Hydroxyapatite particle shape affects screw attachment in cancellous bone when augmented with hydroxyapatite-containing hydrogels.

Screw-bone construct failures are a true challenge in orthopaedic implant fixation, particularly in poor quality bone. Whilst augmentation with bone cement can improve the primary stability of screws, there are cements, e.g. PMMA, that may impede blood flow and nutrients and hamper bone remodelling. In this study, soft, non-setting biomaterials based on Hyalectin gels and hydroxyapatite (HA) particles with different morphological parameters were evaluated as potential augmentation materials, using a lapine ex vivo bone model. The pull-out force, stiffness, and work to fracture were considered in evaluating screw attachment. The pull-out force of constructs reinforced with Hyalectin containing irregularly shaped nano-HA and spherically shaped micro-HA particles were found to be significantly higher than the control group (no augmentation material). The pull-out stiffness increased for the micro-HA particles and the work to fracture increased for the irregular nano-HA particles. However, there were no significant augmentation effect found for the spherical shaped nano-HA particles. In conclusion, injectable Hyalectin gel loaded with hydroxyapatite particles was found to have a potentially positive effect on the primary stability of screws in trabecular bone, depending on the HA particle shape and size.

An Injectable, Shape-Retaining Collagen Hydrogel Cross-linked Using Thiol-Maleimide Click Chemistry for Sealing Corneal Perforations.

Injectable hydrogels show great promise in developing novel regenerative medicine solutions and present advantages for minimally invasive applications. Hydrogels based on extracellular matrix components, such as collagen, have the benefits of cell adhesiveness, biocompatibility, and degradability by enzymes. However, to date, reported collagen hydrogels possess severe shortcomings, such as nonbiocompatible cross-linking chemistry, significant swelling, limited range of mechanical properties, or gelation kinetics unsuitable for in vivo injection. To solve these issues, we report the design and characterization of an injectable collagen hydrogel based on covalently modified acetyl thiol collagen cross-linked using thiol-maleimide click chemistry. The hydrogel is injectable for up to 72 h after preparation, shows no noticeable swelling, is transparent, can be molded in situ, and retains its shape in solution for at least one year. Notably, the hydrogel mechanical properties can be fine-tuned by simply adjusting the reactant stoichiometries, which to date was only reported for synthetic polymer hydrogels. The biocompatibility of the hydrogel is demonstrated in vitro using human corneal epithelial cells, which maintain viability and proliferation on the hydrogels for at least seven days. Furthermore, the developed hydrogel showed an adhesion strength on soft tissues similar to fibrin glue. Additionally, the developed hydrogel can be used as a sealant for repairing corneal perforations and can potentially alleviate the off-label use of cyanoacrylate tissue adhesive for repairing corneal perforations. Taken together, these characteristics show the potential of the thiol collagen hydrogel for future use as a prefabricated implant, injectable filler, or as sealant for corneal repair and regeneration.

The role of nanochitin in biologically-active matrices for tissue engineering-where do we stand?

Our regard to the use of chitin as a material has drastically changed since its discovery, 210 years ago. From an intractable material because of its insolubility in common solvents, it became one of the most important raw materials serving as a source of chitosan (its main derivative), and more recently, as source of nanometric forms: nanocrystals and nanofibers. Nanoscale chitin forms are remarkable high-value compounds for nanomaterials' development, due to their intrinsic biological and mechanical properties, as well as their potential as eco-friendly components to valorize the plentiful by-products of the seafood industry. Lately, these nanochitin forms have been widely used as nanofillers in polymer nanocomposites, and in particular, in natural biologically-active matrices for the development of biomaterials. The recent progresses achieved in the last two decades concerning the use of nanoscale chitin in biologically-active matrices for tissue engineering is highlighted in this review. First, an overview on the use of nanochitin in the different biomedical fields is presented and discussed. Then, the state-of-the-art regarding the development of biomaterials based on chitin nanocrystals or nanofibers is described in the context of the role of nanochitin in biologically-active matrices namely polysaccharides (chitin, chitosan, cellulose, hyaluronic acid, alginate), proteins (silk, collagen, gelatin) and others (lignin). Finally, major conclusions and perspectives on the use of nanochitin as an increasingly important raw material are described.

Influence of extracellular matrix composition on tumour cell behaviour in a biomimetic in vitro model for hepatocellular carcinoma.

The tumor micro-environment (TME) of hepatocellular carcinoma (HCC) consists out of cirrhotic liver tissue and is characterized by an extensive deposition of extracellular matrix proteins (ECM). The evolution from a reversible fibrotic state to end-stage of liver disease, namely cirrhosis, is characterized by an increased deposition of ECM, as well as changes in the exact ECM composition, which both contribute to an increased liver stiffness and can alter tumor phenotype. The goal of this study was to assess how changes in matrix composition and stiffness influence tumor behavior. HCC-cell lines were grown in a biomimetic hydrogel model resembling the stiffness and composition of a fibrotic or cirrhotic liver. When HCC-cells were grown in a matrix resembling a cirrhotic liver, they increased proliferation and protein content, compared to those grown in a fibrotic environment. Tumour nodules spontaneously formed outside the gels, which appeared earlier in cirrhotic conditions and were significantly larger compared to those found outside fibrotic gels. These tumor nodules had an increased expression of markers related to epithelial-to-mesenchymal transition (EMT), when comparing cirrhotic to fibrotic gels. HCC-cells grown in cirrhotic gels were also more resistant to doxorubicin compared with those grown in fibrotic gels or in 2D. Therefore, altering ECM composition affects tumor behavior, for instance by increasing pro-metastatic potential, inducing EMT and reducing response to chemotherapy.

Exploring the energy landscape of a SAM-I riboswitch.

SAM-I riboswitches regulate gene expression through transcription termination upon binding a S-adenosyl-L-methionine (SAM) ligand. In previous work, we characterized the conformational energy landscape of the full-length Bacillus subtilis yitJ SAM-I riboswitch as a function of Mg2+ and SAM ligand concentrations. Here, we have extended this work with measurements on a structurally similar ligand, S-adenosyl-L-homocysteine (SAH), which has, however, a much lower binding affinity. Using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling (HMM) analysis, we identified major conformations and determined their fractional populations and dynamics. At high Mg2+ concentration, FRET analysis yielded four distinct conformations, which we assigned to two terminator and two antiterminator states. In the same solvent, but with SAM added at saturating concentrations, four states persisted, although their populations, lifetimes and interconversion dynamics changed. In the presence of SAH instead of SAM, HMM revealed again four well-populated states and, in addition, a weakly populated 'hub' state that appears to mediate conformational transitions between three of the other states. Our data show pronounced and specific effects of the SAM and SAH ligands on the RNA conformational energy landscape. Interestingly, both SAM and SAH shifted the fractional populations toward terminator folds, but only gradually, so the effect cannot explain the switching action. Instead, we propose that the noticeably accelerated dynamics of interconversion between terminator and antiterminator states upon SAM binding may be essential for control of transcription.

The Role of Patch Testing with Indian Cosmetic Series in Patients with Facial Pigmented Contact Dermatitis in India.

BACKGROUND: Pigmented contact dermatitis (PCD) is a non-eczematoid variant of contact dermatitis, mainly characterised by hyperpigmentation. It occurs due to contact with a low amount of allergen over a long duration of time. PCD is frequently seen in Indians but is often misdiagnosed or underdiagnosed because of the asymptomatic nature of the entity. The aetiology and the allergens implicated in PCD in the Indian subcontinent is still an enigma because of the limited studies done. MATERIALS AND METHODS: This was an institution-based cross-sectional study, done at a tertiary hospital. Patch testing with Indian Cosmetic Series was conducted in a standardised method. Readings were taken at 48 hrs/72 hrs and on the 7th day [Figure 2a and b]. The International Contact Dermatitis Research Group (ICDRG) scoring system was used to grade the readings. RESULTS: Out of the 38 biopsy proven cases of PCD, 18 (47%) showed lichenoid features, 17 (45%) showed spongiotic features, 3 (8%) showed a mixed lichenoid and spongiotic pattern. Among total 1216 (32 patches × 38 patients) patch applied, 42 (3.4%) showed positivity in 30 patients. Among allergen categories, colorant (PPD) was found to be most common (37%) followed by fragrances (18%), preservatives (15%), anti-microbial (11%) and emulsifier and anti-oxidants (each 8%). CONCLUSION: It is important to identify the allergens implicated in PCD to help in better management of the condition. Patch testing proves to be a non invasive, low cost method and its role is indispensable in identifying the correct allergen.

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties.

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma micro-environment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the in vivo situation compared to traditional 2D cultures. Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.

Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels.

Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell-cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca2+ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies.

Injectable Shape-Holding Collagen Hydrogel for Cell Encapsulation and Delivery Cross-linked Using Thiol-Michael Addition Click Reaction.

Injectable hydrogels based on extracellular matrix-derived polymers show much promise in the field of tissue engineering and regenerative medicine. However, the hydrogels reported to date have at least one characteristic that limits their potential for clinical use, such as excessive swelling, complicated and potentially toxic cross-linking process, or lack of shear thinning and self-healing properties. We hypothesized that a collagen hydrogel cross-linked using thiol-Michael addition click reaction would be able to overcome these limitations. To this end, collagen was modified to introduce thiol groups, and hydrogels were prepared by cross-linking with 8-arm polyethylene glycol-maleimide. Rheological measurements on the hydrogels revealed excellent shear-thinning and self-healing properties. Additionally, only minimal swelling (6%) was observed over a period of 1 month in an aqueous buffer solution. Finally, tests using mesenchymal stromal cells and endothelial cells showed that the hydrogels are cell-compatible and suitable for cell encapsulation and delivery. Thus, the reported thiolated-collagen hydrogel cross-linked using thiol-Michael addition click reaction overcomes most of the challenges in the injectable hydrogel design and is an excellent candidate for cell delivery in regenerative medicine and tissue engineering applications. The hydrogel reported here is the first example of a self-healing hydrogel containing covalent cross-links.

The multi-state energy landscape of the SAM-I riboswitch: A single-molecule Förster resonance energy transfer spectroscopy study.

RNA (ribonucleic acid) molecules are highly flexible biopolymers fluctuating at physiological temperatures among many different conformations that are represented by minima in a hierarchical conformational free energy landscape. Here we have employed single-molecule FRET (smFRET) to explore the energy landscape of the B. subtilis yitJ SAM-I riboswitch (RS). In this small RNA molecule, specific binding of an S-adenosyl-L-methionine (SAM) ligand in the aptamer domain regulates gene expression by inducing structural changes in another domain, the expression platform, causing transcription termination by the RNA polymerase. We have measured smFRET histograms over wide ranges of Mg2+ concentration for three RS variants that were specifically labeled with fluorescent dyes on different sites. In the analysis, different conformations are associated with discrete Gaussian model distributions, which are typically fairly broad on the FRET efficiency scale and thus can be extremely challenging to unravel due to their mutual overlap. Our earlier work on two SAM-I RS variants revealed four major conformations. By introducing a global fitting procedure which models both the Mg2+ concentration dependencies of the fractional populations and the average FRET efficiencies of the individual FRET distributions according to Mg2+ binding isotherms, we were able to consistently describe the histogram data of both variants at all studied Mg2+ concentrations. With the third FRET-labeled variant, however, we found significant deviations when applying the four-state model to the data. This can arise because the different FRET labeling of the new variant allows two states to be distinguished that were previously not separable due to overlap. Indeed, the resulting five-state model presented here consistently describes the smFRET histograms of all three variants as well as their variations with Mg2+ concentration. We also performed a triangulation of the donor position for two of the constructs to explore how the expression platform is oriented with respect to the aptamer.

Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants.

Short collagen-like peptides (CLPs) are being proposed as alternatives to full-length collagen for use in tissue engineering, on their own as soft hydrogels, or conjugated to synthetic polymer for mechanical strength. However, despite intended clinical use, little is known about their safety and efficacy, mechanism of action or degree of similarity to the full-length counterparts they mimic. Here, we show the functional equivalence of a CLP conjugated to polyethylene glycol (CLP-PEG) to full-length recombinant human collagen in vitro and in promoting stable regeneration of corneal tissue and nerves in a pre-clinical mini-pig model. We also show that these peptide analogs exerted their pro-regeneration effects through stimulating extracellular vesicle production by host cells. Our results support future use of CLP-PEG implants for corneal regeneration, suggesting the feasibility of these or similar peptide analogs in clinical application in the eye and other tissues. STATEMENT OF SIGNIFICANCE: Although biomaterials comprising full-length recombinant human collagen and extracted animal collagen have been evaluated and used clinically, these macromolecules provide only a limited number of functional groups amenable to chemical modification or crosslinking and are demanding to process. Synthetic, customizable analogs that are functionally equivalent, and can be readily scaled-up are therefore very desirable for pre-clinical to clinical translation. Here, we demonstrate, using cornea regeneration as our test bed, that collagen-like-peptides conjugated to multifunctional polyethylene glycol (CLP-PEG) when grafted into mini-pigs as corneal implants were functionally equivalent to recombinant human collagen-based implants that were successfully tested in patients. We also show for the first time that these materials affected regeneration through stimulation of extracellular vesicle production by endogenous host cells that have migrated into the CLP-PEG scaffolds.

Collagen-Based Fillers as Alternatives to Cyanoacrylate Glue for the Sealing of Large Corneal Perforations.

PURPOSE: To describe the use of collagen-based alternatives to cyanoacrylate glue for the sealing of acute corneal perforations. METHODS: A collagen analog comprising a collagen-like peptide conjugated to polyethylene glycol (CLP-PEG) and its chemical crosslinker were tested for biocompatibility. These CLP-PEG hydrogels, which are designed to act as a framework for corneal tissue regeneration, were then tested as potential fillers in ex vivo human corneas with surgically created full-thickness perforations. Bursting pressures were measured in each of 3 methods (n = 10 for each condition) of applying a seal: 1) cyanoacrylate glue with a polyethylene patch applied ab externo (gold standard); 2) a 100-µm thick collagen hydrogel patch applied ab interno, and 3) the same collagen hydrogel patch applied ab interno supplemented with CLP-PEG hydrogel molded in situ to fill the remaining corneal stromal defect. RESULTS: Cyanoacrylate gluing achieved a mean bursting pressure of 325.9 mm Hg, significantly higher than the ab interno patch alone (46.3 mm Hg) and the ab interno patch with the CLP-PEG filler (86.6 mm Hg). All experimental perforations were sealed effectively using 100 µm hydrogel sheets as an ab interno patch, whereas conventional ab externo patching with cyanoacrylate glue failed to provide a seal in 30% (3/10) cases. CONCLUSIONS: An ab interno patch system using CLP-PEG hydrogels designed to promote corneal tissue regeneration may be a viable alternative to conventional cyanoacrylate glue patching for the treatment of corneal perforation. Further experimentation and material refinement is required in advance of clinical trials.

Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch.

S-adenosyl-L-methionine (SAM) ligand binding induces major structural changes in SAM-I riboswitches, through which gene expression is regulated via transcription termination. Little is known about the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I riboswitch. Therefore, we have explored its conformational energy landscape as a function of Mg2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling analysis. We resolved four conformational states both in the presence and the absence of SAM and determined their Mg2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds coexist, and SAM binding only gradually shifts the populations toward terminator states. We observed a pronounced acceleration of conformational transitions upon SAM binding, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.

Reduced cytotoxicity and enhanced bioactivity of cationic antimicrobial peptides liposomes in cell cultures and 3D epidermis model against HSV.

Cationic antimicrobial peptides (AMPs) are part of the innate immunity, and act against a wide variety of pathogenic microorganisms by perturbation of the microorganism's plasma membrane. Although attractive for clinical applications, these agents suffer from limited stability and activity in vivo, as well as non-specific interaction with host biological membranes, leading to cytotoxic adverse effects. We hypothesized that encapsulation of AMPs within liposomes could result in reduced cytotoxicity, and with enhanced stability as well as bioactivity against herpes simplex virus 1 (HSV-1). We formulated nano-sized liposomal formulations of LL-37 and indolicidin, and their physicochemical properties, cellular uptake, in vitro cytotoxicity and antiviral efficacy have been determined. Lower cytotoxicity of LL-37 liposomes was found in comparison to indolicidin liposomes attributed to the superior physicochemical properties, and to the different degree of interaction with the liposomal membrane. The disc-like shaped LL-37 liposomes (106.8±10.1nm, shelf-life stability of >1year) were taken up more rapidly and to a significantly higher extent than the free peptide by human keratinocyte cell line (HaCaT), remained intact within the cells, followed by release of the active peptide within the cytoplasm and migration of the vesicles' lipids to the plasma membrane. LL-37 liposomes were found significantly less toxic than both the free agent and liposomal indolicidin. In the new 3D epidermis model (immortalized primary keratinocytes) liposomal LL-37 treatment (>20µM), but not free LL-37, efficiently protected the epidermis, inhibiting HSV-1 infection. This positive antiviral effect was obtained with no cytotoxicity even at very high concentrations (400µM). Thus, the antiviral activity of encapsulated LL-37 was significantly improved, expanding its therapeutic window. Liposomal LL-37 appears to be a promising delivery system for HSV therapy.

Asymptomatic papules over central and pericentral areas of the face.

We report the case of a 24-year-old college girl, presenting with numerous acneiform papular eruptions over the central part of the face.

Zinc-responsive acral hyperkeratotic dermatosis-A novel entity or a subset of some well-known dermatosis?

BACKGROUND: We are reporting a series of interesting cases, which presented to us with psoriasiform lesions distributed over the acral regions of the body. The cases are unusual because they were resistant to conventional treatment modalities like topical corticosteroids, tacrolimus and oral methotrexate but showed significant improvement on oral zinc therapy. MATERIALS AND METHODS: Ten patients with characteristic clinical features of distinctive hyperkeratotic plaque in the acral areas, who were resistant to treatment by different modalities including potent topical steroids and oral methotrexate, were included for detailed investigations. A proper history was taken and relevant laboratory investigations were done which included blood count, urine, liver function, renal function, hepatitis-C virus serology and serum zinc levels. Patients were followed up every 2 weeks. Histopathological examinations of the lesional tissue were done at baseline and after 6 weeks of therapy. Patients were given oral zinc daily and no other treatment during the 6 weeks course. RESULTS: All our patients were non-reactive to hepatitis-C. Of the ten patients only one patient (10%) showed low titer of serum zinc, another (10%) showed higher zinc level, while the rest of the patients had normal zinc level. Five of our patients had chronic renal failure, one had Grave's disease and the remaining had no associated systemic illness. Histopathology mostly showed hyperkeratosis, acanthosis, prominent granular layer, spongiosis and dermal infiltrate. After 6 weeks of follow up, all patients showed rapid and remarkable therapeutic response with zinc. CONCLUSIONS: We here report a series of patients, discernible because of their uniform clinical presentation of acral hypekeratotic plaques and in showing a noticeable response to zinc. Clinical, histopathological and laboratory investigations were done to rule out diseases of similar morphology including psoriasis, acral necrolytic erythema and lichen simplex chronicus. Authors understand that further studies with greater number cases and more detailed investigations are required to establish exact etio-pathogenesis and nomenclature of this distinct subset of patients.