetic scaffold in posterolateral lumbar spine fusion.

Spinal fusion procedures often require the use of bone grafts (autograft or allograft) to help bone healing and to increase stability. However, the application of autografts is frequently limited by donor site morbidity. In recent years, different synthetic bone substitutes have been introduced in the clinical practice to overcome these limitations. The purpose of this paper is to report a case where a biomimetic, synthetic and osteoconductive bone graft substitute was successfully implanted in a patient during lumbar spine arthrodesis. The case of a 58-year-old female subjected to lumbar spine arthrodesis with bone augmentation is described. The bone graft substitute RegenOss® (Finceramica, Faenza, Italy) was implanted during spinal arthrodesis. The successful bone integration was evaluated by X-rays. After 11 months, the patient underwent a second surgery due to spine imbalance; the debris of the bone graft was therefore collected and analyzed by macroscopic evaluation and by histology. The bone substitute was successfully implanted during a spinal arthrodesis procedure. Histologic evaluation of the removed bone graft debris showed the complete resorption of the implant and the formation of new bone, which was well integrated with the host bone. This bone substitute may represent a safe and effective alternative to autologous bone grafts, avoiding adverse events related to donor-site morbidity.

Clinical anatomy of the meniscus in animal models: pros and cons.

Nowadays, despite the possibility to use in vitro or computer models in research, animal models are still essential. Different animal models are available for meniscal repair investigation. Although a unique perfect model for the structure of the human's knee does not exist, the choice of the proper animal model is crucial for a correct research. The principal animal models in the meniscal repair are sheep, goats, pigs and dogs. Each of these has pros and cons for their utilization. Analysing each pro and con is essential for optimizing the choice of the animal model, which depends on the experimental question, avoiding unnecessary waste of resources and minimizing the animal suffering, according to the Russell and Burch's three "Rs" principles (Reduce, Refine and Recycle). In this concise review, we resume the meniscus anatomical features of the main large animals, to help choose the most suitable animal model for subsequent studies on meniscal repair.

Histological assessment of new bone formation with biomimetic scaffold in the presence of bone loss in trauma surgery.

Open reduction and internal fixation (ORIF) surgery may require the use of bone grafts (usually allogeneic). In the context of traumatology surgeries, the use of autologous grafts is almost never used and allogeneic grafts are not always available. In recent years, bone substitutes have been introduced in clinical practice to overcome these limitations. The purpose of this paper is to report two cases in which the use of a bone substitute was used to overcome the bone loss during surgeries of ORIF. Two patients, one with a tibial plateau fracture (Schatzker 6) and one with a proximal humerus fracture (Neer 4), underwent ORIF surgery. In both cases, due to a loss of bone stock, a synthetic bone substitute (OrthOss®) was used. One year after surgery, the complete osseointegration of the synthetic bone substitute was seen, both radiologically and histologically. This bone substitute may represent a safe and effective alternative to autologous bone grafts, avoiding adverse events related to donor-site morbidity.

Decorin age-related variations in the distribution of pig extracellular matrix meniscus.

Menisci act like shock absorbers and transmit load across the tibiofemoral joint by increasing congruency during movements or body weight load. This leads to decreasing the resultant stress on the articular cartilages. The meniscus has a dense extracellular matrix (ECM) composed of water, different types of collagens, and proteoglycans, such as decorin, aggrecan and biglycan. Decorin (DCN) regulates collagen fibrillogenesis acting on collagen fibrils diameter and fibrils orientation to achieve the proper assembly of its network. This work investigates the spatial disposition of this fundamental protein in pig meniscus' matrix by immunohistochemistry and western blot analysis. DCN shows an increasing trend, moving from neonatal to adult pig menisci. Adult meniscus, in porcine species, is the only one that could be considered fully mature and functional, and, even if an increasing trend is seen, no precise phenotypical switch points are seen in the age stages considered in this study.

Animal models for cartilage repair.

Cartilage lesions still represent an unsolved problem: despite the efforts of the basic and translational research, the regeneration of this tissue is far from being reached (1-3). Articular cartilage lesions can be divided in two main groups: superficial or partial defects and full-thickness defects (4, 5). Partial lesions are not able to self-heal because multipotent cells from the bone marrow cannot reach the area leading to a progressive degeneration of the tissue (6). Conversely, full-thickness injuries possess greater chances to heal because subchondral bone involvement allows for the migration of mesenchymal cells, which fill the damaged area (7, 8). However, healing occurs through the formation of a fibrocartilaginous tissue, which has different biomechanical and biological properties (9). Native hyaline cartilage has indeed specific biomechanical properties, which confer resistance to compressive and shear stresses; the reparative fibrocartilaginous tissue lacks these abilities, therefore, the surrounding healthy cartilage progressively degenerates. In the past years, several therapeutic strategies have been developed to restore the damaged cartilage, bone marrow stimulation (chondroabrasion, drilling, micro- or nano-fractures) and more recently, tissue engineering approaches (10-14). Some of these latter procedures have already been applied in clinical practice such as matrix-induced autologous chondrocyte implantation (MACI) (15) or osteochondral scaffold implantation (16). Generally, tissue engineering approaches are based on the combination of three main elements: cells (i.e. primary chondrocytes or multipotent mesenchymal cells), biocompatible scaffolds (i.e. polymers, composites, ceramics) and signaling molecules (i.e. growth factors). Moreover, several culture conditions (i.e. static or dynamic cultures) and biomechanical stimuli can be applied during the in vitro culture to promote tissue maturation (17-19). However, an in vivo culture is mandatory to validate a new engineered construct as the in vitro phase lacks the essential in vivo environmental stimuli and because the in vivo culture allows for the testing of the biocompatibility and safety of a new material (18, 19). Moreover, preclinical animal models are crucial to understand the molecular mechanisms of cartilage lesions favoring the development of new regenerative strategies (20, 21). in vivo studies on animal models should focus on the analysis of the cellular component, analyzing the maintenance of the cellular phenotype and the tumorigenicity; on the evaluation of the biocompatibility, toxicity and degradation of the biomaterial and on the assessment of the engineered construct. In this manuscript, we will review the most common preclinical animal models, which are used to understand cartilage biology and therefore to develop new tissue engineering strategies. We will focus on both small and large animal models highlighting their peculiarities, advantages and drawbacks.

Characterization of different in vitro culture conditions to induce a fibro-chondrogenic differentiation of swine adipose-derived stem cells.

Meniscus repair is still a challenge for orthopaedic surgeons as this tissue has a scarce healing potential due to the limited vascularization and to the lack of progenitor cells. Lately, several tissue engineering strategies combining cells and scaffolds have been developed. Adipose-derived stem cells (ASCs) represent a novel cell source for meniscus repair as they are easy to harvest, and they possess an intrinsic chondrogenic potential. The aim of our study was to analyze and compare the chondrogenic differentiation of swine ASCs cultured in vitro with different supplemented media. We isolated ASCs from swine adipose tissue and we placed in pellet cultures supplemented with either Bone Morphogenetic Protein (BMP)-2 and Transforming Growth Factor (TGF)-ß3 or with Bone Morphogenetic Protein (BMP)-7 and Transforming Growth Factor (TGF)-ß1. Samples were analyzed after 7, 14 or 21 days of culture by biochemical, histological and gene expression analysis. Our preliminary results show that BMP-2 and TGF-ß3 are stronger inducers of chondrogenesis.

Histological changes of the meniscus following an osteochondral lesion.

In the last few years, different tissue engineering strategies have been developed for the repair of osteochondral lesions. When the osteochondral scaffold is implanted on the femoral condyle, the meniscus might be affected by the implant and might undergo a progressive degeneration. The aim of our study is to analyze the morphological changes of the meniscus following an osteochondral lesion and the implant of a biphasic scaffold. A critical osteochondral defect was generated in the medial femoral condyle of mature sheep. Three defects were left untreated, the remaining lesions were divided into three groups and treated with a biphasic substituted formed by collagen type I and Wollastonite or Wollastonite/Hydroxyapatite. Animals were sacrificed after 6 months and menisci were isolated and analyzed by arthro-CT, macroscopic evaluation and histology. The results demonstrated that the osteochondral lesion negatively affects meniscus morphology and that the osteochondral substitute only partially mitigates the meniscus degeneration.

Comparison between different cell sources and culture strategies for tendon tissue engineering.

The aim of this study was to evaluate the effect of an in vitro mechanical stimulation by the use of a bioreactor on an engineered tendon for 7 and 14 days and to analyze the effect of the use of different cell sources: tenocytes, dermal fibroblasts or Adipose-Derived Stem Cells (ASCs), isolated from pig tissues. Histology showed a re-organization of the neo-tissue derived from the three cell populations along the direction of the stimulus. At T7, cells morphology was preserved while an increased cellular suffering at T14 was observed for all cell populations. Tenocytes exhibited higher survival than other cells. A stable immunopositivity for collagen type 1 or 3 at both time points was also observed. In conclusion, dermal fibroblasts and ASCs represent an interesting alternative and in vitro culture with mechanical stimuli may enhance the maturation of a tendon-like tissue.

The analysis of different scaffolds and the benefit of fibrin glue for tendon tissue engineering at different culture times.

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.

The meniscus vascularization: the direct correlation with tissue composition for tissue engineering purposes.

Meniscal lesions still represent an unsolved problem in clinical practice. Like the articular cartilage, meniscus has a scarce healing potential. Thus, when this tissue is damaged, the joint biomechanics is completely altered, leading to the development and progression of premature osteoarthritis. Therefore, in the last years, several tissue-engineering strategies have been developed to regenerate the meniscus with debated results. The comprehension of complex processes underlying meniscus maturation and structure is essential for a correct approach for the generation of a biomimetic meniscal substitute. In this chapter, we will first review the morphology of the meniscus during growth, focusing on the unique pattern of vascularization, and then we will discuss the most common tissue engineering strategies for meniscus repair.

Comparison of three novel biphasic scaffolds for one-stage treatment of osteochondral defects in a sheep model.

In the last years, several tissue engineering techniques have been applied to develop different kinds of osteochondral substitutes to overcome the scarce reparative properties of this tissue. The aim of this study was to generate and compare three biphasic scaffolds in an osteochondral lesion in a large-animal model. A critical osteochondral defect was generated in the medial femoral condyle of 18 skeletally mature sheep. Three defects were left untreated, the remaining lesions were divided into three groups: 5 lesions were treated with a biphasic scaffold made of collagen type I and small cylinders of Magnesium Hydroxyapatite; 5 lesions were treated with a biphasic substituted formed by collagen type I and Wollastonite, 5 lesions were treated with a scaffold made of collagen type I and small cylinders of Wollastonite/Hydroxyapatite. Animals were sacrificed after 3 months and samples were analyzed by CT and MRI, macroscopic evaluation and histology. Our study demonstrated that one of these novel biphasic scaffolds possesses the potential for being applied for one-stage procedures for osteochondral defects.

Cartilage canals in newborn dogs: histochemical and immunohistochemical findings.

Cartilage canals (CCs) are microscopic structures involved in secondary ossification centers (SOCs) development. The features of CCs were investigated in the humeral and femoral proximal epiphyses of small-sized newborn dogs (from premature to 28 days after birth) with histochemical and immunohistochemical approaches. Masson's Trichrome revealed a ring-shaped area around CCs, which changes in colour from green (immature collagen) to red (mature collagen) as ossification progresses; perichondrium staining always matched the ring colour. Safranin-O was always negative. Immunohistochemical analysis revealed immunopositivity for both collagen type I and V around the CCs; collagen type II was negative. CCs count showed a tendency to be higher in the humerus than in the femur. This work enlightened for the first time changes in composition of CCs surrounding matrix during SOCs development in dogs, paving the way to further investigations.

The effects of dietary verbascoside on blood and liver oxidative stress status induced by a high n-6 polyunsaturated fatty acids diet in piglets.

Twenty-four weaned female Hypor piglets (10.9 ± 0.1 kg mean BW) were used to evaluate the antioxidant effect of a natural extract, titrated in verbascoside, on blood and liver oxidative status in relation to a high intake of n-6 PUFA, inducing oxidative stress. Piglets were assigned to 1 of 3 experimental groups; the first group was fed a diet with 9% sunflower oil (T1) and the second received the sunflower oil diet supplemented with 5 mg of verbascoside/kg feed from Verbenaceae extract (Lippia spp.; T2). The third group was fed a control diet (CTR), in which an isoenergetic replacement of oil by starch was done. Blood samples were collected at the beginning and the end of the trial (30 d). At the end of the trial, the animals were slaughtered and the liver specimens were collected. Oxidative stress markers, including total antiradical activity, superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activities, were determined in blood samples. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transferase (GGT) plasma levels were also evaluated. Immunohistochemistry and western blot analyses were performed in liver to evaluate heat shock protein (Hsp) 70, Hsp90, and Kupffer and Ito cell activation. Liver activities of SOD, GPX, and CAT were also determined. Total antiradical activity in blood and red blood cells were affected (P < 0.01) by dietary treatments. The n-6 PUFA supplementation at a high dosage for 30 d induced oxidative stress, decreasing total antiradical activity in blood and red blood cells (CTR vs. T1 + T2; P < 0.01) and plasma CAT activity (CTR vs. T1 + T2; P = 0.088) and increasing ALT value (CTR vs. T1 + T2; P < 0.01). Also, in liver, the CAT and GPX activities tended to be lower in pigs fed n-6 PUFA diets than pigs fed a control diet (CTR vs. T1 + T2; = 0.090 and = 0.085, respectively). The liver samples presented a normal architecture and no Ito and Kupffer cell activations were observed. In liver, the SOD activity tended to be lower in the T1 group (P = 0.064) than in the CTR and T2 groups. Moreover, the level of Hsp70 was higher (P < 0.01) in the T1 group than the CTR and T2 groups. These data suggest that the dose of dietary verbascoside partially restores the antioxidant status of the liver without affecting the systemic responses to oxidative stress induced by a high-fat diet.

Effects of Enteromyxum scophthalmi experimental infection on the neuroendocrine system of turbot, Scophthalmus maximus (L.).

Enteromyxum scophthalmi is an intestinal myxosporean parasite responsible for serious outbreaks in turbot Scophthalmus maximus (L.) culture, in North-western Spain. The disease affects the digestive tract, provokes severe catarrhal enteritis, emaciation and high rates of mortality. The digestive parasitization triggers a response with the coordinate participation of immune and neuroendocrine systems through the action of peptides released by enteroendocrine cells and present in nervous elements, acting as neuro-immune modulators. The present study was designed to assess the response of the turbot neuroendocrine system against E. scophthalmi infection. Immunohistochemical tests were applied to sections of the gastrointestinal tract of uninfected and E. scophthalmi-infected turbot to characterize the presence of bombesin (BOM), glucagon (GLUC), somatostatin (SOM), leu-enkephalin (LEU) and met-enkephalin (MET). The occurrence of E. scophthalmi in the turbot gastrointestinal tract increased the number of enteroendocrine cells immunoreactive to SOM, LEU and MET. On the other hand, BOM and GLUC immunoreactive cells were less numerous in the gastrointestinal tract of the parasitized turbot. Scarce immunoreactivity to BOM, GLUC and SOM was observed in nerve fibres and neurons of the myenteric plexus of control and infected fish. The results indicate that E. scophthalmi infection in turbot induced changes in the neuroendocrine system, with the diminution of the anorexigenic peptides BOM and GLUC; the increase of enkephalins, related to pro-inflammatory processes; and the increase of SOM, which may cause inhibitory effects on the immune response, constituting a compensatory mechanism to the exacerbated response observed in E. scophthalmi-infected turbot.

The risk of misinterpreting genital signs of sexual abuse in cadavers: a case report.

The significance of genital findings in a case of suspected child sexual abuse has been widely debated in the past decades, as shown by the different classifications available in literature. In the case of postmortem examination, the search for signs of sexual abuse is considerably more difficult because of the superimposition of postmortem modifications, which may determine tissue modifications that can be mistaken for traumatic lesions. This study aims at reporting a case where presumed findings of the first autopsy were denied by histological analysis; in detail, what looked like a possible bruise of the hymen was correctly recognized as hypostasis (livor) of the hymenal tissue by histological analysis. This case report suggests caution in the analysis and discussion of genital lesions found during postmortem examination since the superimposition of cadaveric modifications may radically modify the morphology of soft tissues.

Effect of silver nanocoatings on catheters for haemodialysis in terms of cell viability, proliferation, morphology and antibacterial activity.

The onset of infections associated to bacterial proliferation and biofilm formation on indwelling medical devices represents the major risk of morbidity and mortality among patients. In order to contain the risk of infections in clinical practice, there is a growing interest nowadays in silver-based products due to the strong antimicrobial efficacy of silver against a broad spectrum of microorganisms. In this work, temporary catheters for haemodialysis were coated with silver nano-particles through the in situ photo-reduction of a silver salt in alcoholic solution. A homogeneous distribution of silver particles firmly bonded to the substrate was obtained through the adopted technique. An optimisation study was required to define the amount of silver, in order to obtain good efficacy against Gram-positive and Gram-negative bacteria and no cytotoxic effect. At this purpose, three concentrations of silver, 0.1, 0.25 and 0.5 wt%, have been deposited and tested with respect to bacterial reduction percentage and cellular response. Particularly, bacterial enumeration on Escherichia coli and Staphylococcus aureus, and BrdU incorporation, TUNEL assay and Actin staining on a selected primary cell population were performed on catheters treated with the different silver solutions. The silver percentages tested demonstrated strong antibacterial properties together with a good cellular response, thus indicating that the developed product could be proposed in clinical practice and that the lower percentage tested can be preferred with evident advantages in terms of costs.

The use of the anti-Glycophorin A antibody in the detection of red blood cell residues in human soft tissue lesions decomposed in air and water: a pilot study.

The distinction between antemortem and postmortem wounds is one of the most important medico-legal problems. In fresh cadavers the macroscopic examination of haemorrhagic infiltration can be sufficient to reveal the vitality of a wound but in more difficult cases (putrefied corpses) histological and histochemical analyses need to be performed. The scope of this study was to detect the vitality of soft tissue samples in an advanced state of putrefaction using a monoclonal anti-human Glycoforin A antibody in order to evaluate the presence of red blood cells or red blood cell residues. Samples of skin wounds were taken from cadavers with a known time survival between trauma and death, and then submitted to a simulated putrefaction procedure. The skin samples were left to decompose for 30 days in air and in water and analyzed at a time interval of 3-6-15-30 days. These samples were stained with haematoxylin eosin stain, trichrome stain and with immunohistochemical stains. Results showed that in the air red blood cells could be seen for up to six days and granular deposits of Glycophorin reactive material for up to 15 days whereas no red blood cells were recognizable after 30 days. In water red cell putrefaction (or non-detection) was faster than in the air: after six days only amorphous granular deposits slightly reactive to Glycophorin could be seen. Nonetheless results showed that extravasated red blood cell residues can still be detected in skin bruises left to decompose in air and water, for up to at least 15 days in the open air and one week in water.

Role of autologous rabbit adipose-derived stem cells in the early phases of the repairing process of critical bone defects.

Adipose-derived stem cells (ASCs) may represent a novel and efficient tool to promote bone regeneration. In this study, rabbit ASCs were expanded in culture and used for the regeneration of full-thickness bone defects in the proximal epiphysis of tibia of 12 New Zealand rabbits. Defects were implanted with graft material as follows: untreated (control), empty hydroxyapatite (HA) disk, ASCs alone, and HA disk seeded with ASCs. Each isolated ASCs population was tested in vitro: they all showed a high proliferation rate, a marked clonogenic ability, and osteogenic differentiation potential. Eight weeks after implantation, macroscopic analyses of all the samples showed satisfactory filling of the lesions without any significant differences in term of stiffness between groups treated with or without cells (p > 0.05). In both the scaffold-treated groups, a good osteointegration was radiographically observed. Even if HA was not completely reabsorbed, ASCs-loaded HA displayed a higher scaffold resorption than the unloaded ones. Histological analyses showed that the osteogenic abilities of the scaffold-treated defects was greater than those of scaffold-free samples, and in particular new formed bone was more mature and more similar to native bone in presence of ASCs. These results demonstrated that autologous ASCs-HA constructs is a potential treatment for the regeneration of bone defects.

The chemical code of porcine enteric neurons and the number of enteric glial cells are altered by dietary probiotics.

BACKGROUND: The enteric nervous system (ENS) contains chemically coded populations of neurons that serve specific functions for the control of the gastrointestinal tract. The ability of neurons to modify their chemical code in response to luminal changes has recently been discovered. It is possible that enteric neuronal plasticity may sustain the adaptability of the gut to changes in intestinal activity or injury, and that gut neurons may respond to an altered intestinal environment by changing their neuropeptide expression. METHODS: We used immunohistochemical methods to investigate the presence and localization of several neuronal populations and enteric glia in both the small (ileum) and large (cecum) intestine of piglets. We assessed their abundance in submucosal and myenteric plexus from animals treated with the probiotic Pediococcus acidilactici compared with untreated controls. KEY RESULTS: The treated piglets had a larger number of galanin- and calcitonin gene-related peptide (CGRP)-immunoreactive neurons than controls, but this was limited to the submucosal plexus ganglia of the ileum. Moreover, immunohistochemistry revealed that glial fibrillary acidic protein-positive enteric glial cells were significantly higher in the inner and outer submucosal plexuses of treated animals. CONCLUSIONS & INFERENCES: The neuronal and glial changes described here illustrate plasticity of the ENS in response to an altered luminal environment in the gastrointestinal tract.

Endometrial cytology and computerized morphometric analysis of epithelial nuclei: a useful tool for reproductive diagnosis in the bitch.

New diagnostic approaches are required to recognize early canine hypofertility or infertility. We suggest that the identification of different cytologic types, cellular aspects, and nuclear features of the endometrial epithelial cells may be suitable for this purpose. This study was performed on the bitch (Canis familiaris) during the physiologic reproductive cycle and in uterine diseases. We also applied computerized cytomorphometry to evaluate nuclear area, perimeter, diameter, density, aspect, and roundness of endometrial epithelial cells in healthy dogs (N=35) at different stages of the reproductive cycle (before puberty, during proestrus, estrus, diestrus, and anestrus) and in bitches affected by uterine disorders (N=10). The stage of the estrous cycle was determined by vaginal cytology and progesterone evaluation and also confirmed by clinical and histologic observations. Samples for endometrial cytology were collected in vivo by uterine flushing with transcervical uterine cannulation. After uterine sampling, each dog underwent OHE or uterine stump revision. Cytologic analyses were compared with histologic examinations to verify the uterine condition. The uterine cellular population was represented by endometrial epithelial cells, erythrocytes, neutrophils, lymphocytes, eosinophils, macrophages, plasma cells, and cervical or incidental vaginal cells. Bacteria and amorphous material were observed. The proportion of different cells and nuclear features in the cytologic samples varied throughout the stages of the reproductive cycle and between normal and pathologic uterine conditions. The computer-assisted nuclear morphometry, performed in cytologic specimens by means of the six nuclear parameters chosen to evaluate the endometrial epithelial cell population, proved to be useful for determining the stage of the reproductive cycle. Furthermore, this system was demonstrated to be a valid support to diagnose and distinguish uterine disorders.