Development of Novel Polysaccharide Membranes for Guided Bone Regeneration: In Vitro and In Vivo Evaluations.

INTRODUCTION: Guided bone regeneration (GBR) procedures require selecting suitable membranes for oral surgery. Pullulan and/or dextran-based polysaccharide materials have shown encouraging results in bone regeneration as bone substitutes but have not been used to produce barrier membranes. The present study aimed to develop and characterize pullulan/dextran-derived membranes for GBR. MATERIALS AND METHODS: Two pullulan/dextran-based membranes, containing or not hydroxyapatite (HA) particles, were developed. In vitro, cytotoxicity evaluation was performed using human bone marrow mesenchymal stem cells (hBMSCs). Biocompatibility was assessed on rats in a subcutaneous model for up to 16 weeks. In vivo, rat femoral defects were created on 36 rats to compare the two pullulan/dextran-based membranes with a commercial collagen membrane (Bio-Gide®). Bone repair was assessed radiologically and histologically. RESULTS: Both polysaccharide membranes demonstrated cytocompatibility and biocompatibility. Micro-computed tomography (micro-CT) analyses at two weeks revealed that the HA-containing membrane promoted a significant increase in bone formation compared to Bio-Gide®. At one month, similar effects were observed among the three membranes in terms of bone regeneration. CONCLUSION: The developed pullulan/dextran-based membranes evidenced biocompatibility without interfering with bone regeneration and maturation. The HA-containing membrane, which facilitated early bone regeneration and offered adequate mechanical support, showed promising potential for GBR procedures.

Soft Hydrogel Environments that Facilitate Cell Spreading and Aggregation Preferentially Support Chondrogenesis of Adult Stem Cells.

Mesenchymal stem/stromal cells (MSCs) represent a promising cell type for treating damaged synovial joints. The therapeutic potential of MSCs will be facilitated by the engineering of biomaterial environments capable of directing their fate. Here the interplay between matrix elasticity and cell morphology in regulating the chondrogenic differentiation of MSCs when seeded onto or encapsulated within hydrogels made of interpenetrating networks (IPN) of alginate and collagen type I is explored. This IPN system enables the independent control of substrate stiffness (in 2D and in 3D) and cell morphology (3D only). The expression of chondrogenic markers SOX9, ACAN, and COL2 increases when MSCs are cultured onto the soft substrate, which correlates with increased SMAD2/3 nuclear localization, enhanced MSCs condensation, and the formation of larger cellular aggregates. The encapsulation of spread MSCs within a soft IPN increases the expression of cartilage-specific genes, which is linked to cellular condensation and nuclear SMAD2/3 localization. Surprisingly, cells forced to adopt a more rounded morphology within the same soft IPNs expressed higher levels of the osteogenic markers RUNX2 and COL1. The insight provided by this study suggests that a mechanobiology informed approach to biomaterial development will be integral to the development of successful cartilage tissue engineering strategies.

Microbiological Evaluation and Sperm DNA Fragmentation in Semen Samples of Patients Undergoing Fertility Investigation.

Fifteen percent of male infertility is associated with urogenital infections; several pathogens are able to alter the testicular and accessory glands' microenvironment, resulting in the impairment of biofunctional sperm parameters. The purpose of this study was to assess the influence of urogenital infections on the quality of 53 human semen samples through standard analysis, microbiological evaluation, and molecular characterization of sperm DNA damage. The results showed a significant correlation between infected status and semen volume, sperm concentration, and motility. Moreover, a high risk of fragmented sperm DNA was demonstrated in the altered semen samples. Urogenital infections are often asymptomatic and thus an in-depth evaluation of the seminal sample can allow for both the diagnosis and therapy of infections while providing more indicators for male infertility management.

Membranes for Guided Bone Regeneration: A Road from Bench to Bedside.

Bone resorption can negatively influence the osseointegration of dental implants. Barrier membranes for guided bone regeneration (GBR) are used to exclude nonosteogenic tissues from influencing the bone healing process. In addition to the existing barrier membranes available on the market, a growing variety of membranes for GBR with tailorable physicochemical properties are under preclinical evaluation. Hence, the aim of this review is to provide a comprehensive description of materials used for GBR and to report the main industrial and regulatory aspects allowing the commercialization of these medical devices (MDs). In particular, a summary of the main attributes defining a GBR membrane is reported along with a description of commercially available and under development membranes. Finally, strategies for the scaling-up of the manufacturing process and the regulatory framework of the main MD producers (USA, EU, Japan, China, and India) are presented. The description of the regulatory approval process of GBR membranes is representative of the typical path that medium- to high-risk MDs have to follow for an effective medical translation, which is of fundamental importance to increase the impact of biomedical research on public health.

Synthesis of cationic quaternized pullulan derivatives for miRNA delivery.

Pullulan is a natural polysaccharide of potential interest for biomedical applications due to its non-toxic, non-immunogenic and biodegradable properties. The aim of this work was to synthesize cationic pullulan derivatives able to form complexes with microRNAs (miRNAs) driven by electrostatic interaction (polyplexes). Quaternized ammonium groups were linked to pullulan backbone by adding the reactive glycidyltrimethylammonium chloride (GTMAC). The presence of these cationic groups within the pullulan was confirmed by elemental analysis, Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR). The alkylated pullulan was able to interact with miRNA and form stable polyplexes that were characterized regarding size, zeta potential and morphology. The presence of miRNA was confirmed by agarose gel electrophoresis and UV spectrophotometry. In vitro tests on human umbilical vein endothelial cells did not show any cytotoxicity after 1 day of incubation with nanosized polyplexes up to 200 µg/mL. QA-pullulan was able to promote miRNA delivery inside cells as demonstrated by fluorescence microscopy images of labelled miRNA. In conclusion, the formation of polyplexes using cationic derivatives of pullulan with miRNA provided an easy and versatile method for polysaccharide nanoparticle production in aqueous media and could be a new promising platform for gene delivery.

Hydrostatic Pressure Regulates the Volume, Aggregation and Chondrogenic Differentiation of Bone Marrow Derived Stromal Cells.

The limited ability of articular cartilage to self-repair has motivated the development of tissue engineering strategies that aim to harness the regenerative potential of mesenchymal stem/marrow stromal cells (MSCs). Understanding how environmental factors regulate the phenotype of MSCs will be central to unlocking their regenerative potential. The biophysical environment is known to regulate the phenotype of stem cells, with factors such as substrate stiffness and externally applied mechanical loads known to regulate chondrogenesis of MSCs. In particular, hydrostatic pressure (HP) has been shown to play a key role in the development and maintenance of articular cartilage. Using a collagen-alginate interpenetrating network (IPN) hydrogel as a model system to tune matrix stiffness, this study sought to investigate how HP and substrate stiffness interact to regulate chondrogenesis of MSCs. If applied during early chondrogenesis in soft IPN hydrogels, HP was found to downregulate the expression of ACAN, COL2, CDH2 and COLX, but to increase the expression of the osteogenic factors RUNX2 and COL1. This correlated with a reduction in SMAD 2/3, HDAC4 nuclear localization and the expression of NCAD. It was also associated with a reduction in cell volume, an increase in the average distance between MSCs in the hydrogels and a decrease in their tendency to form aggregates. In contrast, the delayed application of HP to MSCs grown in soft hydrogels was associated with increased cellular volume and aggregation and the maintenance of a chondrogenic phenotype. Together these findings demonstrate how tailoring the stiffness and the timing of HP exposure can be leveraged to regulate chondrogenesis of MSCs and opens alternative avenues for developmentally inspired strategies for cartilage tissue regeneration.

Evaluation of bone marrow stem cell response to PLA scaffolds manufactured by 3D printing and coated with polydopamine and type I collagen.

The majority of synthetic polymers used in 3 D printing are not designed to promote specific cellular interactions and hence possess limited bioactivity. Most of the strategies proposed to overcome this limitation demand multiple and expensive processing steps. This study aimed to evaluate the surface modification of 3D-printed poly(lactic acid) (PLA) scaffolds with polydopamine (PDA) coating as an alternative strategy to enhance their bioactivity and to facilitate the immobilization of type I collagen (COL I) onto the implant surface. Physical and chemical properties of PLA scaffolds coated with PDA, COL I or both were evaluated. The response of porcine bone marrow stem cells (MSCs) to the coatings was also investigated. The PDA layer improved COL immobilization onto the surface of the PLA scaffolds by 92%. The combination of PDA and COL functionalizations provided the best conditions for early-stage (<7 days) cell response. In addition, the PDA plus COL surface facilitated the robust deposition of extracellular matrix in the first 14 days of cell culture. Although the behavior of the MSCs appeared to be similar for both uncoated PLA and PDA plus COL-coated scaffolds by day 21, cells seeded onto PDA plus COL scaffolds produced substantially higher amounts of alkaline phosphatase. These results indicate that the osteoinductivity of 3D-printed PLA scaffolds can be enhanced by PDA and type I collagen coatings. This surface modification of polymeric scaffolds represents a promising strategy for bone tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 37-49, 2019.

All that glitters is not gold: sacroiliitis.

OBJECTIVES: The aim of this study was to determine the prevalence of "rheumatic" and "non-rheumatic" changes of the sacro-iliac joints (SIJ). MATERIAL AND METHODS: We performed MRI in 210 patients with suspected inflammatory low back pain. We sorted and analysed the characteristics of sacroiliac bone lesions in "rheumatic" and "non-rheumatic" patients and assessed the diagnostic values of their extent and location. SIJ lesions were classified on the basis of their location into two categories: unilateral and bilateral. Their extent was then measured and assigned to one of two groups: <1 cm or ≥ 1 cm. RESULTS: In 45 cases (21%), the MRI findings matched the clinical diagnosis of "rheumatic" sacroiliitis. Interestingly, in 99 cases (47%) the SIJ changes were classified as "non-rheumatic". L5-S1 degenerative changes, scoliosis and pelvic asymmetry were most frequently encountered as concomitant phenomena in our study. CONCLUSIONS: MRI of the sacroiliac joints in patients suspected of inflammatory low back pain demonstrated more often "non-rheumatic" changes.

Decoration of RGD-mimetic porous scaffolds with engineered and devitalized extracellular matrix for adipose tissue regeneration.

Fat grafting is emerging as a promising alternative to silicon implants in breast reconstruction surgery. Unfortunately, this approach does not provide a proper mechanical support and is affected by drawbacks such as tissue resorption and donor site morbidity. Synthetic scaffolds can offer a valuable alternative to address these challenges, but poorly recapitulate the biochemical stimuli needed for tissue regeneration. Here, we aim at combining the positive features of a structural, synthetic polymer to an engineered, devitalized extracellular matrix (ECM) to generate a hybrid construct that can provide a mix of structural and biological stimuli needed for adipose tissue regeneration. A RGD-mimetic synthetic scaffold OPAAF, designed for soft tissue engineering, was decorated with ECM deposited by human adipose stromal cells (hASCs). The adipoinductive potential of the hybrid ECM-OPAAF construct was validated in vitro, by culture with hASC in a perfusion bioreactor system, and in vivo, by subcutaneous implantation in nude mouse. Our findings demonstrate that the hybrid ECM-OPAAF provides proper mechanical support and adipoinductive stimuli, with potential applicability as off-the-shelf material for adipose tissue reconstruction. STATEMENT OF SIGNIFICANCE: In this study we combined the functionalities of a synthetic polymer with those of an engineered and subsequently devitalized extracellular matrix (ECM) to generate a hybrid material for adipose tissue regeneration. The developed hybrid ECM-OPAAF was demonstrated to regulate human adipose stromal cells adipogenic commitment in vitro and adipose tissue infiltration in vivo. Our findings demonstrate that the hybrid ECM-OPAAF provide proper mechanical support and adipoinductive stimuli and represents a promising off-the-shelf material for adipose tissue reconstruction. We believe that our approach could offer an alternative strategy for adipose tissue reconstruction in case of mastectomy or congenital abnormalities, overcoming the current limitations of autologous fat based strategies such as volume resorption and donor site morbidity.

Magnetic Resonance Imaging in degenerative disease of the lumbar spine: Fat Saturation technique and contrast medium.

PURPOSE: To examine both anterior and posterior elements of the lumbar spine in patients with low back pain using MRI T2-weighted sequences with Fat Saturation (FS) and contrast enhanced T1-weighted sequences with FS. MATERIALS AND METHODS: Two thousand eight hundred and twenty (2820) patients (1628 male, 1192 female, mean age 54) presenting low back pain underwent MRI standard examination (Sagittal T1w TSE and T2w TSE, axial T1 SE) with the addition of sagittal and axial T2w Fat Sat (FS) sequences. Among all the patients, 987 (35%) have been studied adding Contrast Enhanced (CE) T1w FS sequences after administration of contrast medium. RESULTS: Among 987 patients studied with contrast medium, we found: active-inflammatory intervertebral osteochondrosis in 646 (65%) patients; degenerative-inflammatory changes in facet joints (facet joint effusion, synovitis, synovial cysts) in 462 (47%); spondylolysis in 69 (7%); degenerative-inflammatory changes of the flava, interspinous and supraspinous ligaments in 245 (25%); inflammatory changes of posterior perispinal muscles in 84 (8%) patients. CONCLUSIONS: In patients with suspected no-disc-related low back pain, the implementation of T2w FS and CE T1w FS sequences to the standard MR protocol could allow a better identification of degenerative-inflammatory changes more likely associated to the pain.

Computed Tomography and MR Imaging in Spondyloarthritis.

This article provides an overview of the computed tomography (CT) and MR imaging appearances suggestive of spondyloarthritis, with a specific emphasis on the MR imaging findings of vertebral and sacroiliac involvement, and presents relevant clinical features that assist early diagnosis. CT is a sensitive imaging modality for the assessment of structural bone changes, but its clinical utility is limited. MR imaging is the modality of choice for early diagnosis, because of its ability to depict inflammation long before structural bone damage occurs, for monitoring of disease activity, and for evaluating therapeutic response.

Biologically and mechanically driven design of an RGD-mimetic macroporous foam for adipose tissue engineering applications.

Despite clinical treatments for adipose tissue defects, in particular breast tissue reconstruction, have certain grades of efficacy, many drawbacks are still affecting the long-term survival of new formed fat tissue. To overcome this problem, in the last decades, several scaffolding materials have been investigated in the field of adipose tissue engineering. However, a strategy able to recapitulate a suitable environment for adipose tissue reconstruction and maintenance is still missing. To address this need, we adopted a biologically and mechanically driven design to fabricate an RGD-mimetic poly(amidoamine) oligomer macroporous foam (OPAAF) for adipose tissue reconstruction. The scaffold was designed to fulfil three fundamental criteria: capability to induce cell adhesion and proliferation, support of in vivo vascularization and match of native tissue mechanical properties. Poly(amidoamine) oligomers were formed into soft scaffolds with hierarchical porosity through a combined free radical polymerization and foaming reaction. OPAAF is characterized by a high water uptake capacity, progressive degradation kinetics and ideal mechanical properties for adipose tissue reconstruction. OPAAF's ability to support cell adhesion, proliferation and adipogenesis was assessed in vitro using epithelial, fibroblast and endothelial cells (MDCK, 3T3L1 and HUVEC respectively). In addition, in vivo subcutaneous implantation in murine model highlighted OPAAF potential to support both adipogenesis and vessels infiltration. Overall, the reported results support the use of OPAAF as a scaffold for engineered adipose tissue construct.

Muscle acellular scaffold as a biomaterial: effects on C2C12 cell differentiation and interaction with the murine host environment.

The extracellular matrix (ECM) of decellularized organs possesses the characteristics of the ideal tissue-engineering scaffold (i.e., histocompatibility, porosity, degradability, non-toxicity). We previously observed that the muscle acellular scaffold (MAS) is a pro-myogenic environment in vivo. In order to determine whether MAS, which is basically muscle ECM, behaves as a myogenic environment, regardless of its location, we analyzed MAS interaction with both muscle and non-muscle cells and tissues, to assess the effects of MAS on cell differentiation. Bone morphogenetic protein treatment of C2C12 cells cultured within MAS induced osteogenic differentiation in vitro, thus suggesting that MAS does not irreversibly commit cells to myogenesis. In vivo MAS supported formation of nascent muscle fibers when replacing a muscle (orthotopic position). However, heterotopically grafted MAS did not give rise to muscle fibers when transplanted within the renal capsule. Also, no muscle formation was observed when MAS was transplanted under the xiphoid process, in spite of the abundant presence of cells migrating along the laminin-based MAS structure. Taken together, our results suggest that MAS itself is not sufficient to induce myogenic differentiation. It is likely that the pro-myogenic environment of MAS is not strictly related to the intrinsic properties of the muscle scaffold (e.g., specific muscle ECM proteins). Indeed, it is more likely that myogenic stem cells colonizing MAS recognize a muscle environment that ultimately allows terminal myogenic differentiation. In conclusion, MAS may represent a suitable environment for muscle and non-muscle 3D constructs characterized by a highly organized structure whose relative stability promotes integration with the surrounding tissues. Our work highlights the plasticity of MAS, suggesting that it may be possible to consider MAS for a wider range of tissue engineering applications than the mere replacement of volumetric muscle loss.