Preparation and characterization of a thermostable and biodegradable biopolymers using natural cross-linker

The present study describes preparation and characterization of a thermally stable and biodegradable biopolymer using collagen and a natural polymer, alginic acid (AA). Required concentration of alginic acid and collagen was optimized and the resulting biopolymer was characterized for, degree of cross-linking, mechanical strength, thermal stability, biocompatibility (toxicity) and biodegradability. Results reveal, the degree of cross-linking of alginic acid (at 1.5% concentration) with collagen was calculated as 75%, whereas it was 83% with standard cross-linking agent, glutaraldehyde (at 1.5% concentration). The AA cross-linked biopolymer was stable up to 245°C and Exhibits 5-6-fold increase in mechanical (tensile) strength compared to plain collagen (native) materials. However, glutaraldehyde cross-linked material exhibits comparatively less thermal stability and brittle in nature (low tensile strength). With regard to cell toxicity, no cytotoxicity was observed for AA cross-linked material when tested with mesenchymal cells and found degradable when treated with collagenase enzyme. The nature of bonding pattern and the reason for thermal stability of AA cross-linked collagen biopolymer was discussed in detail with the help of bioinformatics. A supplementary file on efficacy of AACC as a wound dressing material is demonstrated in detail with animal model studies.     

Mechanical response of porous scaffolds for cartilage engineering

Mechanical properties of scaffolds seeded with mesenchymal stem cells used for cartilage repair seem to be one of the critical factors in possible joint resurfacing. In this paper, the effect of adding hyaluronic acid, hydroxyapatite nanoparticles or chitosan nanofibers into the cross-linked collagen I on the mechanical response of the lyophilized porous scaffold has been investigated in the dry state at 37 oC under tensile loading. Statistical significance of the results was evaluated using ANOVA analysis. The results showed that the addition of hyaluronic acid significantly (p<0.05) reduced the tensile elastic modulus and enhanced the strength and deformation to failure of the modified cross-linked collagen I under the used test conditions. On the other hand, addition of hydroxyapatite nanoparticles and chitosan nanofibers, respectively, increased the elastic modulus of the modified collagen ten-fold and four-fold, respectively. Hydroxyapatite caused significant reduction in the ultimate deformation at break while chitosan nanofibers enhanced the ultimate deformation under tensile loading substantially (p<0.05). The ultimate tensile deformation was significantly (p<0.05) increased by addition of the chitosan nanofibers. The enhanced elastic modulus of the scaffold was translated into enhanced resistance of the porous scaffolds against mechanical load compared to scaffolds based on cross-linked neat collagen or collagen with hyaluronic acid with similar porosity. It can be concluded that enhancing the rigidity of the compact scaffold material by adding rigid chitosan nanofibers can improve the resistance of the porous scaffolds against compressive loading, which can provide more structural protection to the seeded mesenchymal stem cells when the construct is implanted into a lesion. Moreover, scaffolds with chitosan nanofibers seemed to enhance cell growth compared to the neat collagen I when tested in vitro as well as the scaffold stability, extending its resorption to more than 10 weeks.     

Gold Nanoparticles Attenuates Antimycin A-Induced Mitochondrial Dysfunction in MC3T3-E1 Osteoblastic Cells

Gold nanoparticles have shown promising biological applications due to their unique properties. Understanding the interaction mechanisms between nanomaterials and biological cells is important for the control and manipulation of these interactions for biomedical applications. In the present study, we investigated the effects of gold nanoparticles on the differentiation of osteoblastic MC3T3-E1 cells and antimycin A-induced mitochondrial dysfunction. The results showed that gold nanoparticles (5, 10, and 20 nm) caused a significant elevation of cell growth, alkaline phosphatase activity, collagen synthesis, and osteocalcin content in the cells (P?<?0.05). Moreover, pretreatment with gold nanoparticles prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, cytochrome c release, cardiolipin peroxidation, and reactive oxygen species generation. Taken together, our study indicated that gold nanoparticles may improve the differentiation and have protective effects on mitochondrial dysfunction of osteoblastic cells.     

Enhancing Anti-Tumor Efficacy of Doxorubicin by Non-Covalent Conjugation to Gold Nanoparticles – In Vitro Studies on Feline Fibrosarcoma Cell Lines

BackgroundFeline injection-site sarcomas are malignant skin tumors of mesenchymal origin, the treatment of which is a challenge for veterinary practitioners. Methods of treatment include radical surgery, radiotherapy and chemotherapy. The most commonly used cytostatic drugs are cyclophosphamide, doxorubicin and vincristine. However, the use of cytostatics as adjunctive treatment is limited due to their adverse side-effects, low biodistribution after intravenous administration and multidrug resistance. Colloid gold nanoparticles are promising drug delivery systems to overcome multidrug resistance, which is a main cause of ineffective chemotherapy treatment. The use of colloid gold nanoparticles as building blocks for drug delivery systems is preferred due to ease of surface functionalization with various molecules, chemical stability and their low toxicity.MethodsStability and structure of the glutathione-stabilized gold nanoparticles non-covalently modified with doxorubicin (Au-GSH-Dox) was confirmed using XPS, TEM, FT-IR, SAXRD and SAXS analyses. MTT assay, Annexin V and Propidium Iodide Apoptosis assay and Rhodamine 123 and Verapamil assay were performed on 4 feline fibrosarcoma cell lines (FFS1WAW, FFS1, FFS3, FFS5). Statistical analyses were performed using Graph Pad Prism 5.0 (USA).ResultsA novel approach, glutathione-stabilized gold nanoparticles (4.3 +/- 1.1 nm in diameter) non-covalently modified with doxorubicin (Au-GSH-Dox) was designed and synthesized. A higher cytotoxic effect (p<0.01) of Au-GSH-Dox than that of free doxorubicin has been observed in 3 (FFS1, FFS3, FFS1WAW) out of 4 feline fibrosarcoma cell lines. The effect has been correlated to the activity of glycoprotein P (main efflux pump responsible for multidrug resistance).ConclusionsThe results indicate that Au-GSH-Dox may be a potent new therapeutic agent to increase the efficacy of the drug by overcoming the resistance to doxorubicin in feline fibrosarcoma cell lines. Moreover, as doxorubicin is non-covalently attached to glutathione coated nanoparticles the synthesized system is potentially suitable to a wealth of different drug molecules.     

Assembly of collagen fibril meshes using gold nanoparticles functionalized with tris(hydroxymethyl)phosphine-alanine as multivalent cross-linking agents

We report on the use of tris(hydroxymethyl)phosphine-alanine (THPAL) functionalized gold nanoparticles as a multivalent cross-linking agent to assemble collagen fibrils into a mesh-like structure. Atomic force microscopy (AFM) was used for characterization of the structure after adsorption onto an atomically flat mica substrate, revealing a mesh-like construct in which the collagen fibrils and the gold nanoparticles interact to form interconnected nodes measuring from 100 to 500 nm. As expected, the density of the collagen mesh can be increased with a higher initial concentration of gold nanoparticles. The maximum thickness of the meshes (～ 20 nm) obtained through cross-sectional height measurements confirms that the adsorbed structure consists of a single layer of collagen fibrils/gold nanoparticles assembled in two-dimensions. We propose that the capability of gold nanoparticles functionalized with the THPAL to bind to several collagen fibrils combined with the large persistence length of the fibrils, which was reported to be in the hundreds of nanometer range, are determinant factors for the preferential 2D growth of the mesh in solution.     

Peptide-derivatized shell-cross-linked nanoparticles. 2. Biocompatibility evaluation

The conjugation of the protein transduction domain (PTD) from the HIV-1 Tat protein to shell cross-linked (SCK) nanoparticles is a method to facilitate cell surface binding and transduction. In the previous report, the preparation, derivatization, and characterization of peptide-functionalized SCK nanoparticles were reported in detail. Following assembly, the constructs were evaluated in vitro and in vivo to obtain a preliminary biocompatibility assessment. The effects of SCK exposure on cell viability were evaluated using a metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and a fluorescent apoptosis assay. Furthermore, stages of apoptosis were quantified by flow cytometry. Although higher levels of peptide functionalization resulted in decreased metabolic function as measured by MTT assay, significant apoptosis was not observed below 500 mg/L for all the samples. To evaluate the potential immunogenic response of the peptide-derivatized constructs, a real-time polymerase chain reaction (RT-PCR) system that allows for the in vitro analysis and quantification of the cellular inflammatory responses tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL1-beta) was utilized. The inflammatory response to the peptide-functionalized SCK nanoparticles as measured by RT-PCR show statistically significant increases in the levels of both TNF-alpha and IL1-beta relative to tissue culture polystyrene (TCPS). However, the measured cytokine levels did not preclude the further testing of SCKs in an in vivo mouse immunization protocol. In this limited assay, measured increases in immunoglobulin G (IgG) concentration in the sera were minimal with no specific interactions being isolated, and more importantly, none of the mice (>50) subjected to the three 100 microg immunization protocol have died. Additionally, no gross morphological changes were observed in postmortem organ histology examinations.     

Green-synthesized gold nanoparticles decorated graphene sheets for label-free electrochemical impedance DNA hybridization biosensing

Sensitive electrochemical impedance assay of DNA hybridization by using a novel graphene sheets platform was achieved. The graphene sheets were firstly functionalized with 3,4,9,10-perylene tetracarboxylic acid (PTCA). PTCA molecules separated graphene sheets efficiently and introduced more negatively-charged -COOH sites, both of which were beneficial to the decoration of graphene with gold nanoparticles. Then amine-terminated ionic liquid (NH?-IL) was applied to the reduction of HAuCl? to gold nanoparticles. The green-synthesized gold nanoparticles, with the mean diameter of 3 nm, dispersed uniformly on graphene sheets and its outer layer was positively charged imidazole termini. Due to the presence of large graphene sheets and NH?-IL protected gold nanoparticles, DNA probes could be immobilized via electrostatic interaction and adsorption effect. Electrochemical impedance value increased after DNA probes immobilization and hybridization, which was adopted as the signal for label-free DNA hybridization detection. Unlike previously anchoring DNA to gold nanoparticles, this label-free method was simple and noninvasive. The conserved sequence of the pol gene of human immunodeficiency virus 1 was satisfactorily detected via this strategy.     

Unveiling the anticancer and antimycobacterial potentials of bioengineered gold nanoparticles

Synthesis of gold nanoparticles was carried out using Pongammia pinnata (pongam) leaf extract and their anticancer and antimycobacterial activities were studied. Gold nanoparticle formation was confirmed by UV–vis, XRD and HR-TEM. The anticancer efficacies of the biogenic gold nanoparticles were analyzed using cytotoxicity, cell morphology analysis, oxidative DNA damage, apoptosis detection and toxicity studies. Biogenic gold nanoparticles inhibited breast cancer cell line (MCF-7) proliferation with an efficacy of IC₅₀ of 1.85 μg/mL. The antimycobacterial potential of the biogenic gold nanoparticles was screened against M. tuberculosis by Luciferase Reporter Phage (LRP) assay. The gold nanoparticles showed inhibition against sensitive M. tuberculosis with the minimum inhibitory concentration (MIC) of 10 μg/mL whereas no inhibition was found against the rifampicin resistant M. tuberculosis.     

Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles

The high mortality rate in cancer such as oral squamous cell carcinoma is commonly attributed to the difficulties in detecting the disease at an early treatable stage. In this study, we exploited the ability of gold nanoparticles to undergo coupled surface plasmon resonance and set up strong electric fields when closely-spaced to improve the molecular contrast signal in reflectance-based imaging and also to enhance the Raman signal of bioanalytes in cancer. Colloidal gold nanoparticles were synthesized and conjugated to anti-epidermal growth factor receptor (EGFR) for imaging. A self-assembled surface enhanced Raman scattering (SERS)-active gold nanoparticle monolayer film was also developed as a biosensing surface using a simple drop-dry approach. We have shown that gold nanoparticles could elicit an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies allowed them to map the expression of relevant biomarkers for molecular imaging under confocal reflectance microscopy. We have also shown that the SERS spectra of saliva from the closely-packed gold nanoparticles films was differentiable between those acquired from normal individuals and oral cancer patients, thus showing promise of a simple SERS-based saliva assay for early diagnosis of oral cancer.     

Microquantitation of insoluble tissue collagen (types I and III) by radiodilution assay

The individual collagen types of the extracellular matrix of small tissue samples have been difficult to quantitate accurately both due to their marked insolubility and their relatively low immunogenicity. Thus no microassay with the sensitivity of a radioimmunoassay is currently available for quantitation of insoluble collagen types I and III in extremely small tissue samples. A radiochemical assay has been developed which allows direct processing of small tissue samples containing as little as 1-3 micrograms of a given collagen alpha chain. Unprocessed lyophilized tissues were digested with cyanogen bromide (CNBr) in the presence of a tritiated probe containing a soluble mixture of 3H-alpha 1(I) and 3H-alpha 1(III) collagen previously extracted and purified from tissue minces incubated with [3H]leucine. The resulting mix of radiolabeled peptides was separated on sodium dodecyl sulfate-polyacrylamide gradient gels. Reduction of the specific radioactivity of free leucine in acid hydrolysates of each individual CNBr peptide can be used to quantitate the amount of collagen types I or III in the original sample. Similar radiodilution analysis using a 3H-alpha 2(I) probe indicated a normal 2:1 ratio of alpha chains of type I collagen in the tissues tested. This method is also applicable to cell culture, easily measuring the collagen associated with fibroblast cell layers or medium in individual microtiter wells. When applied to various tissues of known collagen-type composition, it provides reproducible results which compare well with values published in the literature.     

Stability,Intracellular Delivery,and Release of siRNA from Chitosan Nanoparticles Using Different Cross-Linkers

Chitosan (CS) nanoparticles have been extensively studied for siRNA delivery; however, their stability and efficacy are highly dependent on the types of cross-linker used. To address this issue, three common cross-linkers; tripolyphosphate (TPP), dextran sulphate (DS) and poly-D-glutamic acid (PGA) were used to prepare siRNA loaded CS-TPP/DS/PGA nanoparticles by ionic gelation method. The resulting nanoparticles were compared with regard to their physicochemical properties including particle size, zeta potential, morphology, binding and encapsulation efficiencies. Among all the formulations prepared with different cross linkers, CS-TPP-siRNA had the smallest particle size (ranged from 127 ± 9.7 to 455 ± 12.9 nm) with zeta potential ranged from +25.1 ± 1.5 to +39.4 ± 0.5 mV, and high entrapment (>95%) and binding efficiencies. Similarly, CS-TPP nanoparticles showed better siRNA protection during storage at 4˚C and as determined by serum protection assay. TEM micrographs revealed the assorted morphology of CS-TPP-siRNA nanoparticles in contrast to irregular morphology displayed by CS-DS-siRNA and CS-PGA-siRNA nanoparticles. All siRNA loaded CS-TPP/DS/PGA nanoparticles showed initial burst release followed by sustained release of siRNA. Moreover, all the formulations showed low and concentration-dependent cytotoxicity with human colorectal cancer cells (DLD-1), in vitro. The cellular uptake studies with CS-TPP-siRNA nanoparticles showed successful delivery of siRNA within cytoplasm of DLD-1 cells. The results demonstrate that ionically cross-linked CS-TPP nanoparticles are biocompatible non-viral gene delivery system and generate a solid ground for further optimization studies, for example with regard to steric stabilization and targeting.     

Collagen cross-linking. Isolation of cross-linked peptides from collagen of chicken bone

Cross-linked peptides were isolated from chicken bone collagen that had been digested with CNBr or with bacterial collagenase. Analyses of (3)H radioactivity in disc electrophoretic profiles of the CNBr peptides from bone collagens that had been treated with NaB(3)H indicated that a major site of intermolecular cross-linking in chicken bone collagen is located between the carboxy-terminal region of an alpha1 chain and a small CNBr peptide, probably situated near the amino-terminus of an alpha1 or alpha2 chain in an adjacent collagen molecule. A small amount of this cross-linked CNBr peptide was isolated from a CNBr digest of chicken bone collagen by column chromatography. Amino acid analysis showed that the CNBr peptide, alpha1CB6B, the carboxy-terminal peptide of the alpha1 chain, was the major CNBr peptide in the preparation, and the reduced cross-linking components were identified as hydroxylysinohydroxynorleucine (HylOHNle), with a smaller amount of hydroxylysinonorleucine (HylNle). However, the composition and the low recovery of the cross-linking amino acids suggested that the preparation was a mixture of CNBr peptides alpha1CB6B and alpha1CB6B cross-linked to a small CNBr peptide whose identity could not be determined. A small cross-linked peptide was isolated from chicken bone collagen that had been reduced with NaB(3)H(4) and digested with bacterial collagenase. This peptide was the major cross-linked peptide in the digest and contained a stoicheiometric amount of the reduced cross-linking compounds. A peptide which had the same amino acid composition, but contained the cross-linking compounds in their reducible forms, was isolated from a collagenase digest of chicken bone collagen that had not been treated with NaBH(4). The absence of the reduced cross-links from this peptide indicates that, at least for the cross-linking site from which the peptide derives, natural reduction is not a significant pathway for biosynthesis of stable cross-links. However, most of the reducible cross-linking component in the peptide appeared to stabilize in the bone collagen by rearrangement from aldimine to ketoamine form.     

Chemiluminescence flow biosensor for glucose based on gold nanoparticle-enhanced activities of glucose oxidase and horseradish peroxidase

In this work, a novel chemiluminescence (CL) flow biosensor for glucose was proposed. Glucose oxidase (GOD), horseradish peroxidase (HRP) and gold nanoparticles were immobilized with sol-gel method on the inside surface of the CL flow cell. The CL detection involved enzymatic oxidation of glucose to d-gluconic acid and H(2)O(2), and then the generated H(2)O(2) oxidizing luminol to produce CL emission in the presence of HRP. It was found that gold nanoparticles could remarkably enhance the CL respond of the glucose biosensor. The enhanced effect was closely related to the sizes of gold colloids, and the smaller the size of gold colloids had the higher CL respond. The immobilization condition and the CL condition were studied in detail. The CL emission intensity was linear with glucose concentration in the range of 1.0 x 10(-5)molL(-1) to 1.0 x 10(-3)molL(-1), and the detection limit was 5 x 10(-6)molL(-1) (3sigma). The apparent Michaelis-Menten constant of GOD in gold nanoparticles/sol-gel matrix was evaluated to be 0.3mmolL(-1), which was smaller than that of GOD immobilized in sol-gel matrix without gold nanoparticles. The proposed biosensor exhibited short response time, easy operation, low cost and simple assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum.     

Cross-linked collagen sponges loaded with plant polyphenols with inhibitory activity towards chronic wound enzymes

Collagen sponges loaded with polyphenols from Hamamelis virginiana were investigated as active materials for chronic wound dressings, evaluating in vitro the inhibition of two major enzymes that impair the wound healing process - myeloperoxidase (MPO) and collagenase. Prior to polyphenols loading, collagen was cross-linked with genipin to improve its biostability. The effect of genipin cross-linking and polyphenol concentration in the development of mechanically and enzymatically stable sponges was studied. The tensile strength of the cross-linked collagen increased with the increase of the cross-linking degree, coupled to decrease in the elongation and the swelling capacity of the sponges. The stability of the sponges to collagenase digestion reached maximum when 1 mM genipin was used. However, the biostability decreased more than 10-fold after loading the sponges with polyphenols (0.5 mg/mL), nevertheless, this effect was partially overcome using higher concentration of polyphenols (1 and 2 mg/mL) to inhibit collagenase. Moreover, the polyphenols released from the sponges were sufficient for complete inhibition of MPO activity. No considerable cytotoxicity of the genipin cross-linked collagen loaded with polyphenols was observed evaluating the NIH 3T3 fibroblasts viability.     

Two-dimensional peptide mapping of cross-linked type IX collagen in human cartilage

Type IX collagen is a quantitatively minor component of hyaline cartilage that is essential for the normal structural integrity of the tissue. Purification and analysis are difficult because the mature protein is insoluble as a cross-linked integral component of the fibrillar matrix. In order to view a peptide map of the total pool of type IX collagen in a cartilage sample, a selective method based on Western blot analysis was developed for displaying collagen IX peptides in a cyanogen bromide digest of tissue. Digests were partially resolved by reverse-phase HPLC, individual fractions were run on SDS-PAGE and then transblotted to membrane, and the collagen IX fragments were revealed using an anti-collagen IX rabbit antiserum. All major CB-peptides from alpha1(IX), alpha2(IX), and alpha3(IX) chains in the resulting two-dimensional display were identified by amino-terminal sequence analysis. Cross-linked peptides originating from sites of covalent interaction between collagen types IX and II and between IX and IX were also defined. By comparison with an analysis of soluble type IX collagen from chondrocyte culture medium, the results showed that the pool of type IX collagen molecules in fetal and adult human cartilage is extensively cross-linked intermolecularly at sites previously revealed by other methods using purified protein. This sensitive, direct method has the potential to screen for abnormalities in the content and properties of type IX collagen in tissue samples, for example, in the study of heritable chondrodysplasia syndromes and the pathogenesis of cartilage destruction in osteoarthritis.     

Binding potency of peptide fragments of type 1 collagen cross-linked N-telopeptide measured by an enzyme-linked immunosorbant assay

Osteoporosis represents a major healthcare problem affecting elderly person. Urinary level of the crosslinked N-telopeptide of type I collagen is a sensitive marker of bone resorption. Ten overlapping peptides covering the N-telopeptide of alpha-2 type I collagen were synthesized, purified, and assayed for their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibody by using a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). Peptides 1, 2, and 3, containing the N-terminal sequence of N-telopeptide, showed higher binding potency than peptides 4-10, suggesting that these peptides may contain binding sites for anti-NTX antibodies, and can serve as the lead for further preparation of their antibodies in order to develop novel bioassays for monitoring the bone loss in humans.     

Cross-linking of collagen I by tissue transglutaminase provides a promising biomaterial for promoting bone healing

Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and β1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of β1 and β3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing.     

Intracellular gold nanoparticles enhance non-invasive radiofrequency thermal destruction of human gastrointestinal cancer cells

Background  

Novel approaches to treat human cancer that are effective with minimal toxicity profiles are needed. We evaluated gold nanoparticles (GNPs) in human hepatocellular and pancreatic cancer cells to determine: 1) absence of intrinsic cytotoxicity of the GNPs and 2) external radiofrequency (RF) field-induced heating of intracellular GNPs to produce thermal destruction of malignant cells. GNPs (5 nm diameter) were added to 2 human cancer cell lines (Panc-1, Hep3B). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and propidium iodide-fluorescence associated cell sorting (PI-FACS) assessed cell proliferation and GNP-related cytotoxicity. Other GNP-treated cells were exposed to a 13.56 MHz RF field for 1, 2, or 5 minutes, and then incubated for 24 hours. PI-FACS measured RF-induced cytotoxicity.     

Immunoaffinity Nanoprobe-Based MALDI-TOF MS for Detection of Fragments of N-Telopeptides of Type I Collagen

Osteoporosis is one of the major bone-related diseases. Among the biomarkers of bone resorption, type I collagen cross-linked N-telopeptides (NTx) are terminal metabolites specifically derived from the degradation of bone collagen, thus, the level of NTx in urine has been regarded as a highly specific index for bone resorption. Our previous studies have identified a synthetic peptide fragment in the N-terminal of NTx (peptide P2) with highest affinity for anti-NTx antibodies by using enzyme-linked immunosorbent assay and surface plasma resonance-based methods. The objective of this study is to prepare the mouse anti-P2 polyclonal antibodies (anti-P2 Ab) and develop an immunonanoprobe-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for detection of fragment of NTx of type I collagen. Anti-P2 Ab were prepared, purified, characterized, and used to produce Ab-conjugated magnetic nanoparticles (Ab@MNPs) for specifically isolation of different concentrations of Ab-bound P2 standards. The profile of P2 standards which were bound to Ab–NPs was analyzed by combining immunoaffinity MNPs with MALDI-TOF MS. We demonstrated that an immunoaffinity nanoprobe-based MALDI-TOF MS method for detecting the fragment of NTx, and might provide a tool to discover a promising biomarker of osteoporosis. The potential of this method for early diagnosis of osteoporosis will be further investigated by recruiting osteoporosis patients from our collaborative hospitals.     

Type III Collagen,a Fibril Network Modifier in Articular Cartilage

The collagen framework of hyaline cartilages, including articular cartilage, consists largely of type II collagen that matures from a cross-linked heteropolymeric fibril template of types II, IX, and XI collagens. In the articular cartilages of adult joints, type III collagen makes an appearance in varying amounts superimposed on the original collagen fibril network. In a study to understand better the structural role of type III collagen in cartilage, we find that type III collagen molecules with unprocessed N-propeptides are present in the extracellular matrix of adult human and bovine articular cartilages as covalently cross-linked polymers extensively cross-linked to type II collagen. Cross-link analyses revealed that telopeptides from both N and C termini of type III collagen were linked in the tissue to helical cross-linking sites in type II collagen. Reciprocally, telopeptides from type II collagen were recovered cross-linked to helical sites in type III collagen. Cross-linked peptides were also identified in which a trifunctional pyridinoline linked both an α1(II) and an α1(III) telopeptide to the α1(III) helix. This can only have arisen from a cross-link between three different collagen molecules, types II and III in register staggered by 4D from another type III molecule. Type III collagen is known to be prominent at sites of healing and repair in skin and other tissues. The present findings emphasize the role of type III collagen, which is synthesized in mature articular cartilage, as a covalent modifier that may add cohesion to a weakened, existing collagen type II fibril network as part of a chondrocyte healing response to matrix damage.