Removal Of Cellular Material Research Articles

Comparative Analysis of Commercially Available Extracellular Matrix Soft Tissue Bioscaffolds.

Decellularized extracellular matrix (dECM) products are widely established for soft tissue repair, reconstruction and reinforcement. These regenerative biomaterials mimic native tissue ECM with respect to structure and biology and are produced from a range of tissue sources and species. Optimal source tissue processing requires a balance between removal of cellular material and the preservation of structural and biological properties of tissue ECM. Despite the wide-spread clinical use of dECM products there is a lack of comparative information on these products Structurally, some dECM products showed a well-preserved collagen architecture with a broad porosity distribution, while others showed a significantly altered structure compared with native tissue. Decellularization varied across the products. Some materials surveyed (OFMm, PPN, PPC, OFMo, UBM, SISz, ADM, PADM and BADM) were essentially devoid of nuclear bodies (mean count of <5 cells per high powered field (HPF)), whereas others (SISu and SISb) demonstrated an abundance of nuclear bodies (>50 cells per HPF). Pathology assessment of the products demonstrated that OFMm, OFMo and PADM had the highest qualitative assessment score for collagen fiber orientation and arrangement, matrix porosity, decellularization efficiency, and residual vascular channels scoring 10.5±0.8, 12.8±1.0, and 9.7±0.7 out of a maximum total score of 16, respectively This analysis of commercially available dECM products in terms of their structure and cellularity includes 12 different commercial materials The findings highlight the variability of the products in terms of matrix structure and the efficacy of decellularization.

Re-endothelialization of non-detergent decellularized porcine vessels.

Tissue engineering utilizes an interdisciplinary approach to generate constructs for the treatment and repair of diseased organs. Generation of small vessels as vascular grafts or as envisioned central vessel for vascularized constructs is still a challenge. Here, the decellularization of porcine vessels by a non-detergent based protocol was developed and investigated. Perfusion-decellularization with sodium hydroxide solution resulted in removal of cellular material throughout the whole length of the vessel while preserving structural and mechanical integrity. A re-endothelialization of the retrieved matrix with human umbilical vein endothelial cells and cardiac endothelial cells was achieved through rotation-based seeding employing a custom-made bioreactor. A confluent monolayer was detected on the entire luminal surface. Thus, a non-detergent-based decellularization method allowing the re-endothelialization of the luminal surface was developed in this study, thereby paving the way for future implementation of the resulting construct as vascular graft or as central vessel for tissue engineered constructs in need of a perfusion system with readily available anastomosis sites.

Evaluation of decellularization of porcine pericardium

The porcine pericardium has been used for its great potential as a biological scaffold, produced from the extracellular matrix (ECM) and used mainly in surgeries reconstructive, tissue repair and surgical procedures for corneal reconstruction. The adequate preservation and biocompatibility of the pericardial ECM structure during the decellularization process is fundamental, the biggest challenge being the total removal of cellular material without damage to the structure. All agents used in decellularization change the composition and cause some damage to the ultrastructure. Sodium Dodecil Sulfate (SDS) is the most effective for removing cell residue from tissue compared to other detergents, which is also the most used for the decellularization process. This work aimed to test 3 different concentrations of SDS, in order to assess the concentration (0.1, 0.5 and 1%) that best preserves the structure of the ECM pericardial. In addition, we listed the type of daily wash to make the process more effective (only distilled water or PBS 1x), in order to assess the concentration capable of decellularizing the tissue and better preserving the pericardial ECM. The concentration of SDS at 1%, when compared to the lowest concentrations of 0.1 and 0.5%, was more effective in the decellularization process, however it did not obtain good results in the preservation of the ECM. Regarding daily washing, there was no difference in the frequency assessed in the experimental groups.

Preparation of whole-kidney acellular matrix using peristaltic pump perfusion and identification the composition of matrix

Objective To make a whole-kidney acellular matrix scaffold through the method of peristaltic pump perfusion and to identify whether the kidney acellular matrix reserves extracellular matrix proteins or not. Methods The male Sprague-Dawley (SD) rats, 200~250 g. Intravenous cathers were injected into the renal artery to establish the channels for perfusion, followed by the perfusion of decellularization solution by using peristaltic pumps. After the decellularization, the acellular matrix was observed under microscope after hematoxylin and eosin(HE) staining and fluorescence microscope after DAPI staining. Immunohistochemistry was performed to identify the composition of kidney acellualr matrix. Results The result of HE staining and DAPI staining demonstrate the whole removal of cellular material in kidney acelluar matrix. Immunohistochemistry confirmed the preservation of the natural expression type of extracellular matrix proteins including collagen types I and IV, fibronectin and laminin. Conclusions Peristaltic pump perfusion is a successful method to make the kidney acellular matrix, and this scaffold retains the critical proteins of natural extracellular matrix. Therefore the kidney acellular matrix is an ideal natural scaffold for renal tissue engineering. Key words: Kidney Failure; Peristalsis; Extracellular Matrix

Decellularization and Delipidation Protocols of Bovine Bone and Pericardium for Bone Grafting and Guided Bone Regeneration Procedures.

The combination of bone grafting materials with guided bone regeneration (GBR) membranes seems to provide promising results to restore bone defects in dental clinical practice. In the first part of this work, a novel protocol for decellularization and delipidation of bovine bone, based on multiple steps of thermal shock, washes with detergent and dehydration with alcohol, is described. This protocol is more effective in removal of cellular materials, and shows superior biocompatibility compared to other three methods tested in this study. Furthermore, histological and morphological analyses confirm the maintenance of an intact bone extracellular matrix (ECM). In vitro and in vivo experiments evidence osteoinductive and osteoconductive properties of the produced scaffold, respectively. In the second part of this study, two methods of bovine pericardium decellularization are compared. The osmotic shock-based protocol gives better results in terms of removal of cell components, biocompatibility, maintenance of native ECM structure, and host tissue reaction, in respect to the freeze/thaw method. Overall, the results of this study demonstrate the characterization of a novel protocol for the decellularization of bovine bone to be used as bone graft, and the acquisition of a method to produce a pericardium membrane suitable for GBR applications.

Corneal decellularisation: recycling tissue for transplantation

AbstractPurpose There is a clinical need for reliable, reproducible biomimetic corneas. Decellularised tissues are advantageous compared to synthetic/semi‐synthetic tissues in that the native matrix ultrastructure and intrinsic cues including growth factors, cytokines and glycosaminoglycans (GAGs) may be retained. However, there is currently no reliable, standardised human corneal decellularisation method. Here, we provide a systematic study of commonly used decellularisation methods and assess their appropriateness for corneal applications.Methods Eye‐bank tissue unsuitable for transplantation was used to test decellularisation methods: Dispase removal of the epi‐ and endothelium; Mechanical agitation; Hypertonic NaCl; Ionic detergent (SDS); Non‐ionic detergent (Triton‐X100); all followed by nuclease treatment. Removal of cellular material, preservation of transparency, retention of corneal architecture and GAGs was assessed via histological, immunofluorescence and quantitative analysis. Potential cytotoxicity in vitro of the treated tissue was also assessed.Results No decellularisation technique investigated successfully removed 100% of cellular components. The techniques which had the least residual DNA, SDS and Triton‐X100, were most structurally compromised with reduced GAG content. Dispase treated, NaCl and mechanically agitated corneas had better preservation of structure, transparency and GAGs, but had higher residual DNA.Conclusion The ability to reprocess and regenerate tissues deemed “unsuitable” for transplantation allows us to salvage valuable tissue. However, in order to progress, we may need to take a step back to establish a “decellularisation” criterion; which should balance effective removal of immune reactive material with maintenance of tissue functionality.

Antibodies against Pentraxins and Lupus Nephritis Activity

Impaired apoptosis and dysfunction of the immune cells are considered to be the most important pathogenic mechanisms of systemic lupus erythematosus. Pentraxins, which are natural opsonins, are directly involved in the removal of cellular material by binding to different antigens and initiating and enhancing phagocytosis of damaged cells. Therefore the deficiency of pentraxins is a crucial risk factor for the development and progression of systemic lupus erythematosus. Despite the presence of elevated levels of interleukin-6, which under physiological conditions increases the expression of acute phase protein genes, in systemic lupus a deficiency of C reactive protein and other pentraxins is observed. Several mechanisms responsible for pentraxin deficiency have been postulated, including the impairment of pentraxin synthesis due to mutations in genes, gene inhibition by interferon-α, and removal of pentraxins by autoantibodies. In this review, we summarize the significance of antibodies directed against pentraxins in assessing the activity and severity of systemic lupus erythematosus and lupus nephritis, as well as the usefulness of these antibodies as an additional marker of the response to treatment. The role of antibodies directed against monomeric C reactive protein in the pathogenesis of lupus nephritis is also discussed, as these antibodies are considered as a factor causing damage to the glomerular cells.

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver (1-5). Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment(6-8). However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

Pentraxins – their importance in pathogenesis of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease, whose main pathomechanism is attributed to the disturbed apoptotic process and dysfunction of the immune cells, leading to the accumulation of undegraded cellular matrix. This paper presents molecules such as complement components, pentraxins, and collectins, which are involved in the opsonization and removal of cellular material, and shows how deficiencies in these processes may contribute to SLE development and progression. Many reports indicate the specific role of the pentraxins (C-reactive protein, serum amyloid P, pentraxin 3), which, due to enhancing the phagocytosis of damaged cells and inducing the classical pathway of complement activation, participate in masking antigens from the immune system. The influence of CRP on inhibition of development and progression of kidney disease and decreasing the immune activity markers was demonstrated on the basis of research in experimental, mouse models of SLE. The decreased pentraxin response described in systemic lupus erythematosus patients, despite the presence of high levels of interleukin-6 and other markers of disease activity, is still unclear. Anti-mCRP antibodies bind CRP to form immune complexes, which are deposited in glomeruli and may initiate or exacerbate inflammation. In the literature, the correlation between raised levels of anti-CRP antibodies and clinical and immunological activity of lupus nephritis was proved. It shows their importance as a factor determining the severity of the disease and response to treatment. Novel studies suggest that the low CRP response in SLE is due to interferon-α inhibition of gene expression and CRP synthesis. This suggests that therapeutic targets in systemic lupus erythematosus should also be based on inhibiting the synthesis of interferon-α .

Critical evaluation of solid waste sample processing for DNA-based microbial community analysis

Landfills represent a unique microbial ecosystem and play a significant role in global biogeochemical processes. The study of complex ecosystems such as landfills using DNA-based techniques can be advantageous since they allow for analysis of uncultured organisms and offer higher resolution in measuring demographic and metabolic (functional) diversity. However, sample acquisition and processing from refuse is challenging due to material heterogeneity. Decomposed refuse was used to evaluate the effect of seven sample processing methods on Bacteria and Archaea community structure using T-RFLP. Bias was assessed using measured richness and by comparing community structure using multi-dimensional scaling (MDS). Generally, direct methods were found to be most biased while indirect methods (i.e., removal of cellular material from the refuse matrix before DNA extraction) were least biased. An indirect method using PO₄ buffer gave consistently high bacterial and archaeal richness and also resulted in 28 and 34% recovery of R. albus and M. formicicum spiked into refuse, respectively. However, the highest recovery of less abundant T-RFs was achieved using multiple processing methods. Results indicate differences in measured T-RF diversity from studies of landfill ecosystems could be caused by methodological (i.e., processing method) variation rather than refuse heterogeneity or true divergence in community structure.

828 CEREBROSPINAL FLUID (CSF) PROCOAGULANT IN ACUTE LYMPHOBLASTIC LEUKEMIA

CSF of children with leukemia treated with cranial radiation and intrathecal methotrexate (IT MTX) can shorten the recalcification time of normal plasma. Graeber postulated that this procoagulant activity of CSF reflects release of phospholipid from the demyelinating effects of central nervous system (CNS) prophylaxis. We have confirmed their findings and report further studies of CSF from 28 samples during CNS prophylaxis, 15 samples during treatment with IT MTX alone given for prophylaxis and 14 samples during IT MTX for active CNS leukemia. Procoagulant activity was equally demonstrable in the three patient populations. CSF from these patients shortened the recalcification time of normal plasma and plasmas deficient in factors VIII, IX, X or XII but not those deficient in II, V or X. Procoagulant activity was independent of chamber nucleated cell count but mononuclear cells could be demonstrated in all CSF samples by cytocentrifugation. Removal of cellular material from CSF by millipore filtration or a special modification of cytocentrifugation diminished the expression of procoagulant activity. Ultracentrifugation or millipore filtration of substrate plasma to more effectively remove platelets rendered the plasma insensitive to the CSF procoagulant. Similarly, complete glass or celite activation of platelet-containing plasma produced a plasma no further responsive to CSF. These observations suggest that the CSF procoagulant is a tissue factor (lipoprotein complex) associated with mononuclear cells in the CSF following IT MTX and reflect arachnoiditis rather than demyelination.

Glycoprotein, elastin, and collagen secretion by rat smooth muscle cells.

Smooth muscle cells from rat heart secreted extracellular matrix components at high rates for many generations in culture. The matrix proteins remained anchored to the culture dish and were characterized after removal of cellular material with sodium dodecyl sulfate. Sequential enzyme digestion demonstrated the presence of at least three components, including glycoprotein(s), elastin, and collagen. Prolonged extraction of the matrix with detergent under reducing conditions solubilized a fucosylated glycoprotein having an apparent molecular weight of 250,000 and two other proteins with molecular weights of 72,000 and 45,000, respectively. Sublines derived from discrete colonies of smooth muscle cells synthesized all of the matrix components, and the proportion of collagen secreted by some sublines increased with time in culture. The biosynthesis of a mixed extracellular matrix and the relationships among the component proteins were therefore studied in one system producing milligram quantities of material.

Microbial cell recovery enhancement through flocculation

AbstractThe removal of cellular material from fermentation broths is of importance in many fermentation processes. The present work compares the performance of recently developed polyelectrolyte flocculating additives with traditionally available additives. Objectives are to establish criteria for the choice of a flocculating additive and establish optimum conditions for the formation of stable, fast settling floe, and for quantitative separation of cellular material from the medium. Fermentation broths of actively growing Candida intermedia were used to evaluate the effectiveness of fifty commercial flocculating additives at different dosages and pH values. Certain strong anionic and strong cationic polyelectrolytes and mineral hydrocolloids were found to be most effective in their enhancement of settling rates. Some differences in behavior exist between glucose grown cells, hydrocarbon grown cells, and washed cells in buffer suspension. Flocculation of cells from fermentation broths is concluded to be highly dependent upon adsorbed material. A high charge density to interact or compete with adsorbed material and a solubility in the adsorbed material are important factors in choosing an additive for a given application. The fluid mechanics of a flocculating suspension is an important variable since low shear does not provide adequate contacting between cells for floe formation and high shear leads to floe breakup. An apparatus was constructed to grow floe under constant fluid mechanical conditions both in laminar and turbulent flow regimes. Turbulent shear was found to be very important in forming large, compact floe in cases where irreversible ionic bridging is the mechanism as for the strong anionic polyelectrolyte, polystyrene sulfonate. Adequate mixing is required to disperse the flocculating additive, but the level of turbulence is relatively unimportant in cases where reversible colloidal bridging is the mechanism as for the mineral hydrocolloid, bentonite.

Nature of Calcification in Codiacean Algae: ABSTRACT

The true relations between CaCO3 hard parts and living tissues in the algae are poorly understood. It has been known for many years that calcification in the Rhodophyta (red algae) occurs within the cell walls of the organism, and that calcification of the dasycladacean Chlorophyta (green algae) partly encloses the plant within an external calcareous encrustation. However, the nature of calcification in the very important codiacean algae (siphonaceous algae, Order Caulerpiales of modern algologists) has not been investigated previously. Because it is important to relate preserved calcareous structures to the cell morphology of the parent organism, the genus Halimeda was studied in detail to aid in the interpretation and classification of fossil codiacean materi l. Previous work by Lowenstam showed that calcareous skeletal material of Halimeda is composed of acicular crystals of aragonite, but no mention has been made of the relations between the aragonite and the living cell. Living Halimeda was collected along the Florida Keys and preserved in formaldehyde, impregnated with Vestapol plastic, sectioned with an ultramicrotome, and studied under an electron microscope. The aragonite was found to consist of acicular to blocky crystals ranging in length from 0.10 to 1.0 micron. The crystals occur as distinctly separate individuals suspended in a slurry outside of the cell walls which fuse to form a rigid calcareous structure on the death of the alga. No CaCO3 was seen within the cells or cell walls. Halimeda is an alga composed of a sing e coenocytic, tube-shaped cell which branches repeatedly until the tiny cell tips (utricles) form the outer surface where photosynthesis occurs. Calcification occurs outside the cell walls but inside the uticle layer, forming a calcareous mold of the living cell. Therefore, fossil codiacean algae like Halimeda would not contain preserved parts of the cells or cell walls, but instead would consist of calcareous molds of the cell material. Openings formed by the removal of cellular material usually are infilled with sparry calcite in fossil codiaceans, whereas the intercellular aragonite usually is composed of dense, fine-crystalline CaCO3. Fossil Codiacea may be classified safely on the basis of their external cell and utricle morphology. End_of_Article - Last_Page 518------------