Arrangement Of Collagen Fibers Research Articles

Complex Susceptibilities and Chiroptical Effects of Collagen Measured with Polarimetric Second-Harmonic Generation Microscopy

Nonlinear optical properties of collagen type-I are investigated in thin tissue sections of pig tendon as a research model using a complete polarimetric second-harmonic generation (P-SHG) microscopy technique called double Stokes-Mueller polarimetry (DSMP). Three complex-valued molecular susceptibility tensor component ratios are extracted. A significant retardance is observed between the chiral susceptibility component and the achiral components, while the achiral components appear to be in phase with each other. The DSMP formalism and microscopy measurements are further used to explain and experimentally validate the conditions required for SHG circular dichroism (SHG-CD) of collagen to occur. The SHG-CD can be observed with the microscope when: (i) the chiral second-order susceptibility tensor component has a non-zero value, (ii) a phase retardance is present between the chiral and achiral components of the second-order susceptibility tensor and (iii) the collagen fibres are tilted out of the image plane. Both positive and negative areas of SHG-CD are observed in microscopy images, which relates to the anti-parallel arrangement of collagen fibres in different fascicles of the tendon. The theoretical formalism and experimental validation of DSMP imaging technique opens new opportunities for ultrastructural characterisation of chiral molecules, in particular collagen, and provides basis for the interpretation of SHG-CD signals. The nonlinear imaging of chiroptical parameters offers new possibilities to further improve the diagnostic sensitivity and/or specificity of nonlinear label-free histopathology.

Decellularized cartilage matrix scaffolds with laser-machined micropores for cartilage regeneration and articular cartilage repair

Decellularized allogeneic and xenogeneic articular cartilage matrix scaffolds (CMS) are considered ideal scaffolds for cartilage regeneration owing to their heterogeneous architecture, and biochemical and biomechanical properties of native articular cartilage. However, the dense structure of the articular cartilage extracellular matrix, particularly the arrangement of collagen fibers, limits cellular infiltration, leading to poor cartilage regeneration. In addition, the incomplete removal of xenograft cells is associated with immunogenic reaction in the host. To facilitate the migration of chondrocytes into scaffolds and the rate of decellularization processing, we applied a carbon dioxide laser technique to modify the surface of porcine CMS while retaining major properties of the scaffold. By optimizing the laser parameters, we introduced orderly, lattice-arranged conical micropores of suitable depth and diameter onto the cartilage scaffold surface without affecting the cartilage shape or mechanical properties. We found that laser-modified CMS (LM-CMS) could enhance the degree of decellularization and were conducive to cell adhesion, as compared with the intact CMS. Decellularized scaffolds were seeded with rabbit-derived chondrocytes and cultured for 8 weeks in vitro. We found that cell-scaffold constructs formed cartilage-like tissue within the micropores and on the scaffold surface. In vivo, we found that cell-scaffold constructs subcutaneously implanted into the flanks of nude mice formed ivory-white neocartilage with high contents of DNA and cartilage matrix components, as well as good mechanical strength as compared with native CMS. Furthermore, scaffolds combined with autogenous chondrocytes induced neocartilage and better structural restoration at 8 weeks after transplantation into rabbit knee articular cartilage defects. In conclusion, decellularized xenogeneic CMS with laser-machined micropores offers an ideal scaffold with high fidelity for the functional reconstruction of articular cartilage.

Clinical application of acellular matrix derived from the bubaline diaphragm and caprine rumen for the repair of abdominal wall defects in animals

The abdominal wall hernias resulting due to trauma or other clinical conditions are common in animals. Large hernias required the use of synthetic mesh, which is costly and may result in infection, fistula formation, and pain. Application of biomaterials in hernia repair causes a reduction in pain, reduced recovery time, and rate of recurrence. The study was undertaken to test the acellular bubaline diaphragm matrix (BDiaM) and acellular caprine rumen matrix (CRuM) for the repaired hernia in clinical cases. Fresh bubaline diaphragm and caprine rumen were decellularized using sodium deoxycholate (1% for CRuM and 2% for BDiaM) for 48h. Acellularity was ascertained histologically and by DNA quantification. Histologically, both the matrices showed complete acellularity and orderly arranged collagen fibers after 48 h. The DNA contents were significantly (P0.05) reduced in both the matrices in comparison to the native matrices. The BDiaM and CRuM matrices were applied in eight and nine clinical cases of abdominal wall defects, respectively. Animals with BDiaM and CRuM matrices recovered uneventfully and remained sound at least up to 3 months. Hematological and immunological findings were unremarkable. BDiaM and CRuM matrices showed good results without complications. Keywords:Biocompatibility, Bubaline diaphragm matrix, Caprine rumen matrix, DNA quantification, ELISA, SDS-PAGE

Combination of mesenchymal stem cell-conditioned medium and botulinum toxin type A for treating human hypertrophic scars

Both mesenchymal stem cell-conditioned medium (MSC CM) and Botox have demonstrated therapeutic effects for hypertrophic scar (HS). It is unclear whether a synergistic effect occurs when these treatments are used in combination. We aimed to investigate the therapeutic effects of MSC CM and Botox alone when compared with those of a combined regimen on HS. Fibroblasts from human HS were isolated and treated with Dulbecco's modified Eagle's medium (DMEM), MSCCM, or Botox alone or a combination of MSCCM and Botox. We also used an in vivo HS-buried null mice model to investigate the efficacy of combination treatment. The results demonstrated that the combination of MSC CM and Botox downregulated both mRNA and protein levels of type I collagen, type III collagen, and alpha-smooth muscle actin (α-SMA) in HS fibroblasts. The combined regimen also suppressed fibroblast proliferative activity, increased apoptosis, and displayed significant inhibitory effects on the contractile ability of HS fibroblasts compared to MSC CM, Botox, or DMEM alone. Using an in vivo HS-buried null mice model, significant scar weight reduction, cell apoptosis, and less α-SMA expression were observed from the combined regimen of MSC CM and Botox compared to those from the other groups. The combined regimen also significantly improved arrangement and deposition of collagen fibers. This study demonstrates that a combination of MSC CM and Botox exhibited a significant therapeutic effect compared to monotherapy. Clinical translation of this therapy should be further considered.

Dynamic changes of the extracellular matrix during corneal wound healing

The extracellular matrix (ECM) confers transparency to the cornea because of the precise organization of collagen fibrils and a wide variety of proteoglycans. We monitored the corneal wound healing process after alkali burns in rabbits. We analyzed the location and expression of collagens and proteoglycans, the clinical impact, and the recovery of optical transparency.After the animals received both general and ocular topical anesthesia, the central cornea of the left eye was burned by placing an 8-mm diameter filter paper soaked in 0.5 N NaOH for 60 s. The eyes were evaluated under a surgical microscope at 1, 3, and 6 months after burning. At each time point, the clinical conditions of the burned and control corneas were observed. The arrangement of collagen fibers in the corneal stroma was visualized by Picrosirius-red staining, Gomori's silver impregnation and transmission electronic microscopy. Corneal light transmittance was also measured. Myofibroblasts presence was analyzed by immunohistochemistry. mRNA expression levels of collagen types I and III, lumican, decorin, keratocan and alpha-smooth muscle actin were determined by quantitative real-time polymerase chain reaction.One month after alkali burn, the ECM was disorganized and filled with lacunae containing different types of cells and collagen type III fibers in the wound area. Corneal opacities were present with attendant loss of light transmittance. Collagen and proteoglycan mRNA expression levels were up-regulated. After three months, wound healing progress was indicated by reduced corneal opacity, increased light transmittance, reorganization of collagen fibers and only collagen type I expression levels were at control levels. After six months, the wound area ECM morphology was similar to controls, but transmittance values remained low, denoting incomplete restoration of the stromal architecture.This multidisciplinary study of the stromal wound healing process revealed changes in corneal transmittance, collagen organization, myofibroblasts presence and ECM composition at 1, 3, and 6 months after alkali burning. Documenting wound resolution during the six-month period provided reliable information that can be used to test new therapies.

The Epiligament: Structure, Postnatal Development and Role in Ligament Healing.

While much is known about the ligament, the precise morphology and function of the thin layer of connective tissue lining its surface, termed the epiligament, have not been fully studied yet. Herein, we aimed at reviewing the recent findings on the structural and functional significance of the epiligament in both animal models and human tissue. The epiligament is made up of various connective tissue cells such as fibroblasts, fibrocytes, mast cells, and adipocytes and contains a number of neurovascular bundles. Arrangement of collagen fibers in the epiligament is rather chaotic, in multiple directions, which allows for greater mobility and resistance to stress. Differences in the collagen content and types of enzymes of the group of matrix metalloproteinases between the epiligament and the underlying ligament tissue have been reported and are reviewed herein. While the ligament tissue mainly contains collagen type I, the epiligament is also rich in collagen types III and V. As suggested by a number of studies, the epiligament plays a key role in ligament repair as a donor of cells and matrix metalloproteinases, particularly matrix metalloproteinase-2 and 9, which are essential for scar tissue remodeling. In conclusion, future studies will likely reveal additional functional aspects of the epiligament, which may allow scientists to devise more suitable treatment strategies for damaged ligaments in a world where injuries resulting from sports activities or daily routine have long merited their due attention.

Simvastatin Attenuates Cardiac Fibrosis via Regulation of Cardiomyocyte-Derived Exosome Secretion.

Exosome-mediated communication within the cardiac microenvironment is associated with cardiac fibrosis. Simvastatin (SIM), a potent statin, protects against cardiac fibrosis, but its mechanism of action is unclear. We investigated the inhibitory effects and underlying mechanism of simvastatin in cardiac fibrosis, by regulating exosome-mediated communication. Male Sprague-Dawley rats were treated with angiotensin (Ang) II alone, or with SIM for 28 d. Cardiac fibrosis, expressions of fibrosis-associated proteins and mRNAs, and collagen fiber arrangement and deposition were examined. Protein expressions in exosomes isolated from Ang II-treated cardiomyocytes (CMs) were evaluated using nano-ultra-performance liquid chromatographic system, combined with tandem mass spectrometry. Transformation of fibroblasts to myofibroblasts was evaluated using scanning electron and confocal microscopy, and migration assays. Our results showed that SIM attenuated in vivo expression of collagen and collagen-associated protein, as well as collagen deposition, and cardiac fibrosis. The statin also upregulated decorin and downregulated periostin in CM-derived exosomes. Furthermore, it suppressed Ang II-induced transformation of fibroblast to myofibroblast, as well as fibroblast migration. Exosome-mediated cell-cell communication within the cardiac tissue critically regulated cardiac fibrosis. Specifically, SIM regulated the release of CM exosomes, and attenuated Ang II-induced cardiac fibrosis, highlighting its potential as a novel therapy for cardiac fibrosis.

Cassava biopolymer polyamide hydrogel accelerates healing of induced skin woundsin rats

ABSTRACT This study aimed to macroscopically and microscopically evaluate the healing of skin wounds induced in rats by topical application of cassava polyamide biopolymer hydrogel. In total, 32 rats were used and divided into four groups (n= 8): negative control - saline solution; positive control - use of commercial ointment; experimental group - I - ointment + cassava hydrogel; experimental group - II - cassava hydrogel. A 1cm2 wound induced on the animals dorsum was treated and evaluated. At day 21 post-operation, the animals were sacrificed by anesthetic overdose, and then 1cm2 of cicatricial skin from the wound region was collected. The material was cut to evaluate healing. In the macroscopic evaluation, complete healing was observed at the end of 21 days. Re-epithelialization was observed histologically; the connective tissue in the negative control, positive, and experimental - I groups was characterized by an abundance of collagen fibers, fibroblasts, and blood vessels. In experimental group - II additional healing was observed, as evidenced by the arrangement of collagen fibers and fibroblasts, and the reduction of neoformed vessels. Thus, we concluded that the hydrogel can assist in healing skin wounds, especially in the remodeling phase.

The possible role of folic acid against sodium fluoride induced toxicity in the skin of adult male albino rats.

Background:Exposure of human being to fluoride occurs through various sources as food, water, toothpastes and fluoride additives. This exposure is either to low concentrations for long periods or to high concentrations and both may lead to poisoning. Folic acid (FA) has an important free radical scavenging property which makes it the most potent antioxidants. Moreover, it suppresses apoptosis and boost cell survival. Aim of the work: To evaluate the protective and therapeutic role of FA against sodium fluoride (NaF) induced toxicity on adult male rat skin. Materials and Methods:Forty adult (3 months) male albino rats(weighing 200-220 gm) were equally divided into 4 groups (10 rats each):group 1 (control),group 2 (NaF, for 4 weeks),group 3 (NaF, for 4 weeks followed by FA for another 4 weeks) and group 4 (NaF plus FA for 4 weeks). Both NaF (25mg/Kg b.w.) and FA (8 mg/Kg b.w.) were given orally daily. The rats were sacrificed and samples of the back skin were processed for histological, immunohistochemical (Bcl2 antiapoptotic protein) and morphometric studies. Results:Group 2 revealed signs of degeneration in epidermal cells, expansion of intercellular spaces and destructed organelles. There wasa reduction in epidermal thickness with disturbance in collagen fibers arrangement and weak immunoreactivity of Bcl-2. FA induced relative improvement in the dermal and epidermal structures in group 3 while there was preservation of these structures in group 4. Conclusion:FA ameliorates NaF induced harmful effects on skin in addition to its valuable preventive role.

Histopathological changes in patellar tendon enthesis of rabbit induced by electrical stimulation intensity

BackgroundEnthesis injury is a common problem in athletes and workers, which is considered closely related to overuse. However, the early pathophysiologic changes of osteotendinous junction are not well understood, and moreover, few studies investigated the relationship between intensity and pathological changes. The purpose of this study was to evaluate microstructural changes induced by different loading intensities and to find out the threshold intensity. MethodsForty-eight New Zealand White rabbits were randomly divided into six groups. One control group, the others were electrically stimulated to contract repetitively for 2 h per day, three days a week. 30% of peak tetanic force (7.06 N) was adopted to stimulate the rabbits in the 100% cyclic loading group. Other groups were stimulated with 20%, 40%, 60% and 80% of this force. After four weeks, prepared samples were stained with hematoxylin and eosin. ResultsAfter 4 weeks of cyclic loading, the shape and the distribution of tendon cells in patellar enthesis changed, the arrangement of collagen fibers became disordered and the tidemark had become irregular or even disappeared. Different stimulus intensity caused a significant change of cell density in different groups (F = 10.19, P < 0.001). The cell densities of tendon were 34.3 ± 7.9 cells/100 μm2 (L60), 38.2 ± 5.9 cells/100 μm2 (L80), 43.8 ± 10.3 cells/100 μm2 (L100) respectively, which had significant difference with CON group 22.5 ± 3.5 cells/100 μm2. The thickness of fibrocartilage zone had no significant difference among the groups. ConclusionsThe changes of histomorphology with the increasing exercise intensity elucidated that the degree of enthesis microdamage was directly related to the intensity of exercises. The findings demonstrated that 18% (used in L60 group) of peak tetanic force was the threshold intensity which could induce pathological changes in enthesis in four weeks.

The influence of Cisplatin-based chemotherapy on the osseointegration of dental implants: An in vivo mechanical and histometrical study.

To evaluate the effect of Cisplatin on bone repair and mineralization around implants and on the mechanical properties of bone tissue. Forty-three Wistar rats were randomly divided into two groups: Cisplatin (CIS, medication) and control (CTL, placebo solution), administered once a week for 4weeks. After 4weeks, implants were installed in both tibiae metaphysis. After 30 and 60days, the animals were sacrificed and their femurs and tibiae were removed. Femurs were subjected to mechanical tests and tibiae for removal torque, arrangement and distribution of collagen fibers, morphometrical analyses (bone tissue in contact with the implant surface [BIC] and areal fraction between implant threads occupied by bone tissue [BAFO]) and scanning electron microscopy to calcium and calcium/phosphorus analysis. Data were analyzed by ANOVA or MANOVA, and Tukey or Games-Howell post hoc tests, respectively (α=0.05). The CTL specimens had significantly higher values (0.0001≤p≤0.036) of strength (N), removal torque (N/cm2 ), %BIC, and %BAFO than CIS specimens, being their best results at day 60. No significant differences were found among the groups regarding the values of deformation, percentage of calcium, and calcium/phosphorus ratio. In CIS groups, there was a reduction in the organization of collagen at the bone/implant interface, resulting in a trabecular bone with thin trabeculae and birefringent collagen and irregular arrangement. Cisplatin interfered negatively in the repair and mineralization around dental implants, as well as on the quality of the bone tissue, mainly in the period of 30days after the implant placement.

Moxibustion promoted wound healing in rats with full-thickness cutaneous wounds

To explore the effects of moxibustion intervention on wound healing in rats with full-thickness cutaneous wounds. A total of 28 adult SD male rats were randomly assigned to model group and moxibustion group (n＝14 in each one). The skin wound model was established by removal of a piece of full-thickness skin from the median line of the rats' back (about 2 cm below the shoulder blade). Moxibustion intervention was applied to the surrounding area of the focus for 25 min, once daily for 6 days. The wound healing was observed and photographed after each moxibustion intervention. The wound tissues were harvested after transcardiac perfusion with 4% paraformaldehyde solution on the 2nd and 7thday after modeling, and stained with H．E. and Masson methods for displaying histopathological changes and collagen fiber growth status, respectively. After modeling, the cutaneous wound was healed gradually in both groups, and the wound area was significantly smaller from the 2nd day to the 5th day in the moxibustion group than in the model group (P<0.01, P<0.05). Correspondingly, the wound closure area was significantly bigger from the 2nd to the 5th day in the moxibustion group than in the model group (P<0.01). H．E. and Masson staining showed that the number of the inflammatory cells (monocytes, macrophages, neutrophile, etc.) and the collagen fiber area in the local wound tissue of the moxibustion group were significantly bigger than those of the model group on the 2nd day after intervention (P<0.01). After 6 sessions of moxibustion intervention, the number of fibroblasts and collagen fibers in the moxibustion group were obviously increased than that in the model group, characterized by closer arrangement of fibroblasts and collagen fibers, more and larger of the new-born blood vessels, and thicker of the scab in the wound area. Moxibustion can promote the wound healing by enhancing the growth of collagen fibers and cell proliferation in the traumatic cutaneous area in full-thickness cutaneous wound rats.

EFFECT OF AUTOLOGOUS PLATELET-RICH PLASMA ON ARRANGEMENT OF COLLAGEN FIBERS AT INJURED ACHILLES TENDON ENTHESES IN RABBITS

Objective: To evaluate the effects of autologous platelet-rich plasma on arrangement of collagen fibers at injured Achilles tendon entheses in rabbits.&#x0D; Study Design: Laboratory-based experimental study.&#x0D; Place and Duration of Study: Department of Anatomy, Army Medical College, Rawalpindi, in collaboration with National Institute of Health, Islamabad, from March to May, 2018.&#x0D; Material and Methods: Forty, healthy, male, “New Zealand white rabbits”, 4-6 months of age, weighing 2000- 2500g, were randomly divided into 4 equal groups (A, B, C and D). Group A served as control, while B, C and D were experimental groups. Injuries were surgically induced at Achilles tendon entheses of rabbits of experimental groups, which were treated with injections of autologous platelet-rich plasma, either at the time of inducing injury or two weeks after injury in groups C and D respectively while group B was left untreated. Animals were sacrificed after 12 weeks. Tissues were processed and stained for histological evaluation. Statistical analysis was done using SPSS version 22. A p-value of ≤0.05 was taken as significant.&#x0D; Results: Experimental group B showed marked deterioration in Bonar’s modified score for arrangement of collagen fibers as compared to the control group A. The scores were significantly improved in treatment groups C and D as compared to group B.&#x0D; Conclusions: Injection of autologous platelet-rich plasma effectively improved the Bonar modified score for arrangement of collagen fibers at injured entheses, 12 weeks after injury as compared to non-treatment group.

Stromal Changes In Benign and Malignant Salivary Gland Tumors With Picrosirius Red-A Polarizing Microscopic Study

Objectives: To explore the polarizing color profile in connective tissue stroma of Picro-Sirius Red (PSR) stained sections of benign and malignant salivary gland tumors (SGT) and to assess the possible utility of this method as a diagnostic tool. Materials and Methods: A total number of 60 Paraffin blocks of benign (30 cases) and malignant (30 cases) salivary gland tumors were selected from the archives of our institute and regional cancer hospital. 30 cases of Mucous Extravasation Phenomenon (MEP) were selected as control. Salivary gland tumors and control were stained with PSR stain and examined under polarizing microscope. We counted 50 thin and 50 thick collagen fibers in each section of benign and malignant SGT and were categorized as Greenish Yellow (GY) or Yellowish Orange (YO). Statistical analysis was performed by using oneway ANOVA and Paired t-test. Results: We found similar presentation of polarizing color arrangement for thin collagen fibers (80-85% GY and 15-20% YO) in both benign and malignant SGT and MEP. In malignant SGT, thick collagen fibers exhibited different polarizing color (80% GY) than benign SGT and MEP (12-14% GY). Polarizing color profile of collagen in malignant SGT displayed a different presentation as compared to benign SGT and control (MEP) and differences were statistically significant(p<0.05). Conclusion: This study concluded about the difference of stromal collagen fibers in benign and malignant SGT. Malignant SGT suggesting loosely arranged collagen fibers could be involved in further growth and invasion of disease along with other cellular and molecular events. Keywords: Collagen, Mucous Extravasation Phenomenon, Picro-Sirius Red (PSR) Stain, Polarized Microscopy, Salivary Gland Tumor

Bubaline Aortic Matrix: Histologic, Imaging, Fourier Transform Infrared Spectroscopic Characterization and Application into Cattle Abdominal Hernia Repair

Extracellular matrix (ECM) scaffolds have been shown to promote constructive remodeling response into a host. Minimal disruption of ECM ultrastructure during tissue processing is typically desirable. The purpose of the study was to prepare and characterize bubaline aortic matrix (BAM) for abdominal hernia repair in cattle. The bubaline aorta was decellularized with 1% sodium dodecyl sulfate (SDS) for 24 h followed by 0.25% trypsin for 2 h and again with 1% SDS for 24 h. Native and decellularized aortae were examined for residual cells, collagen structure and integrity by histologic examination and scanning electron microscopy (SEM). Masson’s trichrome and Weigert’s staining, DNA quantification and Fourier transform infrared (FTIR) spectroscopy were used for further characterization of BAM. BAM was used for repair of abdominal hernia in six cattle having mean weight of 132.50 ± 21.86 kg and hernial ring size of 40.55 ± 15.42 cm2. Clinical, hemato-biochemical and antioxidant parameters were evaluated to assess biocompatibility of xenogenic BAM. Histologically, the absence of cells and orderly arranged collagen fibers were observed in treated aorta. SEM confirmed the preservation of collagen structure and integrity. DNA content was significantly (P < 0.001) reduced in BAM (6.47 ± 1.26 ng/mg) as compared to the native aorta (488.11 ± 49.12 ng/mg). DNA extracts from BAM show marked removal of DNA material, with the absence of DNA band in agarose gel. FTIR spectrum of BAM showed all characteristic transmittance peaks of native aorta collagen indicating preserved collagen structure within decellularized aorta. Cattle with the BAM implant recovered uneventfully and remained sound. Hemato-biochemical and antioxidant findings were unremarkable. Bubaline aortic matrix shows excellent repair efficiency and biocompatibility for abdominal hernia repair in cattle without complications.

Direct and inverse identification of constitutive parameters from the structure of soft tissues. Part 2: dispersed arrangement of collagen fibers

This paper investigates on the relationship between the arrangement of collagen fibers within soft tissues and parameters of constitutive models. Starting from numerical experiments based on biaxial loading conditions, the study addresses both the direct (from structure to mechanics) and the inverse (from mechanics to structure) problems, solved introducing optimization problems for model calibration and regression analysis. A campaign of parametric analyses is conducted in order to consider fibers distributionswith different main orientation and angular dispersion. Different anisotropic constitutive models are employed, accounting for fibers dispersion either with a generalized structural approach or with an increasing number of strain energy terms. Benchmark data sets, toward which constitutive models are fitted, are built by employing a multiscale description of fiber nonlinearities and accounting for fibers dispersion with an angular integration method. Results show how the optimalvalues of constitutive parameters obtained from model calibration vary as a function of fibers arrangement and testing protocol. Moreover, the fitting capabilities of constitutive models are discussed. A novel strategy for model calibration is also proposed, in order to obtain a robust accuracy with respect to different loading conditions starting from a low number of mechanical tests. Furthermore, novel results useful for the inverse determination of the mean angle and the variance of fibers distribution are obtained. Therefore, the study contributes: to better design procedures for model calibration; to account for mechanical alterations due to remodeling mechanisms; and to gain structural information in a nondestructive way.

Development and characterization of Lyophilized Transparized Decellularized stroma as a replacement for living cornea in deep anterior lamellar keratoplasty.

Corneal disease is the second cause of blindness in developing countries, where the number of corneal grafts needed by far exceeds the number available. In industrialized countries, although corneas are generally available for keratoplasty, onto inflamed and vascularized host beds they are often rejected despite immune-suppression. A non-immunogenic, transparent, cytocompatible stroma is therefore required, which can be lyophilized for long-term conservation. Decellularization methods were tested on porcine corneal stromas before validation on human corneas. Decellularization and lyophilization led to opacification of the stroma, which could be reversed by soaking in 100% glycerol. Cell-depleted transparized stromas were then lyophilized (LTDC) to allow their long-term conservation and water content was measured. The ultrastructure of LTDC corneas was examined by transmission electron microscopy (TEM). Histocompatibility antigens were undetectable on LTDC stromas by antibody staining. Finally, cytocompatibility of LTDC stromas was demonstrated on an ex vivo model of anterior lamellar keratoplasty. Differential staining was used to monitor colonization of LTDC stromas by cells from the receiving cornea. Only SDS-based decellularization produced acellular porcine stromas. The lowest SDS concentration tested (0.1%) was validated on human corneas. Unlike lyophilized corneas, LTDC stromas without residual water, express no histocompatibility markers, although TEM revealed the presence of cellular debris in an ultrastructural arrangement of collagen fibers very close to that of native corneas. This structure is compatible with colonization by cells from the receiver cornea in an ex vivo lamellar graft model. Our procedure produced non-immunogenic, transparent stromas with conserved ultrastructure compatible with long-term conservation.

Fractal dimension and directional analysis of elastic and collagen fiber arrangement in unsectioned arterial tissues affected by atherosclerosis and aging.

Structural proteins like collagen and elastin are major constituents of the extracellular matrix (ECM). ECM degradation and remodeling in diseases significantly impact the microorganization of these structural proteins. Therefore, tracking the changes of collagen and elastin fiber morphological features within ECM impacted by disease progression could provide valuable insight into pathological processes such as tissue fibrosis and atherosclerosis. Benefiting from its intrinsic high-resolution imaging power and superior biochemical specificity, nonlinear optical microscopy (NLOM) is capable of providing information critical to the understanding of ECM remodeling. In this study, alterations of structural fibrillar proteins such as collagen and elastin in arteries excised from atherosclerotic rabbits were assessed by the combination of NLOM images and textural analysis methods such as fractal dimension (FD) and directional analysis (DA). FD and DA were tested for their performance in tracking the changes of extracellular elastin and fibrillar collagen remodeling resulting from atherosclerosis progression/aging. Although other methods of image analysis to study the organization of elastin and collagen structures have been reported, the simplified calculations of FD and DA presented in this work prove that they are viable strategies for extracting and analyzing fiber-related morphology from disease-impacted tissues. Furthermore, this study also demonstrates the potential utility of FD and DA in studying ECM remodeling caused by other pathological processes such as respiratory diseases, several skin conditions, or even cancer. NEW & NOTEWORTHY Textural analyses such as fractal dimension (FD) and directional analysis (DA) are straightforward and computationally viable strategies to extract fiber-related morphological data from optical images. Therefore, objective, quantitative, and automated characterization of protein fiber morphology in extracellular matrix can be realized by using these methods in combination with digital imaging techniques such as nonlinear optical microscopy (NLOM), a highly effective visualization tool for fibrillar collagen and elastic network. Combining FD and DA with NLOM is an innovative approach to track alterations of structural fibrillar proteins. The results illustrated in this study not only prove the effectiveness of FD and DA methods in extracellular protein characterization but also demonstrate their potential value in clinical and basic biomedical research where protein microstructure characterization is critical.

Pathomorphology of extraplacental membranes in their premature rupture and undifferentiated connectivetissue dysplasia in women

to investigate the morphological features of the extraplacental membranes (EPM) of the placentas obtained from women with undifferentiated connective tissue dystrophy (UCTD) and premature rupture of the fetal membranes (PROFM). EPMs of 65 placentas, including 55 from study group women and 10 from control women (having no manifestations of UCTD and PROFM), underwent morphological examination (visual examination, histological and immunohistochemical studies, and electron microscopy). There was subamniotic edema, disorientation and fragmentation of collagen structures in the compact layer, a structural change in the brush border of the amniotic epithelium, and a predominance of vacuolated cytotrophoblast forms in the structure of EPM of the placentas from of women with UCTD. Structural changes were accompanied by the altered function of EPM cells and identified by immunohistochemistry as higher MMP-9 expression by amniocytes and cytotrophoblast cells. PROFM was typified by the proliferation of amniotic epithelium; by the ordered arrangement of collagen fibers, and by large, longitudinally oriented fibroblasts. The structural changes in UCTD and PROFM were also accompanied by the altered function in the EPM cells as higher MMP-9 expression in amniocytes and cytotrophoblast cells. The latter was accompanied by the enhanced proteolysis of the extracellular matrix and can serve as one of the mechanisms, which triggers PROFM. Impaired histoarchitectonics and higher MMP-9 expression in the EPM cells should be considered as a manifestation of connective tissue disorganization and may be considered as one of the components in the pathogenesis of PROFM in the presence of UCTD.

Bubaline Diaphragm Matrix: Development and Clinical Assessment into Cattle Abdominal Hernia Repair

Abstract The purpose of the study was to develop a xenogenic bubaline diaphragm matrix (BDM) for abdominal hernia repair. A fresh diaphragm was decellularized using aqueous sodium dodecyl sulfate (SDS) solutions (0.5-4% w/v) over a period. Acellularity was confirmed histologically and characterized by Masson’s trichrome staining, scanning electron microscopy (SEM), DNA quantification, agarose gel electrophoresis, and Fourier-transform infrared spectroscopy. The BDM was used for clinical abdominal hernia repair in six cattle. Clinical, hemato-biochemical and antioxidant parameters were evaluated to assess biocompatibility of xenogenic BDM. Histologically, the diaphragm treated with 2% SDS for 48 h showed complete acellularity and orderly arranged collagen fibers. The SEM confirmed preservation of collagen structure and integrity. The DNA content was significantly (P < 0.05) reduced in BDM (33.12 ± 5.40 ng/mg) as compared to the native diaphragm (443.96 ± 162.60 ng/mg). DNA extracts from BDM show considerable removal of DNA material, with absence of DNA band in agarose gel. The FTIR spectrum of BDM has shown all characteristic transmittance peaks of bovine skin collagen indicating preserved collagen structure. Six cattle with BDM implant recovered uneventfully and remained sound at least upto 6 months. Hemato-biochemical and antioxidant findings were unremarkable. Bubaline diaphragm matrix shows excellent repair efficiency and biocompatibility for abdominal hernia repair in cattle without complications.