Different Concentrations Of Collagen Research Articles

Laser-induced forward transfer (LIFT) based bioprinting of the collagen I with retina photoreceptor cells

This study focuses on the 3D bioprinting of retina photoreceptor cells using a laser-induced forward transfer (LIFT) based bioprinting system. Bioprinting has a great potential to mimic and regenerate the human organoid system, and the LIFT technique has emerged as an efficient method for high-resolution micropatterning and microfabrication of biomaterials and cells due to its capability of creating precise, controlled microdroplets. In this study, the parameters for an effective femtosecond laser-based LIFT process for 3D bioprinting of collagen biomaterial were studied. Different concentrations of collagen I solutions were tested and 0.75 mg/ml to 1 mg/ml collagen Ⅰ was identified as the right concentration that can be transferred through the LIFT system. Then, retinal cone cells were mixed with collagen I and Dulbecco's Modified Eagle's Medium (DMEM) and printed drop-by-drop lines. Some important laser parameters such as pulse energy and pulse pick divider were experimented with to form a successful, smooth, high-resolution deposition. The cell viability in the bioink and printed droplet was measured at different time horizons. A general full factorial design of the experiment was used to analyze and observe the relationship between the droplet quality and the LIFT process parameters. Using 15 µJ and 16 µJ pulse energy, the cell-laden bioink was printed successfully. This research study will help to print other retinal neuron cells with collagen Ⅰ in the LIFT system and show the way of constructing layer-by-layer different cell lines that will help to fabricate the retina ultimately.

Chitosan-collagen-hydroxyapatite membranes for tissue engineering

Tissue engineering is growing in developing new technologies focused on providing effective solutions to degenerative pathologies that affect different types of connective tissues. The search for biocompatible, bioactive, biodegradable, and multifunctional materials has grown significantly in recent years. Chitosan, calcium phosphates collagen, and their combination as composite materials fulfill the required properties and could result in biostimulation for tissue regeneration. In the present work, the chitosan/collagen/hydroxyapatite membranes were prepared with different concentrations of collagen and hydroxyapatite. Cell adhesion was evaluated by MTS assay for two in vitro models. Additionally, cytotoxicity of the different membranes employing hemolysis of erythrocytes isolated from human blood was carried out. The structure of the membranes was analyzed by X-rays diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and thermal stability properties by thermogravimetric methods (TGA). The highest cell adhesion after 48 h was obtained for chitosan membranes with the highest hydroxyapatite and collagen content. All composite membranes showed good cell adhesion and low cytotoxicity, suggesting that these materials have a significant potential to be used as biomaterials for tissue engineering.Graphical abstract

The Absence of Syndecan‐1 Leads to More Inflammation in the Mouse Model of Rheumatoid Arthritis

Introduction Syndecan-1 (sdc-1) is a member of a family of transmembrane proteoglycans expressed in numerous cell types. It has been shown to be involved in carcinogenesis, wound healing and inflammation. However, its potential role in the pathophysiology of Rheumatoid Arthritis (RA) has not been well elucidated yet. Many modified mouse strains are relatively resistant to collagen-induced RA, while others could acquire it spontaneously. Such strains have been very useful for assessing therapeutics with anti-inflammatory properties. Aim Evaluate the potential role of sdc-1 in the development of RA using sdc-1 null mice. Hypothesis We hypothesized that inflammation will be more severe in the absence of sdc-1. Methods A total of 18 mice (9 BALB/c wild type and 9 sdc-1 null), 8-10 weeks old, were divided into 6 groups. They were injected with different concentrations of Collagen II (CII) and Complete Freund's Adjuvant (CFA) to induce arthritis. A two-dose regimen, three weeks apart, was followed (Table 1). The mice were monitored three times a week for 70 days and clinical signs of redness and swelling were recorded. Routine histological studies of joint biopsies were also performed using different stains, including Safranin O for cartilage and bone. Results Preliminary data demonstrated the following: Clinical signs and symptoms:redness and swelling of joints appeared earlier in the sdc-1 null group injected with 75µl CII + 75µl CFA compared to the BALB/c group with similar treatment (Figure 1). Histological assessment: the joints of sdc-1 null mice showed more alterations: marked atrophy and loss of articular cartilage, increased infiltration by inflammatory cells specifically macrophages, subchondral bone porosity, changes in the trabecular network as well as bone erosion. All mice treated with collagen showed increased proteoglycan content in the articular cartilage and growth plate. Conclusion Sdc-1 null mice developed a faster and more severe experimental RA; however, more characterization at the macroscopic, microscopic and molecular level is needed. Uncovering the role of sdc-1 in RA paves the way for future investigations that can lead to the emergence of new therapeutic modalities that could be adopted along with conventional treatments.

Development of Bacterial Cellulose Biocomposites Combined with Starch and Collagen and Evaluation of Their Properties.

One of the major benefits of biomedicine is the use of biocomposites as wound dressings to help improve the treatment of injuries. Therefore, the main objective of this study was to develop and characterize biocomposites based on bacterial cellulose (BC) with different concentrations of collagen and starch and characterize their thermal, morphological, mechanical, physical, and barrier properties. In total, nine samples were produced with fixed amounts of glycerol and BC and variations in the amount of collagen and starch. The water activity (0.400–0.480), water solubility (12.94–69.7%), moisture (10.75–20.60%), thickness (0.04–0.11 mm), water vapor permeability (5.59–14.06 × 10−8 g·mm/m2·h·Pa), grammage (8.91–39.58 g·cm−2), opacity (8.37–36.67 Abs 600 nm·mm−1), elongation (4.81–169.54%), and tensile strength (0.99–16.32 MPa) were evaluated and defined. In addition, scanning electron microscopy showed that adding biopolymers in the cellulose matrix made the surface compact, which also influenced the visual appearance. Thus, the performance of the biocomposites was directly influenced by their composition. The performance of the different samples obtained resulted in them having different potentials for application considering the injury type. This provides a solution for the ineffectiveness of traditional dressings, which is one of the great problems of the biomedical sector.

Effect of collagen and EPS components on the viscoelasticity of Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa is an opportunistic pathogen that causes thousands of deaths every year in part due to its ability to form biofilms composed of bacteria embedded in a matrix of self-secreted extracellular polysaccharides (EPS), e-DNA, and proteins. In chronic wounds, biofilms are exposed to the host extracellular matrix, of which collagen is a major component. How bacterial EPS interacts with host collagen and whether this interaction affects biofilm viscoelasticity is not well understood. Since physical disruption of biofilms is often used in their removal, knowledge of collagen's effects on biofilm viscoelasticity may enable new treatment strategies that are better tuned to biofilms growing in host environments. In this work, biofilms are grown in the presence of different concentrations of collagen that mimic in vivo conditions. In order to explore collagen's interaction with EPS, nine strains of P. aeruginosa with different patterns of EPS production were used to grow biofilms. Particle tracking microrheology was used to characterize the mechanical development of biofilms over two days. Collagen is found to decrease biofilm compliance and increase relative elasticity regardless of the EPS present in the system. However, this effect is minimized when biofilms overproduce EPS. Collagen appears to become a de facto component of the EPS, through binding to bacteria or physical entanglement.

Silk fibroin/collagen 3D scaffolds loaded with TiO2 nanoparticles for skin tissue regeneration

One of the biggest present challenges for clinicians is to regenerate the skin tissues which are lost due to trauma or diseases. Although the autografts are considered as the gold standard, yet they have their own limitations. However, a proper biocompatible, flexible, and mechanically stable, moisture retentive, and permeable synthetic membrane could be used instead of autografts. Silk fibroin (SF) is a protein based mechanically strong, biocompatible, biodegradable, and permeable material which has been used in wide range of applications like cosmetics, foods, drugs, tissue engineering, and wound healing. Similarly, collagen (CG) is biocompatible and biodegradable protein-based natural polymer. TiO2 particles have antibacterial properties. Keeping in mind the properties of these materials, we designed SF/CG membranes loaded with TiO2 particles for skin tissue regeneration using freeze drying technique. Starting with degumming of silk from B. mori, SF was extracted and regenerated which was used for further studies. The membranes were fabricated in two series; in first series concentration of TiO2 was optimized only in silk; in second series SF/CG/TiO2 membranes were fabricated with two different concentrations of CG. Membranes were characterized by FTIR and SEM. For the biological use of as-prepared membranes, swelling and degradation studies in H2O2 and lysozyme were carried out. Antibacterial studies were carried out on S. aureus and E. coli. To check the biocompatibility and cell toxicity, Alamar Blue Assay was performed. Attachment of cell with scaffolds was studied with Cell Attachment Assay and photos of cell attachment were recorded with SEM. Overall, the membranes showed good antibacterial activity and biocompatibility. On the basis of these results, the fabricated membranes are potential candidates for skin tissue regeneration and wound healing application.

Effect of different concentrations of collagen, ceramides, N-acetyl glucosamine, or their mixture on enhancing the proliferation of keratinocytes, fibroblasts and the secretion of colagen and/or the expression of mRNA of type I collagen

The objectives of the study are to survey the effects of different concentrations of collagen, ceramides, N-acetyl glucosamine (NAG), or their mixture on the proliferation of keratinocytes, fibroblasts and on the secretion of collagen and/or the expression of mRNA of type I collagen. The optimal concentrations that facilitated the proliferation of fibroblasts were 49, 2.0, and 24.4 μg/mL, respectively for collagen, ceramides, and NAG. The proliferation percentages were 191%, 170%, and 223%, respectively at the same order in compare to the control. The concentration of the mixture containing 6.1, 0.12 and 6.1 μg/mL, respectively of collagen, ceramides, and NAG facilitated the proliferation of fibroblasts. The optimal concentrations that had highest keratinocytes proliferation were 0.76, 0.001, and 0.76 μg/mL, respectively for collagen, ceramides, and NAG. The proliferation percentages were 242%, 203% and 230%, respectively at the same order in compare to the control. The optimal concentration that effects the highest keratinocytes proliferation by the mixture were 0.38, 0.008 and 0.38 μg/mL for collagen, ceramides and NAG, respectively. The concentrations for fibroblasts to express higher amount of type I collagen mRNA were 6.1, 0.49, and 6.1 μg/mL, respectively for collagen, ceramides, and NAG. The expression levels of type I collagen mRNA were 152%, 160% and 220%, respectively at the same order in compare to the control. However, the expressions of type I collagen mRNA were 180% in compare to the control at the concentration of mixture containing 0.76, 0.015 and 0.76 μg/mL, respectively of collagen, ceramides, and NAG.

Imaging Cancer Cells and their Interactions Within 3D Microenvironment - a Quantitative Study using Cryo Soft X-Ray Tomography

One of the most fundamental questions for cancer cell biologists is how the alterations of cellular morphology, as well as the organelles’ spatial re-organization, facilitate the cancer metastasis process. We are using soft x-ray tomography (SXT) to visualize and quantify all organelles in the cell in three dimensions during this collective behavior. This technique has also enabled us to account for the cell heterogeneity and to examine a large number of cells and their interactions within a created micro-environment. SXT is a non-invasive imaging technique with high spatial resolution (∼35nm). It is a quantitative imaging technology that generates high contrast views of organelles within intact cells in their near-native state. Illuminating cells with soft x-ray takes the advantage of “water-window” property that image contrast comes directly from the absorption of cell constituents. The absorption is also linear with the concentration of bio-organic molecules. Each voxel in an organelle has a denoted value called linear absorption coefficient (LAC) that can be used as a fingerprint for different organelles. We have created a 3D microenvironment using different concentrations of collagen that have resulted in different pore sizes for cancer cells (HT1080) to crawl through. With different physical limitations, we observed the distinct cellular morphologies during different states. We can quantitatively study the variaitons in nuclear size and shapes, the mitochrondria spatial distributions and volumes, the number and volume of lipid droplets and analyze the ratios between euchromatin and heterochromatin.

Development of collagen/PVA composites patches for osteochondral defects using a green processing of ionic liquid

Osteochondral defects are still a big challenge for the surgeons because of good biocompatibility and higher mechanical strength requiring issues of the implants. In this study, different concentrations of collagen (dissolved in ionic liquid) up to 60% were blended with polyvinyl alcohol to prepare hydrogels of good mechanical strength, with the best biocompatibility and excellent fluid uptake ability. Ionic liquid was used as a green solvent for dissolution of collagen at a higher concentration as compared to other normal solvents. The prepared hydrogels were characterized with Fourier transform infrared spectroscopy (FTIR) which showed the characteristic peaks assigned to collagen and PVA. The surface morphology was investigated using scanning electron microscopy (SEM) which revealed homogeneity of the composite patches. Thermal gravimetric analysis (TGA) performed for samples show good thermal stabilities. Fluid uptake ability showed the massive uptake of fluid by the hydrogels. Biocompatibility was tested using hemolysis and MTT assay. Electrodynamic fatigue testing system was used for evaluating the mechanical properties and measured the tensile strength in the range of 2.4 to 8.5 MPa. The prepared osteochondral patches show good biocompatibility and mechanical properties.

An in vitro 3D model using collagen coated gelatin nanofibers for studying breast cancer metastasis

The study of breast cancer metastasis is limited due to poor knowledge of molecular progression of breast tumor and varied heterogeneity. For a better understanding of tumor metastasis, a reliable 3D in vitro model bridging the gap between 2D cultures and in vivo animal model studies is essential. Our study is focused on two key points: (i) designing a 3D microenvironment for studying metastasis and (ii) simulating the metastasis milieu by inducing epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET). An electrospun gelatin nanofiber matrix (EGNF) was fabricated using electrospinning and further dip coated with different concentrations of collagen to obtain surface complexity and mechanical properties, similar to connective tissues. Nanofiber matrices were physically characterized by Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and field-emission scanning electron microscopy (FESEM). The FTIR, AFM, and FESEM results indicated the crosslinking and confirmed the presence of pores in the nanofiber matrices. Comparative studies on biocompatibility, cell attachment, and the proliferation of MCF-7 cells on EGNF and collagen coated gelatin nanofibrous matrix (CCGM) revealed higher cellular attachment and proliferation in CCGM. CCGM with human metastatic breast cancer cell line (MCF-7) was taken to study breast cancer metastasis using estrogen (induces EMT) and progesterone (induces MET) hormones for 24 h. Quantitative real-time PCR was used for quantifying the expression of metastasis related genes, and fluorescence microscopy for verifying the invasion of cells to the matrices. The expression of E-cadherin and matrix metalloproteinase 2 (MMP 2) confirmed the occurrence of EMT and MET. Live cell imaging and cellular attachment showed significant increase of cellular invasion in crosslinked 0.15% CCGM that serves as a suitable non-toxic, biocompatible, and affordable scaffold for studying breast cancer metastasis. Our findings suggested that CCGM can be used as a tissue-like 3D model for studying breast cancer metastatic events in vitro.

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix.

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Microgel microenvironment primes adipose-derived stem cells towards an NP cells-like phenotype.

Cell therapy of the degenerated intervertebral disc is limited by the lack of appropriate cell sources, thus new strategies for the differentiation of stem cells towards a nucleus pulposus (NP)-like phenotype need investigation. In the current study, it is hypothesized that spherical niche-like structures composed of type II collagen and hyaluronan (HA) mimic the NP microenvironment and promote the differentiation of adipose-derived stem cells (ADSCs) towards an NP-like phenotype. ADSCs are embedded in microgels of different concentrations of collagen II/HA. Cells' response to the different environments is studied by characterizing differences in cells' viability, morphology, and gene expression. After 21 days of culture, ADSCs maintain ± 80% viability in all the conditions tested. Moreover, microgels with higher concentration of collagen are stable and maintain cells in a rounder shape. In presence of differentiation media, cells are able to differentiate in all the conditions tested, but in a more pronounced manner in the microgel with a higher concentration of collagen. By tuning microgels' properties, it is possible to influence ADSCs' phenotype and ability to differentiate. Indeed, when cultured in high concentrations of collagen, ADSCs expresses high levels of collagen II, aggrecan, SOX9, and low levels of collagen I.

In Vitro Microvessel Growth and Remodeling within a Three-Dimensional Microfluidic Environment

This paper presents in vitro microvascular network formation within 3D gel scaffolds made from different concentrations of type-I collagen, fibrin, or a mixture of collagen and fibrin, using a simple microfluidic platform. Initially, microvascular network formation of human umbilical vein endothelial cells was examined using live time-lapse confocal microscopy every 90 min from 3 h to 12 h after seeding within three different concentrations of collagen gel scaffolds. Among the three conditions of collagen gel scaffolds (2.0 mg/ml, 2.5 mg/ml, and 3.0 mg/ml), the number of skeleton within collagen gel scaffolds was consistently the highest (3.0 mg/ml), followed by those of collagen gel scaffolds (2.5 mg/ml and 2.0 mg/ml). Results demonstrated that concentration of collagen gel scaffolds, which influences matrix stiffness and ligand density, may affect microvascular network formation during the early stages of vasculogenesis. In addition, the maturation of microvascular networks in monoculture under different gel compositions within gel scaffolds (2.5 mg/ml) was examined for 7 d using live confocal microscopy. It was confirmed that pure fibrin gel scaffolds are preferable to collagen gel or collagen/fibrin combinations, significantly reducing matrix retractions during maturation of microvascular networks for 7 d. Finally, early steps in the maturation process of microvascular networks for 14 d were characterized by demonstrating sequential steps of branching, expanding, remodeling, pruning, and clear delineation of lumens within fibrin gel scaffolds. Our findings demonstrate an in vitro model for generating mature microvascular networks within 3D microfluidic fibrin gel scaffolds (2.5 mg/ml), and furthermore suggest the importance of gel concentration and composition in promoting the maturation of microvascular networks.

Knockdown of electron transfer flavoprotein β subunit reduced TGF-β-induced α-SMA mRNA expression but not COL1A1 in fibroblast-populated three-dimensional collagen gel cultures

The inhibition of transforming growth factor β (TGF-β)-induced myofibroblast differentiation is a key objective for the treatment of hypertrophic scarring. We previously reported that knockdown of the electron transfer flavoprotein β subunit (ETFB) reduced mechanoregulated cell number in fibroblast-populated collagen gel cultures [1]. To characterize the effects of ETFB knockdown, we investigated gel contraction, TGF-β-induced collagen, α-SMA mRNA expression and stress fiber formation. Fibroblasts were transfected with negative control or ETFB-specific siRNAs and embedded in collagen gels in an attached or detached condition. The gel contraction assay was performed in three different concentrations of collagen (0.5, 1.0 or 1.5mg/mL) and was analyzed by measuring the changes in the gel area throughout the culture period. The attached collagen gel culture was performed in the presence of rTGF-β and the mRNA levels of α-SMA and COL1A1 were measured by qRT-PCR. The effect of ETFB knockdown on proliferation and stress fiber organization in monolayer cultures was investigated by conducting AlamarBlue assays and phalloidin staining. The transfection of ETFB siRNA did not alter gel contraction compared to the negative control in all collagen concentrations. When the cells were treated with TGF-β under mechanical stress conditions, ETFB knockdown attenuated α-SMA mRNA expression to a level comparable to that observed in the absence of TGF-β. However, no inhibitory effect on COL1A1 mRNA levels was observed. The AlamarBlue assay indicated that the knockdown had no effect on the proliferation of cells cultured on plastic. Phalloidin staining of a monolayer culture showed that ETFB knockdown weakened the stress fiber organization induced by rTGF-β. ETFB knockdown can affect TGF-β-induced tissue remodeling and/or fibrotic processes in vitro.

In Vivo Implantation of Five Different Compositions of Tissue-Engineered Intervertebral Disks in a Rat Tail Model: Preliminary Results

Introduction Degenerative disk disease is a leading cause of disability in the developed countries and over the past few decades, has become a major public health problem worldwide due to the significant improvement of life expectancy. Current medical and surgical treatment strategies for DDD mainly focus on pain and symptom relief, and do not necessarily address the underlying pathology of the disease. In recent years, there has been an increasing interest in developing biological based implants as an alternative, which could restore function and mobility of the spine. Here, we present the preliminary results of our study, in which five different compositions of tissue-engineered intervertebral disks were implanted into the rat caudal spine, and radiographic studies were used to evaluate the results. Materials and Methods Based on MRI and micro-computed tomography imaging of the rat spine, compound tissue-engineered total disk replacement implants (TE-TDR) were designed to a high degree of geometric accuracy. TE-TDRs were composed of a gelatinous nucleus pulposus (NP) surrounded by an aligned collagenous annulus fibrosus. Five different compositions of the TE-TDR were designed, containing different concentrations of collagen in the AF and different concentrations of sheep chondrocytes seeded in their NP. TE-TDR implants were then inserted into the S3/S4 caudal disk space of 37 mature athymic nude rats (RNU) and were maintained on average for 15 weeks. In group 1 (four rats), TE-TDRs contained 10 million sheep cells/mL in their NP and 1 mg/mL collagen in their AF; in a similar way group 2 (four rats) received TE-TDRs with 10 million cells/mL NP and 2 mg/mL collagen AF; group 3 (eight rats) received TE-TDRs with 1 million cells/mL NP and 2 mg/mL collagen AF; in groups 4 and 5, the TE-TDRs' cell and collagen concentrations were similar to those of group 3, however in group 4 (14 rats), the disks were additionally photo cross-linked and cultured in riboflavin within their development process; and in group 5 (seven rats), an additional layer of collagen gel was pipetted around each disk to produce a second lamella. X-rays were performed both after surgery and at the latest follow-up time. Disk height changes were evaluated by calculating the Disk Height Indices (DHI) on X-rays. Based on the rostral healthy disk, a baseline DHI was also calculated for each animal. The results were then presented as percentages, comparing the disk height of the level of interest to the rostral healthy disk height. Statistical analyses were performed comparing the implanted rats with a control group in the previously published data; comparisons were also been made between the different groups of implanted rats. Results Five different compositions of the TE-TDR with differences in their collagen and cell contents were designed and implanted in the S3/S4 caudal disk space of 37 rats; The radiographic results of these five groups were then compared to the previously published data on a control group (six rats), on which simple discectomies were performed. Both after the operation and at the latest follow-up, the mean disk height in implanted animals was significantly higher than the control group (51% and 61% compared to 37%). Interestingly, the disk height in the implanted group at the latest follow-up also showed a significant increase, compared to the postoperative disk height. Comparisons between different implanted groups, showed that group 3 (1 million cells/mL and 2 mg/mL collagen) was associated with best results both postoperatively and at the latest follow-up; also, the worst outcome were observed in group 5 (multilamellar) postoperatively, and in group 4 (cross-linked) at latest follow-up; nonetheless no statistical significance was detected. Conclusion The findings of the present study, while preliminary, suggest that TE-TDR may yield a clinically feasible and efficacious treatment for intervertebral disk disease. Although limited by the sample size, this research will serve as a base for further future studies on biological disk replacement. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

In Vivo Implantation of Five Different Compositions of Tissue-Engineered Intervertebral Disks in a Rat Tail Model: Preliminary Results

IntroductionDegenerative disk disease is a leading cause of disability in the developed countries and over the past few decades, has become a major public health problem worldwide due to the significant improvement of life expectancy. Current medical and surgical treatment strategies for DDD mainly focus on pain and symptom relief, and do not necessarily address the underlying pathology of the disease. In recent years, there has been an increasing interest in developing biological based implants as an alternative, which could restore function and mobility of the spine. Here, we present the preliminary results of our study, in which five different compositions of tissue-engineered intervertebral disks were implanted into the rat caudal spine, and radiographic studies were used to evaluate the results. Materials and MethodsBased on MRI and micro-computed tomography imaging of the rat spine, compound tissue-engineered total disk replacement implants (TE-TDR) were designed to a high degree of geometric accuracy. TE-TDRs were composed of a gelatinous nucleus pulposus (NP) surrounded by an aligned collagenous annulus fibrosus. Five different compositions of the TE-TDR were designed, containing different concentrations of collagen in the AF and different concentrations of sheep chondrocytes seeded in their NP. TE-TDR implants were then inserted into the S3/S4 caudal disk space of 37 mature athymic nude rats (RNU) and were maintained on average for 15 weeks. In group 1 (four rats), TE-TDRs contained 10 million sheep cells/mL in their NP and 1 mg/mL collagen in their AF; in a similar way group 2 (four rats) received TE-TDRs with 10 million cells/mL NP and 2 mg/mL collagen AF; group 3 (eight rats) received TE-TDRs with 1 million cells/mL NP and 2 mg/mL collagen AF; in groups 4 and 5, the TE-TDRs' cell and collagen concentrations were similar to those of group 3, however in group 4 (14 rats), the disks were additionally photo cross-linked and cultured in riboflavin within their development process; and in group 5 (seven rats), an additional layer of collagen gel was pipetted around each disk to produce a second lamella. X-rays were performed both after surgery and at the latest follow-up time. Disk height changes were evaluated by calculating the Disk Height Indices (DHI) on X-rays. Based on the rostral healthy disk, a baseline DHI was also calculated for each animal. The results were then presented as percentages, comparing the disk height of the level of interest to the rostral healthy disk height. Statistical analyses were performed comparing the implanted rats with a control group in the previously published data; comparisons were also been made between the different groups of implanted rats. ResultsFive different compositions of the TE-TDR with differences in their collagen and cell contents were designed and implanted in the S3/S4 caudal disk space of 37 rats; The radiographic results of these five groups were then compared to the previously published data on a control group (six rats), on which simple discectomies were performed. Both after the operation and at the latest follow-up, the mean disk height in implanted animals was significantly higher than the control group (51% and 61% compared to 37%). Interestingly, the disk height in the implanted group at the latest follow-up also showed a significant increase, compared to the postoperative disk height. Comparisons between different implanted groups, showed that group 3 (1 million cells/mL and 2 mg/mL collagen) was associated with best results both postoperatively and at the latest follow-up; also, the worst outcome were observed in group 5 (multilamellar) postoperatively, and in group 4 (cross-linked) at latest follow-up; nonetheless no statistical significance was detected. ConclusionThe findings of the present study, while preliminary, suggest that TE-TDR may yield a clinically feasible and efficacious treatment for intervertebral disk disease. Although limited by the sample size, this research will serve as a base for further future studies on biological disk replacement.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declared

Determination of critical aggregation concentration and aggregation number of acid-soluble collagen from walleye pollock ( Theragra chalcogramma) skin using the fluorescence probe pyrene

The aim of this paper was to investigate the critical aggregation concentration and aggregation number of acid-soluble collagen from walleye pollock ( Theragra chalcogramma) skin using the fluorescence probe pyrene. Results showed that pyrene was fit for studying the aggregation behaviour of collagen in sodium phosphate buffer at pH 7.2. The plots of the pyrene I 1/ I 3 ratio, as a function of the logarithm of total collagen concentration, revealed a typical sigmoidal decrease, the critical aggregation concentration (CAC) from which was determined to be at 0.48 mg/ml. The subsequent transient fluorescence decay study indicated that the aggregation number of collagen was not constant, but varied with different concentrations of collagen. The structure of the aggregates tended to be stable, when the collagen concentration exceeded 1.2 mg/ml.

Effect of multislice acquisition on T1 and T2 measurements of articular cartilage at 3T

The aim of this study was to investigate the effect of magnetization transfer on multislice T1 and T2 measurements of articular cartilage. A set of phantoms with different concentrations of collagen and contrast agent (Gd-DTPA2-) were used for the in vitro study. A total of 20 healthy knees were used for the in vivo study. T1 and T2 measurements were performed using fast-spin-echo inversion-recovery (FSE-IR) sequence and multi-spin-echo (MSE) sequence, respectively, in both in vitro and in vivo studies. We investigated the difference in T1 and T2 values between that measured by single-slice acquisition and that measured by multislice acquisition. Regarding T1 measurement, a large drop of T1 in all slices and also a large interslice variation in T1 were observed when multislice acquisition was used. Regarding T2 measurement, a substantial drop of T2 in all slices was observed; however, there was no apparent interslice variation when multislice acquisition was used. This study demonstrated that the adaptation of multislice acquisition technique for T1 measurement using FSE-IR methodology is difficult and its use for clinical evaluation is problematic. In contrast, multislice acquisition for T2 measurement using MSE was clinically applicable if inaccuracies caused by multislice acquisition were taken into account.

Antibodies to CD18 influence neutrophil migration through extracellular matrix.

Mac-1 (CD11b/CD18) is known to be involved in neutrophil (PMN) adhesion to endothelial cells and extracellular matrix. Although antibodies to CD 18 are being tested for therapy in humans, their role in PMN migration through the extracellular matrix is unknown. We used direct visualization to quantify PMN motility through reconstituted, three-dimensional gels of collagen type I. Gels were prepared with different concentrations of collagen (ranging from 0.1 to 1.0 mg/mL) and PMN migration was examined in the presence and absence of antibodies to CD18 (anti-CD18), with and without stimulation by N-formyl peptides. In low-concentration gels (<0.6 mg/mL), anti-CD18 had a significant influence on PMN migration, increasing motility in unstimulated PMN by 90% at 0.3 mg/mL collagen, and decreasing motility in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN by 70% at 0.4 mg/mL collagen. But antiCD18 had no effect on the rate of cell migration through high-concentration collagen gels (>0.6 mg/mL). PMN migration through collagen gels is CD18-dependent but only under conditions of high hydration, suggesting that CD18-mediated effects (e.g., adhesion to gel fibers) are only important when the fiber density is relatively low. Anti-CD18 inhibited, but did not eliminate, the adhesion of fMLP-stimulated PMN to the surface of collagen gels, suggesting that cells use multiple mechanisms for gaining traction within the gel. Because of the multiple modes of interaction between motile cells and the deformable fiber matrix, blockade of one component, such as CD18, can enhance the rate of cell migration under one set of conditions, and inhibit under another.