Cross-linked Collagen Matrix Research Articles

Enhanced Wound Healing Potential of Primary Human Oral Fibroblasts and Periodontal Ligament Cells Cultured on Four Different Porcine-Derived Collagen Matrices.

Xenogenic collagen-based matrices represent an alternative to subepithelial palatal connective tissue autografts in periodontal and peri-implant soft tissue reconstructions. In the present study, we aimed to investigate the migratory, adhesive, proliferative, and wound-healing potential of primary human oral fibroblasts (hOF) and periodontal ligament cells (hPDL) in response to four commercially available collagen matrices. Non-crosslinked collagen matrix (NCM), crosslinked collagen matrix (CCM), dried acellular dermal matrix (DADM), and hydrated acellular dermal matrix (HADM) were all able to significantly enhance the ability of hPDL and hOF cells to directionally migrate toward the matrices as well as to efficiently repopulate an artificially generated wound gap covered by the matrices. Compared to NCM and DADM, CCM and HADM triggered stronger migratory response. Cells grown on CCM and HADM demonstrated significantly higher proliferative rates compared to cells grown on cell culture plastic, NCM, or DADM. The pro-proliferative effect of the matrices was supported by expression analysis of proliferative markers regulating cell cycle progression. Upregulated expression of genes encoding the adhesive molecules fibronectin, vinculin, CD44 antigen, and the intracellular adhesive molecule-1 was detected in hPDL and hOF cells cultured on each of the four matrices. This may be considered as a prerequisite for good adhesive properties of the four scaffolds ensuring proper cell–matrix and cell–cell interactions. Upregulated expression of genes encoding TGF-β1 and EGF growth factors as well as MMPs in cells grown on each of the four matrices provided support for their pro-proliferative and pro-migratory abilities. The expression of genes encoding the angiogenic factors FGF-2 and VEGF-A was dramatically increased in cells grown on DADM and HADM only, suggesting a good basis for accelerated vascularization of the latter. Altogether, our results support favorable influence of the investigated collagen matrices on the recruitment, attachment, and growth of cell types implicated in oral soft tissue regeneration. Among the four matrices, HADM has consistently exhibited stronger positive effects on the oral cellular behavior. Our data provide solid basis for future investigations on the clinical application of the collagen-based matrices in surgical periodontal therapy.

Augmentation of soft tissue volume at pontic sites: a comparison between a cross-linked and a non-cross-linked collagen matrix

AimTo assess histopathological and histomorphometric outcomes of soft tissue volume augmentation procedures at pontic sites using a volume-stable cross-linked collagen matrix (VCMX) and a non-cross-linked collagen matrix (XCM).Materials and methodsIn twelve adult beagle dogs, the mandibular premolars and first molar were hemisected and the mesial root extracted. Soft tissue augmentation was randomly performed using VCMX, XCM, or a sham-operated control. Sacrifice was performed after 4, 8, and 26 weeks. Non-decalcified sections were analyzed for histopathologic and histomorphometric measurements at four different levels below the crest (1.5, 2.5, 3.5, and 5.5 mm).ResultsGroup VCMX presented a greater overall amount of soft tissue at all healing time points, more pronounced fibroblast ingrowth, vascularization, and a substantial new collagen deposition. Over time, group XCM demonstrated faster signs of degradation compared with group VCMX. Four weeks after augmentation, group VCMX yielded a higher mean ridge width compared with groups XCM (2.22 mm VCMX, 0.89 mm XCM (at 2.5 mm); 2.05 mm VCMX, 0.80 mm XCM (at 3.5 mm) p < 0.05) and sham (0.59 mm sham (at 1.5 mm); 0.48 mm (at 2.5 mm); 0.44 mm (at 3.5 mm) p < 0.05). After healing periods of 8 and 26 weeks, measurements in group VCMX remained significantly higher compared with group sham both at 8 weeks (levels of 1.5 mm, 2.5 mm and 5.5 mm) and at 26 weeks (levels of 1.5 mm, 3.5 mm and 5.5 mm) (p < 0.05).ConclusionThe use of a cross-linked collagen matrix resulted in a greater and more stable ridge width over time compared with control groups.Clinical relevanceSoft tissue volume augmentation at pontic sites is more effective when using a cross-linked compared with a non-cross-linked collagen matrix.

Adsorption and Release of Growth Factors from Four Different Porcine-Derived Collagen Matrices.

Xenogeneic acellular collagen matrices represent a safe alternative to autologous soft tissue transplants in periodontology and implant dentistry. Here, we aimed to investigate the adsorption and release of growth factors from four porcine-derived collagen matrices using enzyme-linked immunosorbent assay. Non-crosslinked collagen matrix (NCM), crosslinked collagen matrix (CCM), dried acellular dermal matrix (DADM), and hydrated acellular dermal matrix (HADM) adsorbed each of the following growth factors, TGF-β1, FGF-2, PDGF-BB, GDF-5 and BMP-2, with an efficiency close to 100%. Growth factor release for a 13-day period was in the range of 10–50% of the adsorbed protein, except for the BMP-2 release that was in the range of 5–7%. Generally, protein release occurred in two phases. Phase I was arbitrary defined by the highest release from the matrices, usually within 24 h. Phase II, spanning the period immediately after the peak release until day 13, corresponded to the delayed release of the growth factors from the deeper layers of the matrices. HADM showed significantly (P < 0.001) higher TGF-β1, FGF-2, and PDGF-BB release in phase II, compared to the rest of the matrices. NCM exhibited significantly (P < 0.001) higher FGF-2 release in phase II, compared to CCM and DADM as well as a characteristic second peak in PDGF-BB release towards the middle of the tested period. In contrast to NCM and HADM, CCM and DADM showed a gradual and significantly higher release of GDF-5 in the second phase. Several burst releases of BMP-2 were characteristic for all matrices. The efficient adsorption and sustained protein release in the first 13 days, and the kinetics seen for HADM, with a burst release within hours and high amount of released growth factor within a secondary phase, may be beneficial for the long-term tissue regeneration following reconstructive periodontal surgery.

Effects of soft tissue grafting prior to orthodontic treatment on preventing gingival recession in dogs.

PurposeThis study was conducted to assess the efficacy of prophylactic gingival grafting in the mandibular anterior labial area for preventing orthodontically induced gingival recession.MethodsEight mongrel dogs received gingival graft surgery at the first (I1) and third (I3) mandibular incisors on both sides based on the following group allocation: AT group (autogenous connective tissue graft on I1), AT-control group (contralateral side in the AT group), CM group (xenogeneic cross-linked collagen matrix graft on I3) and CM-control group (contralateral side in the CM group). At 4 weeks after surgery, 6 incisors were splinted and proclined for 4 weeks, followed by 16 weeks of retention. At 24 weeks after surgery, casts were made and compared with those made before surgery, and radiographic and histomorphometric analyses were performed.ResultsDespite the proclination of the incisal tip (by approximately 3 mm), labial gingival recession did not occur. The labial gingiva was thicker in the AT group (1.85±0.50 mm vs. 1.76±0.45 mm, P>0.05) and CM group (1.90±0.33 mm vs. 1.79±0.20 mm, P>0.05) than in their respective control groups.ConclusionsThe level of the labial gingival margin did not change following labial proclination of incisors in dogs. Both the AT and CM groups showed enhanced gingival thickness.

Polypropylene Mesh Predicts Mesh/Suture Exposure After Sacrocolpopexy Independent of Known Risk Factors: A Retrospective Case-Control Study.

The aim of this study was to determine if ultralightweight polypropylene mesh reduced the risk of mesh/suture exposure after sacrocolpopexy compared with heavier-weighted polypropylene. Bivariate and multivariate analyses were used to interpret data from 133 cases and 261 control subjects to evaluate independent predictors of mesh/suture exposure after sacrocolpopexy from 2003 to 2013. Multivariate logistic regression revealed that prior surgery for incontinence (odds ratio [OR], 2.87; 95% confidence interval [CI], 1.19-6.96), porcine acellular cross-linked collagen matrix with medium-weight polypropylene mesh (OR, 4.95; 95% CI, 1.70-14.42), other polypropylene mesh (OR, 6.73; 95% CI, 1.12-40.63), nonabsorbable braided suture for vaginal mesh attachment (OR, 4.52; 95% CI, 1.53-15.37), and immediate perioperative complications (OR, 3.64; 95% CI, 1.53-13.37) were independent risk factors for mesh/suture exposure. After multivariate analysis, ultralightweight polypropylene mesh was no longer associated with decreased rates of mesh/suture exposure after controlling for known risk factors identified during bivariate analysis (P = 0.423). Both mesh choice and suture selection remained independent predictors of mesh/suture exposure, with heavier meshes increasing and monofilament suture decreasing rates of mesh/suture exposure. Based on this study, surgeons may consider use of delayed-absorbable, monofilament suture over nonabsorbable braided suture for attachment of vaginal mesh to reduce the risk of mesh/suture exposure when using mesh.

The fibrotic tumor stroma.

Intratumoral fibrosis results from the deposition of a cross-linked collagen matrix by cancer-associated fibroblasts (CAFs). This type of fibrosis has been shown to exert mechanical forces and create a biochemical milieu that, together, shape intratumoral immunity and influence tumor cell metastatic behavior. In this Review, we present recent evidence that CAFs and tumor cells are regulated by provisional matrix molecules, that metastasis results from a change in the type of stromal collagen cross-link, and that fibrosis and inflammation perpetuate each other through proteolytic and chemotactic mediators released into the tumor stroma. We also discuss aspects of the emerging biology that have potential therapeutic value.

The Biological Basis for Cardiac Repair After Myocardial Infarction: From Inflammation to Fibrosis.

In adult mammals, massive sudden loss of cardiomyocytes after infarction overwhelms the limited regenerative capacity of the myocardium, resulting in the formation of a collagen-based scar. Necrotic cells release danger signals, activating innate immune pathways and triggering an intense inflammatory response. Stimulation of toll-like receptor signaling and complement activation induces expression of proinflammatory cytokines (such as interleukin-1 and tumor necrosis factor-α) and chemokines (such as monocyte chemoattractant protein-1/ chemokine (C-C motif) ligand 2 [CCL2]). Inflammatory signals promote adhesive interactions between leukocytes and endothelial cells, leading to extravasation of neutrophils and monocytes. As infiltrating leukocytes clear the infarct from dead cells, mediators repressing inflammation are released, and anti-inflammatory mononuclear cell subsets predominate. Suppression of the inflammatory response is associated with activation of reparative cells. Fibroblasts proliferate, undergo myofibroblast transdifferentiation, and deposit large amounts of extracellular matrix proteins maintaining the structural integrity of the infarcted ventricle. The renin-angiotensin-aldosterone system and members of the transforming growth factor-β family play an important role in activation of infarct myofibroblasts. Maturation of the scar follows, as a network of cross-linked collagenous matrix is formed and granulation tissue cells become apoptotic. This review discusses the cellular effectors and molecular signals regulating the inflammatory and reparative response after myocardial infarction. Dysregulation of immune pathways, impaired suppression of postinfarction inflammation, perturbed spatial containment of the inflammatory response, and overactive fibrosis may cause adverse remodeling in patients with infarction contributing to the pathogenesis of heart failure. Therapeutic modulation of the inflammatory and reparative response may hold promise for the prevention of postinfarction heart failure.

Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase

The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

Sequential healing of onlay bone grafts using combining biomaterials with cross-linked collagen in dogs.

The aim of this study was to observe the sequential healing of onlay grafts in terms of the volumetric and histologic changes using different bone substances and absorbable membranes according to the presence of collagen cross-linking. Four groups involving onlay grafting with different materials were randomly assigned to both sides of the maxillae of 15 beagle dogs: (i) control group without any treatment; (ii) group NN, bovine hydroxyapatite incorporated into a non-cross-linked collagen matrix (BHC)+non-cross-linked collagen membrane (NCCM); (iii) group NC, BHC+cross-linked collagen membrane (CCM); and (iv) group CC, porcine hydroxyapatite incorporated into a cross-linked collagen matrix+CCM. Radiographic and histological analyses were performed after three different healing periods: 4, 8 and 12weeks. At week 4, the bone substances were well localized under the barrier membrane in groups NC and CC, while the bone substances became spread out and flattened in group NN. Similarly, the augmented height was significantly greater in groups NC and CC (2.55 and 2.51mm, respectively; median) than in group NN (1.96mm, P<0.001, both). The percentages of newly formed bone were significantly higher at week 12 than at weeks 4 and 8 in all of the groups. The NCCM showed an earlier angiogenesis pattern than the CCM; however, earlier degradation was observed at week 12. Combining biomaterials with cross-linked collagen might contribute to maintaining its initial morphology with excellent biocompatibility in early healing period of lateral onlay grafts.

Cross-linked xenogenic collagen implantation in the sheep model for vaginal surgery.

The properties of meshes used in reconstructive surgery affect the host response and biomechanical characteristics of the grafted tissue. Whereas durable synthetics induce a chronic inflammation, biological grafts are usually considered as more biocompatible. The location of implantation is another determinant of the host response: the vagina is a different environment with specific function and anatomy. Herein, we evaluated a cross-linked acellular collagen matrix (ACM), pretreated by the anti-calcification procedure ADAPT® in a sheep model for vaginal surgery. Ten sheep were implanted with a cross-linked ACM, and six controls were implanted with a polypropylene (PP; 56 g/m2) control. One implant was inserted in the lower rectovaginal septum, and one was used for abdominal wall defect reconstruction. Grafts were removed after 180 days; all graft-related complications were recorded, and explants underwent bi-axial tensiometry and contractility testing. Half of ACM-implanted animals had palpable induration in the vaginal implantation area, two of these also on the abdominal implant. One animal had a vaginal exposure. Vaginal ACMs were 63 % less stiff compared to abdominal ACM explants (p = 0.01) but comparable to vaginal PP explants. Seven anterior vaginal ACM explants showed areas of graft degradation on histology. There was no overall difference in vaginal contractility. Considering histologic degradation in the anterior vaginal implant as representative for the host, posterior ACM explants of animals with degradation had a 60 % reduced contractility as compared to PP (p = 0.048). Three abdominal implants showed histologic degradation; those were more compliant than non-degraded implants. Vaginal implantation with ACM was associated with graft-related complications (GRCs) and biomechanical properties comparable to PP. Partially degraded ACM had a decreased vaginal contractility.

Bone micro-fragility caused by the mimetic aging processes in α-klotho deficient mice: In situ nanoindentation assessment of dilatational bands

The nanoscale structure-function relationship is a key determinant of bone toughness or micro-fragility. The loss of bone toughness during the aging process has been accepted based on empirical evidence, but this concept has not yet been fully supported by evidence at the material level. Here, we demonstrate a reduction in bone toughening mechanism in mimetic aged cortical bone obtained from α-klotho deficient (α-klotho−/−) mice and assessed by in situ dynamic mechanical analysis. The strain-rate nanoindentation tests showed enhanced stiffening of the wild-type calvarial bone and a large dimensional recovery during rapid loading following the constant displacement test. Such strain-dependent stiffening was likely associated with nanoscale dilatational bands and subsequent strain-energy transfer to the superior wild-type cross-linked collagen matrix network. The absence of dilatational bands formed by hydroxyapatite crystals and non-collagenous proteins in the α-klotho−/− bone samples likely diminished the intrinsic bone toughening mechanisms almost independent of viscoelastic behaviors. Such nanoscale structural alternations that occur during aging processes lead to crack propagation and result in overall bone fractures under large external stresses. In addition, dynamic mechanical analysis using instrumented nanoindentation was useful for the evaluation of bone mechanical properties in this pathological model of a genetic knockout mouse.

Effect of platelet‐derived growth factor‐BB on tissue integration of cross‐linked and non‐cross‐linked collagen matrices in a rat ectopic model

To test whether or not the network structure and the addition of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) to a chemically cross-linked collagen matrix (CCM)- and a non-cross-linked collagen matrix (NCCM)-influenced tissue integration, angiogenesis, and matrix degradation. Four treatment modalities were randomly assigned to four unconnected pouches in the back of 50 rats: (i) CCM-S (soaked in saline), (ii) CCM-P (plus rhPDGF-BB), (iii) NCCM-S (soaked in saline), and (iv) NCCM-P (plus rhPDGF-BB). The animals were sacrificed at 2, 4, 8, 16, and 24 weeks. Descriptive histology and histomorphometric assessments were performed thereby evaluating matrix thickness, the number of vessels (angiogenesis), and connective tissue formation. Means and standard deviations were calculated. Robust linear mixed modeling was used to test the effect of group (NCCM vs CCM), rhPDGF-BB, and time point of sacrifice (2, 4, and 8 weeks). The thickness of NCCM groups revealed stability (range 440-570 μm) over 8 weeks, while the matrices were no longer present at 16 and 24 weeks. CCM matrices demonstrated a maximal thickness at 2 weeks (2689 ± 187 μm for CCM-S and 2693 ± 389 μm for CCM-P), a decrease of roughly 40% at 8 weeks, but were still present at 16 and 24 weeks. Vascularization of NCCM gradually increased over time with a peak (mean 17.0; SD 1.7) for NCCM-S and NCCM-P (22.0 ± 34.8) at 8 weeks. Angiogenesis in CCM was significantly more pronounced at early time points with a peak at 2 weeks (29.3 ± 16.8 for CCM-S and 30.3 ± 18.4 for CCM-P). No statistically significant effect of rhPDGF-BB was observed for any of the evaluated parameters (all P > 0.05). The compact layer (in NCCM) delayed angiogenesis and connective tissue formation, while the spongeous cross-linked matrix of CCM facilitated early vascularization and demonstrated network presence over a longer time span.

Endonasal Septal Perforation Repair Using an Interpositional Porcine Dermal Collagen Implant

Objectives: Describe our novel minimally invasive technique for closure of nasal septal perforations. Methods: We describe our experience in a series of patients from 2 London teaching hospitals treated using the same surgical technique. Closure of the defect was undertaken using one inferiorly based nasal floor mucosal flap and a contra-lateral superiorly based vascularized septal mucosal flap. This was achieved by a closed endonasal approach with a porcine dermal collagen sheet interpositional graft (Permacol) placed on the more concave side of the perforation. The mucosal flaps are used to cover the repair and sutured in place. Results: Septal perforations of up to 2-cm diameter were successfully repaired in greater than 90% of cases over a 36-month follow-up period. Conclusions: Our novel technique has proved effective in closure of septal perforations. Permacol is a durable graft, utilizing acellular cross-linked collagen matrix, used commonly in abdominal hernia repair. We found Permacol to be a biocompatible graft with the ability to resist dessication if there is slow re-epithelization or partial exposure, making it a robust and easy to use interpositional graft with no morbidity from graft harvesting. The use of local intranasal mucosal advancement flaps allows a minimally invasive approach without any external incisions or scars. Potential limitations are the size of the pedicled flaps for very large perforations, or the lengths of the flaps for very anterior defects. For the majority of septal perforations the authors feel this technique offers an effective minimally invasive option with low morbidity.

Analysis of variations in porosity of metal crosslinked collagen matrix

ABSTRACTThe fundamental knowledge of the porous nature of crosslinked collagen matrix such as tanned leather is an aid to design appropriate chemicals for leather making. It would also help to target a particular area of matrix to improve its uniformity and other functional properties. The purpose of this study is to analyze the variations in pore sizes of chromium crosslinked collagen matrix, chrome tanned leather, from different animal species and different areas of the same species. In this study, chrome tanned leather from goat and sheep were investigated for surface area, pore size, and distribution. Thermoporometry results show that average pore radius of goat leather is around 2–30 nm and that of sheep is 2–20 nm. Nitrogen adsorption result shows that average surface area of goat (8.24 m2/g) leather is higher than sheep (6.73 m2/g), but the average pore diameter of goat (289 nm) is smaller than sheep (385 nm) leather. It has been found that more numbers of smaller pores are present in goat than sheep leather and all the leather samples including goat and sheep obeyed type‐III adsorption isotherm. Capillary flow porometry analysis gives the smallest, largest, and mean‐flow‐diameter of through‐pores. The average size of largest throat pore diameter of sheep (1313 nm) is smaller than that of goat (1385 nm) leather. In general, the pore volume distribution of sheep leather is higher than that of goat leather. Morphological analysis using scanning electron microscopy shows that pore mouth of goat is deeper than that of sheep. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40835.

Rectovaginal fistula: a new technique and preliminary results using collagen matrix biomesh

A rectovaginal fistula (RVF) is a debilitating condition that is difficult to treat. Many available techniques are invasive and involve extensive surgery. A local procedure with good closure rates would be preferable as a first step in the treatment of RVF. The aim of this study was the development of a local technique for the closure of RVF with good closure rates to prevent the use of more invasive procedures. This was a pilot study. Patients with RVF who had undergone multiple operations in the pelvic area, local radiotherapy, chemotherapy or had been diagnosed with Crohn's disease were included in the study. All had a history of surgery for RVF. A cross-linked collagen matrix biomesh was placed in the rectovaginal septum using a transperineal or a transvaginal approach. The main outcome measure in this study was the closure rate reported as absence of the fistula at 1year. Twelve patients were included in the study. Absence of fistula at 1year was 0.64 (95% confidence interval 0.30-0.85). Three patients (25.0%) developed a recurrence, two were reoperated on with a gracilis flap transposition, and one was treated with laparoscopic ligation. In one patient (8.3%), the fistula failed to close within 3months after the mesh placement. Our technique shows promising results. A local and simple technique with acceptable closure and morbidity rates, like our local repair with biomesh, would be ideal as a first step in treating RVFs. Long-term results are needed.

Morphological and chemical characterization of cross-linked dentin collagen matrix

Morphological and chemical characterization of cross-linked dentin collagen matrix

Relationship among Bone Mineral Density Reduction, Hearing Loss, and Balance Disorders in Osteoporotic Patients

Osteoporosis is a disease that weakens bones and is known to be “silent,” but it does not mean that it should be ignored. According to some researches, osteoporosis causes more than 8.9 million fractures every year, resulting in an osteoporotic fracture every 3 s and by 2050 the world-wide occurrence of hip fracture is estimated to increase by 310% in men and 240% in women (Gullberg et al., 1997; Johnell and Kanis, 2006). The main aspect of osteoporosis is losing bone mineral density (BMD). Mainly bones consist of minerals such as a matrix of hydroxyapatite (calcium phosphate) and other minerals which are embedded in a cross-linked collagen matrix. Normally, there is a delicate balance between bone formation and bone re-absorption, which if bone re-absorption becomes dominant osteopenia and osteoporosis may occur (Larsen, 2007). In another word, in osteoporotic patients due to reduction of bone formation in comparison with bone re-absorption, low bone mineral is stored and as a result BMD loss occurs (Bone et al., 2000; Marie, 2013). On the other hand, according to some researches, it has been shown that there is a correlation between BMD loss in osteoporotic patients and conductive hearing loss; in those patients over a specific age, changing the structure of the ossicles or hormonal mechanism in hearing may correlate BMD loss with hearing loss (Shafer, 2006; Babich et al., 2009). In fact, the hearing and balance systems are connected inside the inner ear and hearing loss can affect balance system. Basically, Balance is a complex that receives sensory information from a variety of organs (vision, auditory, and joints) and integrates it to inform the body of where it is. Furthermore, information from the vestibular system of the inner ear (semicircular canals, the saccule and the utricle) is sent to the brain. Semicircular canals in the inner ear are able to sense changes in movement of the body. Due to these changes, endolymph within the canals moves inner ear’s hair cells. As a result, position of the head is sensed by hair cells. Normally, the information coming from the ears perfectly matches the information coming from the eyes and the sensors in the joints and balance complex completes (Standring, 2004; Wedro, 2009). What is more, there are some evidences that show due to metabolic dysfunction in bones – osteoporosis – there is a correlation between hearing loss and balance disorders (Zatonski et al., 2012). In conclusion, we hypothesize that in osteoporotic patients, who have low BMD, hearing loss occurs and as a result balance problems happen. In spite of the fact that some researchers have shown that osteoporotic patients have balance disorders and hearing loss (Henkin et al., 1972; Matsuo et al., 2005), the possible relationship with BMD loss and hearing loss has not been clarified up to now (Figure ​(Figure1).1). In fact, our methodology toward this study would be based on clinical records of patients. Basically, patients’ BMD score, history of falling (summary of their status of stability), and their auditory test score is needed to provide our hypothesis. Last but not least, it is proposed that using some specific earphones which intensify the sound, by compensating the hearing loss in patients may reduce risk of falls in osteoporotic patients who suffer from balance problems, but obviously earphones are not preventive enough for falling. Surely experimental researches and clinical trials are needed to validate our hypothesis. Figure 1 Relationship between BMD loss and balance disorders in osteoporotic patients.

Tissue integration of collagen‐based matrices: an experimental study in mice

To test whether or not tissue integration, biodegradation, and new blood vessel formation in two collagen-based matrices depend on the level of chemical cross-linking. Two collagen matrices with high (CM1) and low (CM2) levels of chemical cross-linking were randomly implanted in two pouches in 14 athymic nude mice. Three and 6 weeks later, the animals were euthanized. Histologic and histomorphometric measurements were performed on paraffin-embedded sections. Both collagen matrices integrated well into the surrounding soft tissues. The level of cross-linking and duration of implantation had an effect on the formation of new blood vessels. More blood vessels (n = in absolute numbers) were found in outer compartments compared to the central compartments of the matrices, reaching 5.6 (CM2) vs. 4.3 (CM1) at 3 weeks, and 5.3 (CM2) vs. 7.3 (CM1) at 6 weeks. Similarly, connective tissue formation increased for both matrices between 3 and 6 weeks, whereas the amount of remaining collagen network gradually decreased over time being more pronounced for CM1 (-50%) compared to CM2 (-15%). The degree of cross-linking was negatively correlated for all outcome measures resulting in improved tissue integration, superior matrix stability and enhanced angiogenic patterns for the less cross-linked collagen matrix (CM2) in this experimental study in mice.

165 T-Cell Derived Microparticles Induce Fibrolytic Activation of Hepatic Stellate Cells via ICAM-1 (CD54) Dependent Uptake and Emmprin (CD147)

Background: Targeting extracellular matrix stability is an attractive antifibrotic strategy. Prior studies have suggested a role for tissue transglutaminase-mediated collagen crosslinking in matrix stabilization, but the extent and mechanisms remain poorly understood. We therefore used a quantitative biochemical approach to characterize dynamic changes in collagen crosslinking during progression and reversal of CCL4-induced liver fibrosis in wildtype mice and in mice deficient in transglutaminase 2 (TG2), the predominant transglutaminase isoform in the liver. Materials and Methods: Liver fibrosis was induced in TG2-/mice and wildtype littermates (C57BL6) by repeated gavage with carbon tetrachloride (CCL4) 3x per week for 1, 3, 6 and 12 weeks. Fibrosis reversal was monitored for up to 36 weeks after CCL4 withdrawal. Collagen crosslinking was measured by sequential extraction using neutral salt, acetic acid and pepsin. Collagen was quantified biochemically via hydroxyproline in the livers, in the extracts and in residual liver tissue. This was correlated to histological evolution and transcript levels of fibrosis related genes. Results: In normal liver, neutral salt, acetic acid and pepsin solubilized 3.2, 1.2% and 91% of total collagen, respectively, while 4.5% remained insoluble, representing a highly cross-linked collagenous matrix. CCL4 administration for 12w led to development of cirrhosis and a 6-fold increase in total liver collagen. Collagen solubility during fibrosis progression revealed progressive cross-linking, with an increase of the insoluble collagen fraction to 27% (6-fold) at week 12 at the expense of the pepsin-soluble fraction. The saltand acid-soluble collagen fractions, representing freshly synthesized collagen, remained constant at 5% of total collagen. The extent of matrix deposition and stabilization remained constant up to 36 weeks after discontinuation of CCL4. Furthermore, no difference in collagen solubility and no fibrosis reversal or change in matrix-related transcripts were found in TG2-/vs. wildtype mice during all time points of progression or follow-up. Conclusions: 1) Quantitative measurement of fibrotic matrix stability revealed a significant increase of collagen crosslinking, which parallels fibrosis progression and limits its reversal. 2) Matrix stabilization in fibrotic liver is independent of transglutaminase-mediated cross-linking since it was unaffected in TG2-/mice 3) This TG2independent collagen cross-linking is an important target for future antifibrotic therapies.

162 Progressive Matrix Crosslinking Limits Reversibility of Liver Fibrosis and is Independent of Tissue Transglutaminase

Background: Targeting extracellular matrix stability is an attractive antifibrotic strategy. Prior studies have suggested a role for tissue transglutaminase-mediated collagen crosslinking in matrix stabilization, but the extent and mechanisms remain poorly understood. We therefore used a quantitative biochemical approach to characterize dynamic changes in collagen crosslinking during progression and reversal of CCL4-induced liver fibrosis in wildtype mice and in mice deficient in transglutaminase 2 (TG2), the predominant transglutaminase isoform in the liver. Materials and Methods: Liver fibrosis was induced in TG2-/mice and wildtype littermates (C57BL6) by repeated gavage with carbon tetrachloride (CCL4) 3x per week for 1, 3, 6 and 12 weeks. Fibrosis reversal was monitored for up to 36 weeks after CCL4 withdrawal. Collagen crosslinking was measured by sequential extraction using neutral salt, acetic acid and pepsin. Collagen was quantified biochemically via hydroxyproline in the livers, in the extracts and in residual liver tissue. This was correlated to histological evolution and transcript levels of fibrosis related genes. Results: In normal liver, neutral salt, acetic acid and pepsin solubilized 3.2, 1.2% and 91% of total collagen, respectively, while 4.5% remained insoluble, representing a highly cross-linked collagenous matrix. CCL4 administration for 12w led to development of cirrhosis and a 6-fold increase in total liver collagen. Collagen solubility during fibrosis progression revealed progressive cross-linking, with an increase of the insoluble collagen fraction to 27% (6-fold) at week 12 at the expense of the pepsin-soluble fraction. The saltand acid-soluble collagen fractions, representing freshly synthesized collagen, remained constant at 5% of total collagen. The extent of matrix deposition and stabilization remained constant up to 36 weeks after discontinuation of CCL4. Furthermore, no difference in collagen solubility and no fibrosis reversal or change in matrix-related transcripts were found in TG2-/vs. wildtype mice during all time points of progression or follow-up. Conclusions: 1) Quantitative measurement of fibrotic matrix stability revealed a significant increase of collagen crosslinking, which parallels fibrosis progression and limits its reversal. 2) Matrix stabilization in fibrotic liver is independent of transglutaminase-mediated cross-linking since it was unaffected in TG2-/mice 3) This TG2independent collagen cross-linking is an important target for future antifibrotic therapies.