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S J Warner

Publications and source records attributed to S J Warner.

At least 19 recordsLinked to original sources
PubMed2005

Analysis of the shelf life of a two-solution bone cement.

Two-solution bone cement consists of methyl methacrylate monomer and poly(methyl methacrylate) polymer dissolved together to yield a viscous solution. Two solutions are used such that the initiator, benzoyl peroxide (BPO), is placed in one solution and the activator, N,N, dimethyl-para-toluidine, is placed in the other. This approach to bone cement provides for a simplified use during surgery and eliminates some of the sources of porosity formation. However, the BPO-containing solution cement will spontaneously polymerize over time and will limit the useful shelf life of this component of the system. The activator-containing component is much more stable and is not as susceptible to spontaneous polymerization. In making two-solution cements, it is envisioned that antibiotics may be incorporated and that the polymer may be sterilized using gamma(gamma)-irradiation. Therefore, this study investigated the shelf life of the initiator-containing solution bone cement and studied the effects of initiator concentration, gamma-irradiation, gentamicin addition, and the role of storage temperature. Isothermal differential scanning calorimetry (Iso-DSC) techniques were used to monitor the polymerization of BPO-containing solutions. It was found that the shelf life was highly temperature dependent and followed an Arrhenius expression where refrigeration storage (4 degrees C) yielded approximately a 12-month storage time, while 70 degrees C storage results in setting in about 5-7 min. gamma-irradiation and gentamicin addition did not significantly affect the shelf life. Initiator concentration affected storage time with higher levels resulting in shorter shelf life.

Benzoyl Peroxide↗
PubMed2000

A family of human RNA-binding proteins related to the Drosophila Bruno translational regulator.

The post-transcriptional regulation of gene expression by RNA-binding proteins is an important element in controlling both normal cell functions and animal development. The diverse roles are demonstrated by the Elav family of RNA-binding proteins, where various members have been shown to regulate several processes involving mRNA. We have identified another family of RNA-binding proteins distantly related to the Elav family but closely related to Bruno, a translational regulator in Drosophila melanogaster. In humans, six Bruno-like genes have been identified, whereas other species such as Drosophila, Xenopus laevis, and Caenorhabditis elegans have at least two members of this family, and related genes have also been detected in plants and ascidians. The human BRUNOL2 and BRUNOL3 are 92% identical in the RNA-binding domains, although the BRUNOL2 gene is expressed ubiquitously whereas BRUNOL3 is expressed predominantly in the heart, muscle, and nervous system. Both of these proteins bind the same target RNA, the Bruno response element. The RNA-binding domain that recognizes the Bruno response element is composed of two consecutive RNA recognition motifs at the amino terminus of vertebrate Bruno protein. The possible involvement of the Bruno family of proteins in the CUG repeat expansion disease myotonic dystrophy is discussed.

Amino Acid Sequence↗
PubMed1989

Human vascular smooth muscle cells. Target for and source of tumor necrosis factor.

TNF-alpha (also known as cachectin) may produce many of its important effects in vivo by actions on blood vessels. Endothelial cells are well known to respond to TNF-alpha. We investigated whether vascular smooth muscle cells (SMC), the most abundant cell type in most vessels, also respond to TNF-alpha and the related cytokine lymphotoxin (TNF-beta). Both human rTNF-alpha and beta (0.1 to 100 ng/ml) induced transient accumulation of IL-1 mRNA by adult human vascular SMC that peaked between 1 and 4 h. The inhibitor of RNA synthesis actinomycin D (1 microgram/ml) blocked the induction of IL-1 mRNA, whereas inhibition of protein synthesis with cycloheximide (1 microgram/ml) resulted in a marked "superinduction" of both IL-1 alpha and IL-1 beta mRNA species. TNF-alpha treatment also increased intracellular biologically active IL-1 and subsequent release of IL-1 activity from SMC. Metabolic labeling and immunoprecipitation with specific antibodies demonstrated de novo synthesis of IL-1 alpha and IL-1 beta precursors in TNF-treated or lymphotoxin-treated SMC. TNF-alpha also activated other SMC functions including the concentration-dependent release of PGE2 from SMC, and time-dependent induction of the gene for (2'-5')-oligoadenylate synthetase, an enzyme thought to mediate the anti-viral and anti-proliferative actions of IFN. We also explored whether SMC, which both produce and respond to IL-1, might also express either of the TNF genes. Bacterial LPS (10 micrograms/ml) caused slight accumulation of TNF-alpha transcripts. Incubation of SMC for 4 h with inhibitors of protein synthesis alone caused little or no elevation of TNF-alpha mRNA, but simultaneous addition of LPS ("superinduction" conditions) induced large amounts of TNF-alpha (but not TNF-beta) mRNA. Cells treated with anisomycin (1 microgram/ml) and LPS, then washed to remove this reversible inhibitor of protein synthesis, released TNF-alpha into the medium, as assessed by the L929 cytotoxicity assay and by metabolic labeling and immunoprecipitation. Thus, SMC both respond to both TNF and lymphotoxin and can produce TNF-alpha, a cytokine with numerous effects on vascular cells of potential significance in the pathophysiology of septic shock and other inflammatory conditions.

2',5'-Oligoadenylate Synthetase↗
PubMed1989

Observations on human smooth muscle cell cultures from hyperplastic lesions of prosthetic bypass grafts: production of a platelet-derived growth factor-like mitogen and expression of a gene for a platelet-derived growth factor receptor--a preliminary study.

Prosthetic bypass grafts placed to the distal lower extremity often fail because of an occlusive tissue response in the perianastomotic region. The origin of the cells that comprise this occlusive lesion and the causes of the cellular proliferation are not known. To increase our understanding of this process we cultured cells from hyperplastic lesions obtained from patients at the time of reexploration for lower extremity graft failure, and we studied their identity and growth factor production in tissue culture. These cultures contain cells that express muscle-specific actin isoforms, shown by immunohistochemical staining, consistent with vascular smooth muscle origin. These cultures also released material that stimulated smooth muscle cell growth. A portion of this activity was similar to platelet-derived growth factor, since preincubation with antibody-to-human platelet-derived growth factor partially blocked the mitogenic effect of medium conditioned by human anastomotic hyperplastic cells. These conditioned media also contained material that competed with platelet-derived growth factor for its receptor, as measured in a radioreceptor assay. Northern blot analysis showed that these cells contain messenger RNA that encodes the A chain but not the B chain of platelet-derived growth factor. In addition, these cells contain messenger RNA that encodes a platelet-derived growth factor receptor. We conclude that cultured smooth muscle cells from human anastomotic hyperplastic lesions express genes for platelet-derived growth factor A chain and a platelet-derived growth factor receptor and secrete biologically active molecules similar to platelet-derived growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Immune interferon inhibits proliferation and induces 2'-5'-oligoadenylate synthetase gene expression in human vascular smooth muscle cells.

Proliferation of vascular smooth muscle cells (SMC) contributes to formation of the complicated human atherosclerotic plaque. These lesions also contain macrophages, known to secrete SMC mitogens, and T lymphocytes. Many of the SMC in the lesions express class II major histocompatibility antigens, an indication that activated T cells secrete immune IFN-gamma locally in the plaque. We therefore studied the effect of IFN-gamma on the proliferation of cultured SMC derived from adult human blood vessels. IFN-gamma (1,000 U/ml) reduced [3H]thymidine (TdR) incorporation into DNA by SMC stimulated with the well-defined mitogens IL 1 (from 15.3 +/- 0.7 to 6.2 +/- 0.7 dpm X 10(-3)/24 h) or platelet-derived growth factor (PDGF) (from 18.5 +/- 1.0 to 7.3 +/- 0.7 dpm X 10(-3)/24 h). Kinetic and nuclear labeling studies indicated that this effect of IFN-gamma was not due to altered thymidine transport or specific radioactivity of TdR in the cell. In longer term experiments (4-16 d) IFN-gamma prevented net DNA accumulation by SMC cultures stimulated by PDGF. IFN-gamma also delayed (from 30 to 60 min) the time to peak level of c-fos RNA in IL 1-treated SMC. It is unlikely that cytotoxicity caused these effects of IFN-gamma, as the inhibition of growth was reversible and we detected no cell death in SMC cultures exposed to this cytokine. Activation of 2'-5' oligoadenylate synthetase gene expression may mediate certain antiproliferative and antiviral effects of interferons. Both IFN-gamma and type I IFNs (IFN-alpha or IFN-beta) induced 2'-5' oligoadenylate synthetase mRNA and enzyme activity in SMC cultures, but with concentration dependence and time course that may not account for all of IFN-gamma's cytostatic effect on SMC. The accumulation of SMC in human atherosclerotic lesions is a long-term process that must involve altered balance between growth stimulatory and inhibitory factors. The cytostatic effect of IFN-gamma on human SMC demonstrated here may influence this balance during human atherogenesis, because T cells present in the complicated atherosclerotic plaque likely produce this cytokine.

2',5'-Oligoadenylate Synthetase↗
PubMed1988

Production of platelet-derived growth factor-like mitogen by smooth-muscle cells from human atheroma.

Proliferation of vascular smooth-muscle cells occurs during the development of atherosclerosis and the remodeling of arteries that accompanies chronic systemic or pulmonary hypertension. To help define the signals that initiate this abnormal growth, we cultured smooth-muscle cells from human atherosclerotic plaques. These cells (n = 9) released material into their culture medium that stimulated the proliferation of aortic smooth-muscle cells to a mean (+/- SD) level 5.1 +/- 1 times that in control medium. Part of this activity was due to molecules that resemble a mitogen first isolated from platelets and known as platelet-derived growth factor (PDGF), since these cells released PDGF measured in a radioreceptor assay (355 +/- 117 pg per milliliter per 48 hours; n = 6) and since anti-PDGF antibody neutralized 38 +/- 7 percent of this mitogenic activity (range, 13 to 60 percent; n = 6 carotid-plaque isolates). Two human genes encode distinct PDGF subunits that form dimers in different combinations to create biologically active PDGF. Cells cultured from human atheroma contained mRNAs for the PDGF A chain (16 of 17 isolates) but none (of 13) that encoded PDGF B chain (the c-sis proto-oncogene product). We conclude that smooth-muscle cells from diseased human arteries can secrete mitogenic activity, some of which resembles PDGF, and that these cells express the gene for the PDGF A chain selectively. This capacity to produce an endogenous, potentially self-stimulatory (autocrine) growth factor may help to explain how replication of smooth-muscle cells can begin, even while the endothelial barrier remains morphologically intact, early in atherogenesis.

Aged↗
PubMed1988

Interleukin 1: a mitogen for human vascular smooth muscle cells that induces the release of growth-inhibitory prostanoids.

There is much interest in defining the signals that initiate abnormal proliferation of cells in a variety of states characterized by the presence of mononuclear phagocytes. Since IL-1 is a major secretory product of activated human monocytes we examined whether this cytokine can stimulate the growth of human vascular smooth muscle cells (SMC). Neither recombinant IL-1 (rIL-1) alpha (less than or equal to 5.0 ng/ml) nor beta (less than or equal to 100 ng/ml) stimulated SMC growth during 2-d incubations under usual conditions. IL-1 did stimulate SMC to produce prostanoids such as PGE1 or PGE2 that can inhibit SMC proliferation. When prostaglandin synthesis was inhibited by indomethacin or aspirin both rIL-1 alpha and beta (greater than or equal to 1 ng/ml) markedly increased SMC growth. In longer-term experiments (7-28 d) rIL-1 stimulated the growth of SMC even in the absence of cyclooxygenase inhibitors. The addition of exogenous PGE1 or PGE2 (but not PGF1 alpha, PGF2 alpha, PGI2) to indomethacin-treated SMC blocked their mitogenic response to rIL-1. Antibody to IL-1 (but not to platelet-derived growth factor [PDGF]) abolished the mitogenic response of SMC to rIL-1. Exposure of SMC to rIL-1 or PDGF caused rapid (maximal at 1 h) and transient (baseline by 3 h) expression of the c-fos proto-oncogene, determined by Northern analysis. We conclude that IL-1 is a potent mitogen for human SMC. Endogenous prostanoid production simultaneously induced by IL-1 appears to antagonize this growth-promoting effect in the short term (2 d) but not during more prolonged exposures. IL-1 produced by activated monocytes at sites of tissue inflammation or injury may thus mediate both positive and negative effects on SMC proliferation that are temporally distinct.

6-Ketoprostaglandin F1 alpha↗
PubMed1987

Interleukin 1 induces interleukin 1. I. Induction of circulating interleukin 1 in rabbits in vivo and in human mononuclear cells in vitro.

Interleukin 1 (IL-1) plays an important role in host defense mechanisms by increasing body temperature, inducing the synthesis of a variety of lymphokines and hepatic acute phase proteins and acting as a chemoattractant for lymphocytes. However, in some microenvironments such as injured tissue or joint spaces, elevated IL-1 levels may contribute to pathologic processes, for example, proliferation and fibrosis of tissue involved in pannus formation as well as degradation of matrix and abnormal tissue architecture. To investigate potential mechanisms that may lead to excessive production of IL-1, we have examined the ability of IL-1 to participate in an amplification event by inducing its own gene expression leading to synthesis of biologically active IL-1. When injected into rabbits, recombinant human IL-1-alpha induced biphasic fevers, and during the second temperature elevation 3 hr later, a circulating pyrogenic material was detected by passive transfer of plasma to other rabbits. Induction of the biphasic fever was not caused by endotoxin contamination of the recombinant IL-1. The 3-hr circulating pyrogen was heat-labile and was not residual injected IL-1-alpha. Chromatographic separation of this plasma and biologic assay suggested that it was new IL-1 of rabbit origin. We next incubated human blood mononuclear cells with recombinant IL-1-alpha and measured the intracellular and extracellular levels of IL-1 by bioassay using the D10.G4.1 murine T cell line. In order to control for the carryover of recombinant IL-1-alpha used to stimulate the mononuclear cells (MNC), we used neutralizing antibodies that were specific for IL-1-alpha or IL-1-beta. The results of these neutralizations showed that recombinant human IL-1-alpha induces the synthesis of IL-1-beta in human MNC in vitro. These results were verified with a radioimmunoassay specific for IL-1-beta. At concentrations of 100 ng/ml, IL-1-alpha induced prostaglandin E2 production in the MNC culture, and this was associated with decreased production of immunoreactive IL-1-beta. Adding indomethacin to the cultures prevented the decreased production of IL-1-beta induced by high concentrations of IL-1-alpha. Using nonadherent MNC, we observed an increase in IL-1-beta as well as IL-1-alpha mRNA after 4 hr of exposure to recombinant IL-1-alpha. These results demonstrate that IL-1-alpha induces biologically active and immunoreactive IL-1-beta from MNC in vitro and that the same concentrations of IL-1-alpha induce gene expression for both forms of IL-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Interleukin 1 induces interleukin 1. II. Recombinant human interleukin 1 induces interleukin 1 production by adult human vascular endothelial cells.

Interleukin 1 (IL-1) alters several potentially pathogenic endothelial cell (EC) functions. The authors report here that recombinant human IL-1 (rIL-1) alpha (0.1 to 10 ng/ml) or IL-1-beta (1 to 100 ng/ml) induce concentration- and time-dependent increases in IL-1-beta mRNA levels in EC derived from adult human saphenous vein. rIL-1 induced IL-1-alpha mRNA only in EC treated concomitantly with cycloheximide (2 micrograms/ml). IL-1-beta mRNA production began within 1 hr of exposure to rIL-1, peaked after 24 hr, and declined thereafter. Actinomycin D prevented the appearance of IL-1 mRNA in rIL-1-treated EC. rIL-1 also induced the release of biologically active IL-1 from EC, which was inhibited by cycloheximide (1 microgram/ml). When compared on the basis of their activity in the thymocyte costimulation assay, rIL-1-alpha and rIL-1-beta were equipotent as inducers of IL-1 production by EC. EC stimulated with rIL-1 produced prostaglandin E2, which inhibits IL-1 production by other cell types and also decreases the responsiveness of thymocytes to IL-1. When EC were exposed to rIL-1 in the presence of indomethacin (1 microgram/ml), which blocked prostaglandin E2 production, greater amounts of rIL-1-induced IL-1 release were detected, although the inhibitor did not affect IL-1-beta mRNA levels. IL-1-induced IL-1 production was unlikely to be caused by endotoxin contamination of tissue culture media or IL-1 preparations, because the lipopolysaccharide (LPS) antagonist polymyxin B (10 micrograms/ml) blocked LPS-induced IL-1 production by EC but did not affect IL-1 release in response to rIL-1-beta (100 ng/ml). The IL-1-inducing property of rIL-1-beta was heat-labile, whereas heated LPS stimulated EC IL-1 production. The source of IL-1 in our cultures was not monocyte/macrophages, as treatment of EC with monoclonal antibody to the monocyte antigen Mo2 under conditions that lysed adherent peripheral blood monocytes did not affect production of IL-1 by EC in response to LPS (1 microgram/ml) or rIL-1-beta (100 ng/ml). IL-1 elicits a coordinated program of altered endothelial function that increases adhesiveness for leukocytes and coagulability. IL-1-induced IL-1 gene expression in human adult EC could thus provide a positive feedback mechanism in the pathogenesis of vascular disease including atherosclerosis, vasculitis, and allograft rejection.

Cells, Cultured↗
PubMed1987

Human interleukin 1 induces interleukin 1 gene expression in human vascular smooth muscle cells.

The recognition that cells of the vascular wall can secrete cytokines such as IL-1 suggests new mechanisms for initiating or sustaining inflammatory responses in blood vessels. We report that purified human monocyte-derived IL-1 or recombinant human IL-1 (rIL-1 beta and rIL-1 alpha) induce cultured human smooth muscle cells derived from veins or arteries to synthesize IL-1 beta mRNA and produce and release biologically active IL-1. rIL-1 beta also stimulated the production of PGE2 by smooth muscle cells. Exposure to rIL-1 beta (1-100 ng/ml), or rIL-1 alpha (0.01-10 ng/ml) increased IL-1 beta mRNA levels within 30 min. Actinomycin D (1 microgram/ml) prevented the induction of IL-1 beta mRNA by rIL-1. IL-1 alpha mRNA was detected in SMC treated with cycloheximide (1 microgram/ml) and rIL-1 beta, or cycloheximide alone. rIL-1 alpha and rIL-1 beta produced maximal levels of IL-1 beta mRNA after 4 h, and intracellular IL-1 biological activity after 6 h of exposure. Release of IL-1 activity in the extracellular medium began after 1 h of incubation with rIL-1 beta or rIL-1 alpha, and continued for up to 24 h. Anti-TNF antiserum that neutralized the biological activity of rTNF did not affect rIL-1-induced production of IL-1 beta mRNA or IL-1 release, suggesting that the release of TNF does not mediate these processes. Several experimental approaches indicated that the release of IL-1 by smooth muscle cells was not due to endotoxin contamination of the IL-1 preparations. Anti-IL-1 antiserum blocked the induction of smooth muscle cell IL-1 gene expression by rIL-1 beta. Polymyxin B did not prevent IL-1-induced IL-1 expression by these cells, but blocked the effect of endotoxin. Heat treatment destroyed the stimulatory capacity of rIL-1 beta, but did not affect the ability of bacterial endotoxin to induce IL-1 expression. The production of IL-1 by human vascular smooth muscle cells was not due to contamination of the cell cultures with blood monocytes, inasmuch as treatment with an antimonocyte antibody (anti-Mo2) and complement did not alter IL-1 beta mRNA content or the amount of IL-1 released from the cells in response to endotoxin, rIL-1 alpha, or rIL-1 beta.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene Expression Regulation↗
PubMed1987

Studies on the molecular nature of human interleukin 1.

Adherent human blood monocytes were stimulated with heat-killed Staphylococcus albus or Escherichia coli lipopolysaccharide in the presence of 35S-methionine-, [3H]leucine-, or 14C-labeled amino acids. After incubation, interleukin 1 (IL 1) activity in the supernatant medium was purified over an anti-human IL 1 immunoadsorbent followed by gel filtration and chromatofocusing. The purity of the IL 1 was assessed by fluorography of one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Isoelectric and chromatofocusing of low m.w. proteins (less than 20,000 m.w.) revealed three charged 18,000 m.w. species of IL 1 with approximate pI's of 7, 6, and 5, with the most abundant form at pI 7. During the purification procedures, lymphocyte co-mitogenic activity, fever in rabbits, and prostaglandin E2 release from dermal fibroblasts co-eluted in the same fractions. In addition, these fractions were active when injected into endotoxin-resistant C3H/HeJ mice for the production of fever, the induction of serum amyloid A protein, a decrease in serum iron concentration, and an increase in the number of circulating neutrophils. Fluorography revealed homogeneous bands with an m.w. of about 18,000 which correlated with these biological activities. The specific activity of the pI 6 or 5 IL 1, as judged by the ratio of T cell co-mitogenic activity to incorporated radiolabeled amino acid, was at least 10-fold greater than that observed for the pI 7 form. This result suggests that the amino acid compositions of the two 18,000 m.w. acidic forms are unrelated to the pI 7 species. These results also demonstrate that the pI 7 human monocyte IL 1 is the predominant 18,000 m.w. form synthesized and, furthermore, that homogeneous pI 7 IL 1 exhibits multiple biological properties on various tissues by modulating immunologic, inflammatory, metabolic, and neurologic functions. Data are also presented for the existence of a high m.w. (32,000) human pro-IL 1 molecule as the predominant monocytic intracellular form. This pro-IL 1 is degraded artifactually during isolation to lower m.w. forms in the presence of an extracellular serine protease activity. These data are consistent with a model for IL 1 secretion in which pro-IL 1 is first synthesized within the cell and is processed during or after extracellular transport.

Biological Assay↗
PubMed1985

Characteristics of lipopolysaccharide interaction with human peripheral-blood monocytes.

The interaction between radioiodinated lipopolysaccharide from Escherichia coli 0111:B4 (125I-LPS) and human peripheral-blood monocytes was studied. The association of 125I-LPS with monocytes at 37 degrees C appeared to depend on binding to the cell membrane with subsequent internalization of the molecule, and was not saturable with time (up to 2 h) or 125I-LPS concentration (up to 10 micrograms/ml). There was no apparent difference in the behaviour of unlabelled LPS and 125I-LPS with respect to monocyte association. 125I-LPS association with monocytes was inhibited by LPS and O-polysaccharide from E. coli 0111:B4 and Salmonella typhi 0901, but not by lipid A or polymyxin B. We propose that the mechanism of human monocyte stimulation by LPS involves polysaccharide-dependent binding to the cell membrane followed by internalization of the LPS molecule. We were unable to demonstrate a specific LPS receptor such as that found on murine B-lymphocytes.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Dose-dependent reduction of lipopolysaccharide pyrogenicity by polymyxin B.

Lipopolysaccharides (LPS) from Gram-negative bacteria are potent pyrogens in mammals. Polymyxin B (PB), a cationic polypeptide antibiotic, binds lipid A, the active moiety of LPS, with high affinity and abrogates several biological responses to LPS. We studied the effect of PB on pyrogenicity of purified LPS from E. coli 0111:B4 in rabbits. PB reduced the pyrogenic response to LPS in a dose-dependent manner at mass ratios (PB:LPS) from 5:1 to 100:1. Previous reports have suggested that PB is effective only at much higher doses. In our hands, PB itself is pyrogenic, unless previously gamma-irradiated. Our results confirm in vivo the anti-endotoxic action of PB.

Animals↗
PubMed1980

Characterization of collagenous meshworks by volume exclusion of dextrans.

The volumes from which 3H-labelled dextrans are excluded by dermal collagenous fibres were calculated by dilution of dextran probes. Five dextrans, of average Stokes' radii 1.72, 2.53, 3.92, 4.54 and 14.24nm, were investigated at concentrations between 0.1 and 3% (w/w). The excluded volume was dependent on dextran concentration only for the two smaller probes. The largest dextran was shown not to bind to the fibres. A plot of the square root of excluded volume against Stokes' radius was linear for the four smallest dextrans, corresponding to the predictions of Ogston's [(1958) Trans. Faraday Soc. 54, 1754--1757] rod-and-sphere model of fibrous exclusion, and suggesting that dextrans of Stokes' radius between 1.72 and 4.54 nm were excluded by a cylindrical solid fibre of radius 2.90 +/- 0.72 nm. Larger molecules were excluded by a structure of much greater size, since the volume exclusion for the largest dextran was only slightly greater than that of the dextran less than one-third its radius. The excluded volume of 3H2O fell slightly below the line describing the dextran data, indicating that water had access to most of the volume not occupied by the collagenous fibres.

Chemical Phenomena↗