Reducible Cross-links Research Articles

Formulation and characterization of insulin nanoclusters for a controlled release

The growing interest in biopharmaceuticals combined with the challenges regarding formulation and delivery continues to encourage the development of new and improved formulations of this class of therapeutics. Nanoclusters (NCs) represent a type of formulation strategy where the biopharmaceutical is clustered in a reversible manner to function as both the therapeutic and the vehicle. In this study, insulin NCs (INCs) were formulated by a new methodology of first crosslinking proteins followed by desolvation. Crosslinking of the protein with the reducible DTSSP crosslinker improved control of the INC synthesis process to give INCs with a mean size of 198 ± 7 nm and a mean zeta potential of −39 ± 1 mV. Crosslinking and clustering of insulin did not induce cytotoxicity or major differences in the biological activity compared to the free unmodified protein. The potency of the crosslinked insulin and the INCs appeared slightly lower than that of the unmodified protein, and significantly higher doses of the INCs compared to the free protein were applied to achieve similar blood sugar lowering effects in vivo. Interestingly, the INCs allowed for high doses to be subcutaneously delivered with prolonged efficacy without being lethal in rats.

Characteristics of Collagen Changes in Small Intestine Anastomoses Induced by High-Frequency Electric Field Welding.

High-frequency electric field welding-induced tissue fusion has been explored as an advanced surgical method for intestinal anastomoses; however, intrinsic mechanisms remain unclear. The aim of this study was to investigate microcosmic changes of collagen within the fusion area, with various parameters. Ex vivo small intestine was fused with mucosa-mucosa. Four levels of compressive pressure (100 kPa, 150 kPa, 200 kPa, 250 kPa) were applied for 10 s in order to fuse the colons under a power level of 140 W. Then, collagen fibers of the fusion area were examined by fibrillar collagen alignment and TEM. Three levels of power (90 W, 110 W, 140 W) and three levels of time (5 s, 10 s, 20 s) were applied in order to fuse colons at 250 kPa, and then collagen within the fusion area was examined by Raman spectroscopy. Fibrillar collagen alignment analysis showed that with the increase in compression pressure, alignment of the collagen in the fusion area gradually increased, and the arrangement of collagen fibers tended to be consistent, which was conducive to the adhesion of collagen fibers. TEM showed that pressure changed the distribution and morphology of collagen fibers. Raman spectroscopy showed that increased power and time within a certain range contributed to collagen cross linking. Peak positions of amide I band and amide III band changed. These results suggested that higher power and a longer amount of time resulted in a decrease in non-reducible cross links and an increase in reducible cross links. Compression pressure, power, and time can affect the state of collagen, but the mechanisms are different. Compressive pressure affected the state of collagen by changing its orientation; power and time denatured collagen by increasing temperature and improved the reducible cross linking of collagen to promote tissue fusion.

Raman Spectroscopy as Noninvasive Method of Diagnosis of Pediatric Onset Inflammatory Bowel Disease

We propose here a spectroscopic method to diagnose and differentiate inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn’s disease (CD) with pediatric onset, in a complete noninvasive way without performing any duodenal biopsy. In particular, the Raman technique was applied to proteic extract from fecal samples in order to achieve information about molecular vibrations that can potentially furnish spectral signatures of cellular modifications occurring as a consequence of specific pathologic conditions. The attention was focused on the investigation of the amide I region, quantitatively accounting the spectral changes in the secondary structures by applying deconvolution and curve-fitting. Inflammation is found to give rise to a significant increasing of the nonreducible (trivalent)/reducible (divalent) cross-linking ratio R of the protein network. This parameter revealed an excellent marker in order to distinguish IBD subjects from non-IBD ones, and, among IBD patients, to differentiate between UC and CD. The proposed methodology was validated by statistical analysis using the receiver operating characteristic (ROC) curve.

Synthesis of micellar-like terpolymer nanoparticles with reductively-cleavable cross-links and evaluation of efficacy in 2D and 3D models of triple negative breast cancer

Triple negative or basal-like breast cancer (TNBC) is characterised by aggressive progression, lack of standard therapies and poorer overall survival rates for patients. The bad prognosis, high rate of relapse and resistance against anticancer drugs have been associated with a highly abnormal loss of redox control in TNBC cells. Here, we developed docetaxel (DTX)-loaded micellar-like nanoparticles (MLNPs), designed to address the aberrant TNBC biology through the placement of redox responsive cross-links designed into a terpolymer. The MLNPs were derived from poly(ethyleneglycol)-b-poly(lactide)-co-poly(N3-α-ε-caprolactone) with a disulfide linker pendant from the caprolactone regions in order to cross-link adjacent chains. The terpolymer contained both polylactide and polycaprolactone to provide a balance of accessibility to reductive agents necessary to ensure stability in transit, but rapid micellar breakdown and concomitant drug release, when in breast cancer cells with increased levels of reducing agents. The empty MLNPs did not show any cytotoxicity in vitro in 2D monolayers of MDA-MB-231 (triple negative breast cancer), MCF7 (breast cancer) and MCF10A (normal breast epithelial cell line), whereas DTX-loaded reducible crosslinked MLNPs exhibited higher cytotoxicity against TNBC and breast cancer cells which present high intracellular levels of glutathione. Crosslinked and non-crosslinked MLNPs showed high and concentration-dependent cellular uptake in monolayers and tumour spheroids, including when assessed in co-cultures of TNBC cells and cancer-associated fibroblasts. DTX loaded crosslinked MLNPs showed the highest efficacy against 3D spheroids of TNBC, in addition the MLNPs also induced higher levels of apoptosis, as assessed by annexin V/PI assays and increased caspase 3/7 activity in MDA-MB-231 cells in comparison to cells treated with DTX-loaded un-crosslinked MLNP (used as a control) and free DTX. Taken together these data demonstrate that the terpolymer micellar-like nanoparticles with reducible crosslinks have high efficacy in both 2D and 3D in vitro cancer models by targeting the aberrant biology, i.e. loss of redox control of this type of tumour, thus may be promising and effective carrier systems for future clinical applications in TNBC.

Light exposure accelerates oxidative protein polymerization in beef stored in high oxygen atmosphere

Protein oxidation of beef patties stored in high oxygen modified atmosphere packaging for 9 days was investigated. Meat was either stored in the dark, under light, or in the dark with addition of FeCl2/H2O2/myoglobin (forced oxidation). SDS–PAGE analysis showed high degree of protein polymerization for meat exposed to light, compared to the other samples. Light exposure induced reducible (disulfide) and non-reducible cross-links, while mainly disulfides were formed in meat stored in the dark. Light exposure was responsible for 58% loss of free thiols (Cys residues). No significant loss of other amino acid residues was observed and none of the most common oxidation products of tryptophan, tyrosine, and phenylalanine were detected. Intrinsic fluorescence measurements of tryptophan showed 27% loss in samples exposed to light, which was ascribed to loss of protein solubility via protein polymerization rather than tryptophan oxidation. Protein carbonyls were mainly detected in forced oxidized samples at Day 0.

Characterisation and quantification of protein oxidative modifications and amino acid racemisation in powdered infant milk formula

Modification of proteins in infant milk formula (IF) is of major concern to the dairy industry and consumers. Thermal treatment is required for microbiological safety, but heat, light, metal-ions and other factors may induce oxidative damage, and be a health risk. In this study protein modifications in IFs were quantified. IFs contained both reducible (disulphide) and non-reducible (di-tyrosine, lanthionine, lysinoalanine) protein cross-links. Dehydroalanine and the cross-linked species lanthionine and lysinoalanine were detected. Protein carbonyls were detected predominantly on high molecular mass materials. Oxidation products of phenylalanine (m-tyrosine), tryptophan (N-formylkynurenine, kynurenine, 3-hydroxykynurenine), tyrosine (di-tyrosine) and methionine (methionine sulphoxide) were detected, consistent with amino acid modification. Higher levels of most of the markers of protein modification were present in the hydrolysed protein brand, when compared to the conventional IF samples, indicative of increased damage during additional processing. Significant levels of racemised (D-) amino acids were present. These data indicate that amino acids in proteins in IFs are modified to a significant extent during manufacture, with hydrolysed IF being particularly prone.

P-8 - Characterization and quantification of protein oxidative modifications and amino acid racemization in powdered infant milk formula

Oxidation of dairy products, and especially infant formulas (IFs), is a major concern to the dairy industry and consumers. Thermal treatment is required for microbiological safety but may contribute to oxidative damage. Such modification may be a health risk for infants with IF as the sole source of nutrition. This study quantifies the extent of modification in commercial IFs. Protein modification in commercial IFs was examined by multiple analytical techniques. Aggregated proteins containing both reducible (disulfide) and non-reducible (di-tyrosine, lanthionine and lysinoalanine) cross-links were detected. Protein carbonyls (

Effect of Oxidation and Protein Unfolding on Cross-Linking of β-Lactoglobulin and α-Lactalbumin.

Oxidation and heat treatment can initiate changes in the amino acid composition, structure, solubility, hydrophobicity, conformation, function, and susceptibility to proteolysis of proteins. These can result in adverse consequences for mammals, plants, foodstuffs, and pharmaceuticals. This study investigated whether and how individual or combined treatment with heat, a commonly encountered factor in industrial processing, and H2O2 alters the structure and composition of two major milk whey proteins, α-lactalbumin and β-lactoglobulin, and mixtures of these. Thermal treatment induced reducible cross-links in isolated β-lactoglobulin, but not isolated α-lactalbumin under the conditions employed. Cross-linking occurred at lower temperatures and to a greater extent in the presence of low concentrations of H2O2. H2O2 did not induce cross-linking in the absence of heat. Mixtures of α-lactalbumin and β-lactoglobulin showed similar behavior, except that mixed α-lactalbumin-β-lactoglobulin dimers were detected. Cross-linking was associated with formation of sulfenic acids (RS-OH species), oxidation of methionine residues, cleavage of disulfide bonds in α-lactalbumin, altered conformation of disulfide bonds in β-lactoglobulin, alterations in the fluorescence intensity and maximum emission wavelength of endogenous tryptophan residues, and binding of the hydrophobic probe 8-anilinonaphthalenesulfonate. These data are consistent with increased unfolding and subsequent aggregation of the protein, with these changes being maximized in the presence of both heat and H2O2. The enhanced aggregation detected with H2O2 is consistent with additional pathways to aggregation above that induced by heat alone. These mechanistic insights provide potential strategies for modulating the extent and nature of protein modification induced by thermal and oxidant treatment.

Disulfide Bond Pattern of Transforming Growth Factor β-Induced Protein.

Transforming growth factor β-induced protein (TGFBIp) is an extracellular matrix protein composed of an NH2-terminal cysteine-rich domain (CRD) annotated as an emilin (EMI) domain and four fasciclin-1 (FAS1-1-FAS1-4) domains. Mutations in the gene cause corneal dystrophies, a group of debilitating protein misfolding diseases that lead to severe visual impairment. Previous studies have shown that TGFBIp in the cornea is cross-linked to type XII collagen through a reducible bond. TGFBIp contains 11 cysteine residues and is thus able to form five intramolecule disulfide bonds, leaving a single cysteine residue available for the collagen cross-link. The structures of TGFBIp and its homologues are unknown. We here present the disulfide bridge pattern of TGFBIp, which was determined by generating specific peptides. These were separated by ion exchange followed by reversed-phase high-performance liquid chromatography and analyzed by mass spectrometry and Edman degradation. The NH2-terminal CRD contains six cysteine residues, and one of these (Cys65) was identified as the candidate for the reducible cross-link between TGFBIp and type XII collagen. In addition, the CRD contained two intradomain disulfide bridges (Cys49-Cys85 and Cys84-Cys97) and one interdomain disulfide bridge to FAS1-2 (Cys74-Cys339). Significantly, this arrangement violates the predicted disulfide bridge pattern of an EMI domain. The cysteine residues in FAS1-3 (Cys473 and Cys478) were shown to form an intradomain disulfide bridge. Finally, an interdomain disulfide bridge between FAS1-1 and FAS1-2 (Cys214-Cys317) was identified. The interdomain disulfide bonds indicate that the NH2 terminus of TGFBIp (CRD, FAS1-1, and FAS1-2) adopts a compact globular fold, leaving FAS1-3 and FAS1-4 exposed.

Key role of cysteine residues and sulfenic acids in thermal- and H2O2-mediated modification of β-lactoglobulin

Oxidation results in protein deterioration in mammals, plants, foodstuffs and pharmaceuticals, via changes in amino acid composition, fragmentation, aggregation, solubility, hydrophobicity, conformation, function and susceptibility to digestion. This study investigated whether and how individual or combined treatment with heat, a commonly encountered factor in industrial processing, and H2O2 alters the structure and composition of the major whey protein β-lactoglobulin. Thermal treatment induced reducible cross-links, with this being enhanced by low H2O2 concentrations, but decreased by high concentrations, where fragmentation was detected. Cross-linking was prevented when the single free Cys121 residue was blocked with iodoacetamide. Low concentrations of H2O2 added before heating depleted thiols, with H2O2 alone, or H2O2 added after heating, having lesser effects. A similar pattern was detected for methionine loss and methionine sulfoxide formation. Tryptophan loss was only detected with high levels of H2O2, and no other amino acid was affected, indicating that sulfur-centered amino acids are critical targets. No protection against aggregation was provided by high concentrations of the radical scavenger 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), consistent with molecular oxidation, rather than radical reactions, being the major process. Sulfenic acid formation was detected by Western blotting and LC–MS/MS peptide mass-mapping of dimedone-treated protein, consistent with these species being significant intermediates in heat-induced cross-linking, especially in the presence of H2O2. Studies using circular dichroism and intrinsic fluorescence indicate that H2O2 increases unfolding during heating. These mechanistic insights provide potential strategies for modulating the extent of modification of proteins exposed to thermal and oxidant treatment.

PP3 - Role of oxidation and thermal unfolding in structural changes to beta-lactoglobulin

Oxidation is a major cause of protein deterioration in mammals, plants, foodstuffs and pharmaceuticals, with this giving rise to changes in amino acid composition, fragmentation, aggregation, solubility, hydrophobicity, conformation, susceptibility to digestion and function. Whilst it is well established that these processes have negative impacts on product quality and human health, the mechanisms that generate these alterations are incompletely understood. In this project we are examining whether and how unfolding and oxidation induce changes in the structure of beta-lactoglobulin, to determine whether unfolding alters the rate and mechanism by which samples undergo oxidation, and vice versa. Gaining an understanding of how this occurs is critical to the development of methods to stop undesirable changes, particularly in dairy products where heat treatment is widely used, and oxidation is often encountered. Heat treatment has been shown to result in unfolding of beta-lactoglobulin as assessed by circular dichroism measurements. This effect was increased, in a dose-dependent manner, in the presence of added H 2 O 2 . Addition of high doses of H 2 O 2 before heating also resulted in a shift of the intrinsic (Trp-derived) protein fluorescence maximum to higher wavelengths. Thiol quantification, using 5,5’-dithio-(2-nitrobenzoic acid), revealed that H 2 O 2 depletes thiols in a dose-dependent manner. Using SDS-PAGE, it was shown that whilst heating induced reducible cross-links; pretreatment with high levels of H 2 O 2 prevented the formation of these species. With lower concentrations of H 2 O 2 , reducible cross-links were generated during subsequent heating, consistent with a key role for disulphide formation in heat-induced cross-link formation, with H 2 O 2 diminishing aggregate formation. Altogether, these data indicate that both thermal treatment and oxidation can induce modifications to the protein via competing processes, and alter the vulnerability of the protein to aggregation.

A New Method for Quantitative Immunoblotting of Endogenous α-Synuclein

β-Sheet-rich aggregates of α-synuclein (αSyn) are the hallmark neuropathology of Parkinson’s disease and related synucleinopathies, whereas the principal native structure of αSyn in healthy cells - unfolded monomer or α-helically folded oligomer - is under debate. Our recent crosslinking analysis of αSyn in intact cells showed that a large portion of endogenous αSyn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying αSyn Western blot signals among samples, as crosslinked αSyn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased αSyn immunoreactivity even after cleavage with the reducing agent β-mercaptoethanol. The effect was observed with all αSyn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous αSyn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of αSyn after DSP/β-mercaptoethanol treatment, in contrast to a marked loss of αSyn without this treatment. The treatment also enhanced immunodetection of the homologs β- and γ-synuclein and of histones, another group of small, lysine-rich proteins. We conclude that by neutralizing positive charges and increasing protein hydrophobicity, amine crosslinker treatment promotes adhesion of αSyn to blotting membranes. These data help explain the recent report of fixing αSyn blots with paraformaldehyde after transfer, which we find produces similar but weaker effects. DSP/β-mercaptoethanol treatment of Western blots should be particularly useful to quantify low-abundance αSyn forms such as extracellular and post-translationally modified αSyn and splice variants.

Novel Reduction-Responsive Cross-Linked Polyethylenimine Derivatives by Click Chemistry for Nonviral Gene Delivery

Novel reducible disulfide-containing cross-linked polyethylenimines (PEI-SS-CLs) were synthesized via click chemistry and evaluated as nonviral gene delivery vectors. First, about four azide pendant groups were introduced into a low-molecular-weight (LMW) PEI (1.8 kDa) to get an azide-terminated PEI. Then, click reaction between a disulfide-containing dialkyne cross-linker and the azide functionalized LMW PEI resulted in a high-molecular-weight disulfide-containing cross-linked PEI composed of LMW constitute via a reducible cross-linker. The synthesized polymers were characterized by (1)H NMR, FTIR, and size-exclusion chromatography (SEC). It was shown that the obtained disulfide-containing cross-linked PEIs were able to condense plasmid DNA into positively charged nanoparticles. The degradation of the disulfide cross-linked polymers PEI-SS-CLs induced by DTT was confirmed by a gel retardation assay and SEC analysis. In vitro experiments revealed that the reducible PEI-SS-CLs were less cytotoxic and more effective in gene transfection (in both the presence and absence of serum) than the control nondegradable 25-kDa PEI. This study demonstrates that a reducibly degradable cationic polymer composed of LMW PEI cross-linked via a disulfide-containing linker possesses both higher gene transfection efficiency and lower cytotoxicity than PEI (25 kDa). These polymers are therefore attractive candidates for further in vivo evaluations.

Cell-surface Processing of the Metalloprotease Pro-ADAMTS9 Is Influenced by the Chaperone GRP94/gp96

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs 9 (ADAMTS9) is a highly conserved metalloprotease that has been identified as a tumor suppressor gene and is required for normal mouse development. The secreted ADAMTS9 zymogen undergoes proteolytic excision of its N-terminal propeptide by the proprotein convertase furin. However, in contrast to other metalloproteases, propeptide excision occurs at the cell surface and leads to decreased activity of the zymogen. Here, we investigated the potential cellular mechanisms regulating ADAMTS9 biosynthesis and cell-surface processing by analysis of molecular complexes formed by a construct containing the propeptide and catalytic domain of pro-ADAMTS9 (Pro-Cat) in HEK293F cells. Cross-linking of cellular proteins bound to Pro-Cat followed by mass spectrometric analysis identified UDP-glucose:glycoprotein glucosyltransferase I, heat shock protein gp96 (GRP94), BiP (GRP78), and ERdj3 (Hsp40 homolog) as associated proteins. gp96 and BiP were present at the cell surface in an immunoprecipitable complex with pro-ADAMTS9 and furin. Treatment with geldanamycin, an inhibitor of the HSP90alpha family (including gp96), led to decreased furin processing of pro-ADAMTS9 and accumulation of the unprocessed pro-ADAMTS9 at the cell surface. gp96 siRNA down-regulated the levels of cell-surface pro-ADAMTS9 and furin, whereas the levels of cell-surface pro-ADAMTS9, but not of cell-surface furin, were decreased upon treatment with BiP siRNA. These data identify for the first time the cellular chaperones associated with secretion of an ADAMTS protease and suggest a role for gp96 in modulating pro-ADAMTS9 processing.

Molecular changes in fibrillar collagen in myxomatous mitral valve disease

Myxomatous mitral valve disease (MMVD) is the single most common acquired cardiac disease of dogs and is a disease of significant veterinary importance. It also bears close similarities to mitral valve prolapse in humans and therefore is a disease of emerging comparative interest. We have previously mapped the structure of collagen fibrils in valve leaflets using synchrotron X-rays and have demonstrated changes in collagen structure associated with the regions of disease. Differential scanning calorimetry (DSC),biochemical assay of collagen content, high-performance liquid chromatography (HPLC), and neutron diffraction were combined with further analysis of our previous X-ray data to elucidate molecular changes in fibrillar collagen in mild to moderately affected MMVD dogs. Comparing diseases and adjacent grossly uninvolved areas in the same leaflets, there was a 20% reduction in collagen fibrils, but only a 10% depletion of collagen content. The enthalpy of collagen denaturation was reduced in affected areas. Chromatography showed a 25% decrease in mature nonreducible covalent cross-links in the affected samples, and neutron diffraction data showed fewer reducible immature covalent cross-links in grossly uninvolved tissue samples. Mild to moderate MMVD in the dog is associated with a marginal decline in collagen content in overtly diseased areas of valves, but more importantly is associated with an increase in immature collagen content. These changes will contribute to the mechanical dysfunction of the leaflet, and this study provides important information on the structure-mechanical alterations associated with this disease. The data suggests MMVD involves a dyscollagenesis process in the development of valve pathology.

Oxidation of myosin by haem proteins generates myosin radicals and protein cross-links

Previous studies have reported that myosin can be modified by oxidative stress and particularly by activated haem proteins. These reactions have been implicated in changes in the properties of this protein in food samples (changes in meat tenderness and palatability), in human physiology (alteration of myocyte function and force generation) and in disease (e.g. cardiomyopathy, chronic heart failure). The oxidant species, mechanisms of reaction and consequences of these reactions are incompletely characterized. In the present study, the nature of the transient species generated on myosin as a result of the reaction with activated haem proteins (horseradish peroxidase/H2O2) and met-myoglobin/H2O2) has been investigated by EPR spectroscopy and amino-acid consumption, product formation has been characterized by HPLC, and changes in protein integrity have been determined by SDS/PAGE. Multiple radical species have been detected by EPR in both the presence and the absence of spin traps. Evidence has been obtained for the presence of thiyl, tyrosyl and other unidentified radical species on myosin as a result of damage-transfer from oxidized myoglobin or horseradish peroxidase. The generation of thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation on myosin, product generation and cross-link induction are consistent with these processes being interlinked. These changes are consistent with the altered function and properties of myosin in muscle tissue exposed to oxidative stress arising from disease or from food processing.

Characterization of Mandibular Bones in Senile Osteoporotic Mice

At present, little is known about the age-related changes in jaw bones. The aim of this study was to characterize the mandibles of 6 month-old senile osteoporotic mice, SAMP6, and compare with those of age-matched controls, SAMR1. In comparison to SAMR1, SAMP6 showed thinner cortical bone, lower bone volume, and poorly organized collagen matrix. The collagen fibril diameter in SAMP6 was significantly smaller than that of SAMR1. In SAMP6 both collagen content and cross-links were lower than those of SAMR1, but the ratio of the major mature cross-link (pyridinoline) to its precursor reducible cross-link (dehydrodihydroxylysinonorleucine/its ketoamine) was higher in comparison to SAMR1. In addition, the extent of lysine hydroxylation of collagen was higher in SAMP6 than that of SAMR1. These results indicate that not only the quantity of collagen but also its quality are altered in SAMP6 and may result in the age-associated osteoporotic defects of mandibles.

PDGF‐BB released in tendon repair using a novel delivery system promotes cell proliferation and collagen remodeling

The purpose of this study was to promote fibroblast proliferation and collagen remodeling in flexor tendon repair through sustained delivery of platelet derived growth factor (PDGF-BB). The release kinetics of PDGF-BB from a novel fibrin matrix delivery system was initially evaluated in vitro. After the in vivo degradation rate of the fibrin matrix was determined using fluorescently tagged fibrin, PDGF-BB was delivered to the site of flexor tendon repair in vivo in a canine model. The effect of PDGF-BB on intrasynovial tendon healing was studied using histology-based assays (cell density, proliferation, and type I collagen expression) and by measuring total DNA levels and reducible collagen crosslink levels. The fibrin matrix delivery system provided sustained release of PDGF-BB in vitro at a rate modulated by the ratio of heparin to growth factor. In vivo, the fibrin matrix remained at the repair site for more than 10 days. Delivery of PDGF-BB led to a qualitative increase in cell density, cell proliferation, and type I collagen mRNA expression. PDGF-BB also led to statistically significant increases in total DNA (20% increase at 7 days, 18% increase at 14 days) and reducible collagen crosslinks (30% increase at 7 days). Sustained delivery of growth factors may be achieved using a novel fibrin-based delivery system. PDGF-BB delivery increased cell proliferation and matrix remodeling and thus may accelerate flexor tendon healing.

Biochemical Characterization of Collagen in Alveolar Mucosa and Attached Gingiva of Pig

Alveolar mucosa and attached gingiva are two continuous but functionally distinct connective tissues covering alveolar bone of the jaw. In this study, the major matrix component of these tissues, collagen, was biochemically characterized and compared. The tissues were obtained from mature pigs and analyzed for collagen content, amino acid composition, collagen types, collagen cross-linking, and gene expression. We found that alveolar mucosa is primarily composed of fibrillar collagens and the collagen content is higher than attached gingiva. The content of type III relative to type I collagen was higher in alveolar mucosa when compared with attached gingiva. The collagen cross-linking pattern also was distinct between the two tissues demonstrating that alveolar mucosa contained fewer reducible cross-links but more nonreducible cross-links in comparison to attached gingiva. The mRNA expression level of type I collagen in alveolar mucosa was significantly lower than that of attached gingiva. These results indicate that alveolar mucosa is a fibrillar collagen-rich tissue and, in comparison to gingival tissue, remodels slowly.

Degree of Mineralization-related Collagen Crosslinking in the Femoral Neck Cancellous Bone in Cases of Hip Fracture and Controls

Based on the present definition of osteoporosis, both bone density and quality are important factors in the determination of bone strength. Collagen crosslinking is a determinant of bone quality. Cross-links can form enzymatically by the action of lysyl oxidase or non-enzymatically, resulting in advanced glycation end products. Collagen crosslinking is affected by tissue maturation as well as the degree of mineralization. Homocysteine and vitamin B6 (pyridoxal) are also regulatory factors of collagen crosslinking. We elucidate the relationship between the degree of mineralization and collagen cross-links in cancellous bone from hip fracture cases. We also determined plasma levels of homocysteine and pyridoxal. Twenty-five female intracapsular hip fracture cases (78 +/- 6 years) and 25 age-matched postmortem controls (77 +/- 6 years) were included in this study. Collagen crosslinking was analyzed after each bone specimen was fractionated into low (1.7-2.0 g/ml) and high (>2.0 g/ml) density fractions. The content of enzymatic (immature reducible and mature nonreducible cross-links) and nonenzymatic cross-link (pentosidine) were determined. In the controls, there was no difference in total enzymatic cross-links between low and high density bone, while pentosidine content was significantly higher in high density bone. In the fracture cases, not only reduced enzymatic cross-links in high density bone and increased pentosidine in both low and high density bone, but also higher plasma homocysteine and lower pyridoxal levels were evident compared with the controls. These results indicate that detrimental crosslinking in both low and high mineralized bone result in impaired bone quality in osteoporotic patients.