Consequences of incorporating thiaproline and its oxidized derivatives into collagen triple helices.

(2R)-4-thiaproline (Thp) is an analog of proline, replacing Cγ in the pyrrolidine ring with sulfur. Its thiazolidine ring easily interconverts between endo and exo puckers due to a small energy barrier, which leads to destabilize polyproline helices. Collagen, composed of three polyproline II helices, mainly consists of X-Y-Gly triplets, where X is often proline and Y is frequently (2S,4R)-hydroxyproline. In this study, we incorporated Thp into either position-X or position-Y to investigate the consequences of such a replacement on the triple helix. Circular dichroism and differential scanning calorimetry analyses showed that the Thp-containing collagen-mimetic peptides (CMPs) can fold into stable triple helices, in which the substitution at position-Y exhibits a larger destabilization effect. Additionally, we also prepared the derivative peptides by oxidizing Thp in the peptide to N-formyl-cysteine or S,S-dioxide Thp. The results showed that the oxidized derivatives at position-X only slightly affect collagen stability, but those at position-Y induce a large destabilization effect. The consequences of incorporating Thp and its oxidized derivatives into CMPs are position dependent. Computational results suggested that the ease of interconversion between exo and endo puckers for Thp and the twist conformation of S,S-dioxide Thp may cause the destabilization effect at position-Y. We have revealed new insights into the impacts of Thp and its oxidized derivatives on collagen and demonstrated that Thp can be used to design collagen-related biomaterials.

Peptoid Residues Make Diverse, Hyperstable Collagen Triple-Helices.

As the only ribosomally encoded N-substituted amino acid, proline promotes distinct secondary protein structures. The high proline content in collagen, the most abundant protein in the human body, is crucial to forming its hallmark structure: the triple-helix. For over five decades, proline has been considered compulsory for synthetic designs aimed at recapitulating collagen's structure and properties. Here we describe that N-substituted glycines (N-glys), also known as peptoid residues, exhibit a general triple-helical propensity similar to or greater than proline, enabling synthesis of stable triple-helical collagen mimetic peptides (CMPs) with unprecedented side chain diversity. Supported by atomic-resolution crystal structures as well as circular dichroism and computational characterizations spanning over 30 N-gly-containing CMPs, we discovered that N-glys stabilize the triple-helix primarily by sterically preorganizing individual chains into the polyproline-II helix. We demonstrated that N-glys with exotic side chains including a "click"-able alkyne and a photosensitive side chain enable CMPs for functional applications including the spatiotemporal control of cell adhesion and migration. The structural principles uncovered in this study open up opportunities for a new generation of collagen-mimetic therapeutics and materials.

A graphene oxide-aided triple helical aggregation-induced emission biosensor for highly specific detection of charged collagen peptides.

Aggregation-induced emission (AIE) probes have emerged as promising "turn-on" sensing tools for DNA and proteins, and the AIE biosensors conjugated with graphene oxide (GO) have shown improved selectivity. Collagen is an essential structural protein in the human body, and its degraded products are involved in a plethora of severe diseases. Collagen has a high content of charged amino acids, while EOG represents one of the most abundant charged triplets in Type I collagen. We, herein, for the first time report the construction of a GO-aided AIE biosensor for the detection of charged collagen peptides. We have shown that an AIE fluorophore TPE conjugated with a triple helical peptide TPE-PRG possesses strong fluorescence due to the restriction of intramolecular rotation of TPE in the trimer state. The adsorption of the probe TPE-PRG by GO leads to efficient fluorescence quenching, while the addition of target collagen peptide EOG releases the probe peptide from the GO surface and recovers its fluorescence. We have demonstrated that the TPE-PRG/GO complex provides a highly specific "turn-on" sensing platform for the target collagen peptide with a typical charged amino acid-rich sequence. The assay has shown little interference from other biomolecules, and it can also effectively distinguish the target charged collagen peptide from its single amino acid mutant type. The development of robust analytical assays for charged collagen peptides could pronouncedly extend our capability to investigate the pathology of collagen diseases, showing great potential for their molecular diagnosis.

Zinc Silicate/Nano-Hydroxyapatite/Collagen Scaffolds Promote Angiogenesis and Bone Regeneration via the p38 MAPK Pathway in Activated Monocytes.

Recent studies show that biomaterials are capable of regulating immune responses to induce a favorable osteogenic microenvironment and promote osteogenesis and angiogenesis. In this study, we investigated the effects of zinc silicate/nanohydroxyapatite/collagen (ZS/HA/Col) scaffolds on bone regeneration and angiogenesis and explored the related mechanism. We demonstrate that 10ZS/HA/Col scaffolds significantly enhanced bone regeneration and angiogenesis in vivo compared with HA/Col scaffolds. ZS/HA/Col scaffolds increased tartrate-resistant acid phosphatase (TRAP)-positive cells, nestin-positive bone marrow stromal cells (BMSCs) and CD31-positive neovessels, and expression of osteogenesis (Bmp-2 and Osterix) and angiogenesis-related (Vegf-α and Cd31) genes increased in nascent bone. ZS/HA/Col scaffolds with 10 wt % ZS activated the p38 signaling pathway in monocytes. The monocytes subsequently differentiated into TRAP+ cells and expressed higher levels of the cytokines SDF-1, TGF-ß1, VEGF-α, and PDGF-BB, which recruited BMSCs and endothelial cells (ECs) to the defect areas. Blocking the p38 pathway in monocytes reduced TRAP+ differentiation and cytokine secretion and resulted in a decrease in BMSC and EC homing and angiogenesis. Overall, these findings demonstrate that 10ZS/HA/Col scaffolds modulate monocytes and, thereby, create a favorable osteogenic microenvironment that promotes BMSC migration and differentiation and vessel formation by activating the p38 signaling pathway.

Biomimetic mineralizable collagen hydrogels for dynamic bone matrix formation to promote osteogenesis.

The simulation of the native bone matrix formation process is crucial for the construction of the cellular microenvironment for bone regeneration. However, it is still challenging to design bioactive materials that simultaneously mimic the composition and dynamic mineralization process of the bone matrix, let alone realize osteoinduction by a biomimetic dynamic microenvironment. In this study, we prepared a biomimetic mineralizable collagen hydrogel (CAV) and explored the effects of a dynamic mineralized matrix on the osteogenesis of stem cells both in vitro and in vivo. We showed the feasibility of the biomimetic CAV hydrogel to induce mineralization in simulated media including simulated body fluid (SBF), glycerol phosphate calcium salt hydrate (CaGP) solution and cell co-cultured systems. The participation of cells in the mineralization process is more likely to induce matrix remodeling due to the synergistic effects of CAV mineralization and cellular secretion, resulting in higher matrix strength. We also demonstrated that the biomimetic mineralized hydrogel could up-regulate osteogenic genes and protein expression of bone marrow mesenchymal stem cells (BMSCs), thus enhancing osteogenesis in vivo. The interactions between the mineralizable hydrogel and cells play an important role in regulating dynamic matrix mineralization and osteogenesis. Our findings prove that the biomimetic mineralizable hydrogel is a promising candidate for implantable orthopedic applications and provides essential implications for the future design of materials for bone regeneration.

Modification of chitosan grafted with collagen peptide by enzyme crosslinking.

Free radicals are closely related to the occurrence and development of aging, cancer and inflammation. In this paper, the microbial transglutaminase (MTGase) was used as a catalyst to graft the collagen peptide (COP) molecules on the amino group of carboxymethyl chitosan sulfate (CMCS) to improve the antioxidant effects. FT-IR and NMR spectroscopy were used to confirm the successful grafting of COP to CMCS. Degree of substitution (DS) of CMCS-COP could be controlled by adjusting the reaction conditions. With the increase of concentration, the ability of each sample on scavenging capacity and reducibility tends to increase obviously. The results of anticoagulant experiments showed that the ability of CMCS and CMCS-COP with three different degrees of substitution on activated partial thrombin time (APTT) and prothrombin time (PT) values were all increased to compare with the control group. No relevant cytotoxicity against NIH-3T3 mouse fibroblasts was found for the copolymers. These results suggested that CMCS-COP would appear to be a promising candidate for wound dressing application.

Preparation and in vitro characterization of cross-linked collagen-gelatin hydrogel using EDC/NHS for corneal tissue engineering applications.

Corneal disease is considered as the second leading cause of vision loss and keratoplasty is known as an effective treatment for it. However, the tissue engineered corneal substitutes are promising tools in experimental in vivo repair of cornea. Selecting appropriate cell sources and scaffolds are two important concerns in corneal tissue engineering. The object of this study was to investigate biocompatibility and physical properties of the bio-engineered cornea, fabricated from type-I collagen (COL) and gelatin (Gel). Two gelatin based hydrogels cross-linked with EDC/NHS were fabricated, and their physicochemical properties such as equilibrium water content, enzymatic degradation, mechanical properties, rheological, contact angle and optical properties as well as their ability to support human bone-marrow mesenchymal stem cells (hBM-MSCs) survival were characterized. The equilibrium water content and enzymatic degradation of these hydrogels can be easily controlled by adding COL. Our findings suggest that incorporation of COL-I increases optical properties, hydrophilicity, stiffness and Young's modulus. The viability of hBM-MSCs cultured in Gel and Gel: COL was assessed via CCK-8 assay. Also, the morphology of the hBM-MSCs on the top of Gel and Gel: COL hydrogels were characterized by phase-contrast microscopy. This biocompatible hydrogel may promise to be used as artificial corneal substitutes.

Biomimetic hydrogels direct spinal progenitor cell differentiation and promote functional recovery after spinal cord injury.

OBJECTIVE: Demyelination that results from disease or traumatic injury, such as spinal cord injury (SCI), can have a devastating effect on neural function and recovery. Many researchers are examining treatments to minimize demyelination by improving oligodendrocyte availability in vivo. Transplantation of stem and oligodendrocyte progenitor cells is a promising option, however, trials are plagued by undirected differentiation. Here we introduce a biomaterial that has been optimized to direct the differentiation of neural progenitor cells (NPCs) toward oligodendrocytes as a cell delivery vehicle after SCI. APPROACH: A collagen-based hydrogel was modified to mimic the mechanical properties of the neonatal spinal cord, and components present in the developing extracellular matrix were included to provide appropriate chemical cues to the NPCs to direct their differentiation toward oligodendrocytes. The hydrogel with cells was then transplanted into a unilateral cervical contusion model of SCI to examine the functional recovery with this treatment. Six behavioral tests and histological assessment were performed to examine the in vivo response to this treatment. MAIN RESULTS: Our results demonstrate that we can achieve a significant increase in oligodendrocyte differentiation of NPCs compared to standard culture conditions using a three-component biomaterial composed of collagen, hyaluronic acid, and laminin that has mechanical properties matched to those of neonatal neural tissue. Additionally, SCI rats with hydrogel transplants, with and without NPCs, showed functional recovery. Animals transplanted with hydrogels with NPCs showed significantly increased functional recovery over six weeks compared to the media control group. SIGNIFICANCE: The three-component hydrogel presented here has the potential to provide cues to direct differentiation in vivo to encourage regeneration of the central nervous system.

Calcium deposition in photocrosslinked poly(Pro-Hyp-Gly) hydrogels encapsulated rat bone marrow stromal cells.

Reproducing the features of the extracellular matrix is important for fabricating three-dimensional (3D) scaffolds for tissue regeneration. A collagen-like polypeptide, poly(Pro-Hyp-Gly), is a promising material for 3D scaffolds because of its excellent physical properties, biocompatibility, and biodegradability. In this paper, we present a novel photocrosslinked poly(Pro-Hyp-Gly) hydrogel as a 3D scaffold for simultaneous rat bone marrow stromal cell (rBMSC) encapsulation. The hydrogels were fabricated using visible-light photocrosslinking at various concentrations of methacrylated poly(Pro-Hyp-Gly) (20-50 mg/ml) and irradiation times (3 or 5 min). The results show that the rBMSCs encapsulated in the hydrogels survived 7 days of incubation. Calcium deposition on the encapsulated rBMSCs was assessed with scanning electron microscope observation, Alizarin Red S, and von Kossa staining. The most strongly stained area was observed in the hydrogel formed with 30 mg/ml of methacrylated poly(Pro-Hyp-Gly) with 5-min irradiation. These findings demonstrate that poly(Pro-Hyp-Gly) hydrogels support rBMSC viability and differentiation, as well as demonstrating the feasibility of using poly(Pro-Hyp-Gly) hydrogels as a cytocompatible, biodegradable 3D scaffold for tissue regeneration.

New composite materials prepared by calcium phosphate precipitation in chitosan/collagen/hyaluronic acid sponge cross-linked by EDC/NHS.

Nowadays, fabrication of composite materials based on biopolymers is a rising field due to potential for bone repair and tissue engineering application. Blending of different biopolymers and incorporation of inorganic particles in the blend can lead to new materials with improved physicochemical properties and biocompatibility. In this work 3D porous structures called scaffolds based on chitosan, collagen and hyaluronic acid were obtained through the lyophilization process. Scaffolds were cross-linked by EDC/NHS. Infrared spectra for the materials were made, the percentage of swelling, scaffolds porosity and density, mechanical parameters, thermal stability were studied. Moreover, the scaffolds were used as matrixes for the calcium phosphate in situ precipitation. SEM images were taken and EDX analysis was carried out for calcium and phosphorous content determination in the scaffold. In addition, the adhesion and proliferation of human osteosarcoma SaOS-2 cells was examined on obtained scaffolds. The results showed that the properties of 3D composites cross-linked by EDC/NHS were altered after the addition of 1, 2 and 5% hyaluronic acid. Mechanical parameters, thermal stability and porosity of scaffolds were improved. Moreover, calcium and phosphorous were found in each kind of scaffold. SEM images showed that the precipitation was homogeneously carried in the whole volume of samples. Attachment of SaOS-2 cells to all modified materials was better compared to unmodified control and proliferation of these cells was markedly increased on scaffolds with precipitated calcium phosphate. Obtained materials can provide the support useful in tissue engineering and regenerative medicine.

Production of low molecular collagen peptides-loaded liposomes using different charged lipids.

To encapsulate water soluble collagen peptides, liposomes loaded with peptides were assembled using a combination of thin film hydration and ultrasonication emulsification techniques. The influence of lipid charge, duration and power of ultrasonication, and collagen peptide concentration were evaluated. Layered liposomes loaded with collagen peptides, charged lipids, chitosan (+) or low-methoxyl pectin (-) were produced using the layer-by-layer electrostatic deposition method. For the liposomes loaded with collagen peptides, the most efficient and dependable manufacturing method was variation of the ultrasonication duration, which was capable of producing smaller sizes (through increasing ultrasonication duration) and liposomes loaded with peptides with >60% encapsulation efficiency. For layered liposomes loaded with collagen peptides, charged lipids were determined to be more effective in the production of smaller liposomes than charged biopolymers. In addition, layered and non-layered liposomes loaded with peptides with a particle size <100nm were physically stable during storage, regardless of storage temperature and time.

Enzymatic synthesis of N-succinyl chitosan-collagen peptide copolymer and its characterization.

Collagen peptide (COP) grafted N-succinyl chitosan (NSC) was prepared by using microbial transglutaminase (MTGase) as biocatalyst. The catalyzed reaction displayed high efficiency, high selectivity, mild reaction condition and environmental friendliness. The degree of substitution (DS) of N-succinyl chitosan-collagen peptide (NSC-COP)depended on the reaction time, the reaction temperature, the mass ratio of COP to NSC and the mass ratio of MTGase to NSC. NSC-COP showed excellent moisture absorption and retention properties. Antioxidant activities of varying DS and concentration of NSC-COP were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl. Methylthiazol tetrazolium (MTT) assay exhibited that at a suitable concentration NSC-COP with different DS value could promote L929 mouse fibroblasts effectively. The animal experiment indicated that the wound covered with NSC-COP were completely filled with new epithelium within 2 weeks without any significant adverse side reactions. Therefore, the results may contribute to finding the application of NSC-COP in pharmaceutical and biomedical fields.

Supra-molecular assembly of a lumican-derived peptide amphiphile enhances its collagen-stimulating activity.

C16-YEALRVANEVTLN, a peptide amphiphile (PA) incorporating a biologically active amino acid sequence found in lumican, has been examined for its influence upon collagen synthesis by human corneal fibroblasts in vitro, and the roles of supra-molecular assembly and activin receptor-like kinase ALK receptor signaling in this effect were assessed. Cell viability was monitored using the Alamar blue assay, and collagen synthesis was assessed using Sirius red. The role of ALK signaling was studied by receptor inhibition. Cultured human corneal fibroblasts synthesized significantly greater amounts of collagen in the presence of the PA over both 7-day and 21-day periods. The aggregation of the PA to form nanotapes resulted in a notable enhancement in this activity, with an approximately two-fold increase in collagen production per cell. This increase was reduced by the addition of an ALK inhibitor. The data presented reveal a stimulatory effect upon collagen synthesis by the primary cells of the corneal stroma, and demonstrate a direct influence of supra-molecular assembly of the PA upon the cellular response observed. The effects of PA upon fibroblasts were dependent upon ALK receptor function. These findings elucidate the role of self-assembled nanostructures in the biological activity of peptide amphiphiles, and support the potential use of a self-assembling lumican derived PA as a novel biomaterial, intended to promote collagen deposition for wound repair and tissue engineering purposes.

Synthesis and characterization of ibandronate-loaded silica nanoparticles and collagen nanocomposites.

Non-porous bare silica nanoparticles, amine modified silica nanoparticles and mesoporous particles, were evaluated as carriers for sodium ibandronate. The synthesized nanoparticles were characterized by SEM, TEM, DLS and porosity. Then, their capacity to incorporate a bisphosphonate drug (sodium ibandronate) and the in vitro release behavior was analyzed by capillary electrophoresis. Mesoporous and amine-modified particles showed higher levels of drug incorporation, 44.68 mg g(-1) and 28.90 mg g(-1), respectively. The release kinetics from the two types of particles was similar following a first order kinetics. However, when these particles were included into collagen hydrogels only mesoporous nanoparticles had a sustained release for over 10 days. The biocompatibility of mesoporous particles towards Saos-2 cells was also evaluated by the MTT assay observing an increase in cell viability for concentrations lower than 0.6 mg ml(-1) of particles and a decrease for concentrations over 1.2 mg ml(-1). Furthermore, when these particles were incubated with mesenchymal cells it was observed that they had the capacity to promote the differentiation of the cells with a significant increase in the alkaline phosphatase activity.

Synthesis and characterization of monodisperse poly(ethylene glycol)-conjugated collagen pentapeptides with collagen biosynthesis-stimulating activity.

Although collagen pentapeptide (Lys-Thr-Thr-Lys-Ser, KTTKS) has received a great deal of attention owing to its collagen biosynthesis-stimulating effects, its enzymatic instability in the skin is an obstacle to effective topical application. PEGylation is a useful approach for improving the chemical and biological stability of peptides. However, the polydispersity of poly(ethylene glycol) (PEG) produces conjugates with different molecular sizes, which may create difficulties in chemical characterization and purity control, and in variability of biological properties. To overcome these difficulties, monodisperse PEG was site-specifically conjugated to the N-terminal amine of KTTKS to produce a single molecular conjugate, enabling more complete chemical characterization and more exact product specifications. PEG-KTTKS conjugates prepared using monodisperse PEG with two different molecular weights, monodisperse PEG220 and PEG572, were characterized by mass spectrometry. These monodisperse PEG-KTTKS conjugates showed no cytotoxicity (1-100 µM) and stimulated collagen biosynthesis in human skin fibroblasts. They also had high stability against proteolytic enzymes in rat skin. This study demonstrates the usefulness of monodisperse PEG for preparing chemically defined conjugates and suggests that monodisperse PEG-KTTKS would be a good candidate for use as a collagen biosynthesis-stimulating agent.

Radiation cross-linked collagen/dextran dermal scaffolds: effects of dextran on cross-linking and degradation.

Ionizing radiation effectively cross-links collagen into network with enhanced anti-degradability and biocompatibility, while radiation-cross-linked collagen scaffold lacks flexibility, satisfactory surface appearance, and performs poor in cell penetration and ingrowth. To make the radiation-cross-linked collagen scaffold to serve as an ideal artificial dermis, dextran was incorporated into collagen. Scaffolds with the collagen/dextran (Col/Dex) ratios of 10/0, 7/3, and 5/5 were fabricated via (60)Co γ-irradiation cross-linking, followed by lyophilization. The morphology, microstructure, physicochemical, and biological properties were investigated. Compared with pure collagen, scaffolds with dextran demonstrated more porous appearance, enhanced hydrophilicity while the cross-linking density was lower with the consequence of larger pore size, higher water uptake, as well as reduced stiffness. Accelerated degradation was observed when dextran was incorporated in both the in vitro and in vivo assays, which led to earlier integration with cell and host tissue. The effect of dextran on degradation was ascribed to the decreased cross-linking density, looser microstructure, more porous and hydrophilic surface. Considering the better appearance, softness, moderate degradation rate due to controllable cross-linking degree and good biocompatibility as well, radiation-cross-linked collagen/dextran scaffolds are expected to serve as promising artificial dermal substitutes.

Injectable biodegradable hybrid hydrogels based on thiolated collagen and oligo(acryloyl carbonate)-poly(ethylene glycol)-oligo(acryloyl carbonate) copolymer for functional cardiac regeneration.

Injectable biodegradable hybrid hydrogels were designed and developed based on thiolated collagen (Col-SH) and multiple acrylate containing oligo(acryloyl carbonate)-b-poly(ethylene glycol)-b-oligo(acryloyl carbonate) (OAC-PEG-OAC) copolymers for functional cardiac regeneration. Hydrogels were readily formed under physiological conditions (37°C and pH 7.4) from Col-SH and OAC-PEG-OAC via a Michael-type addition reaction, with gelation times ranging from 0.4 to 8.1 min and storage moduli from 11.4 to 55.6 kPa, depending on the polymer concentrations, solution pH and degrees of substitution of Col-SH. The collagen component in the hybrid hydrogels retained its enzymatic degradability against collagenase, and the degradation time of the hydrogels increased with increasing polymer concentration. In vitro studies showed that bone marrow mesenchymal stem cells (BMSCs) exhibited rapid cell spreading and extensive cellular network formation on these hybrid hydrogels. In a rat infarction model, the infarcted left ventricle was injected with PBS, hybrid hydrogels, BMSCs or BMSC-encapsulating hybrid hydrogels. Echocardiography demonstrated that the hybrid hydrogels and BMSC-encapsulating hydrogels could increase the ejection fraction at 28 days compared to the PBS control group, resulting in improved cardiac function. Histology revealed that the injected hybrid hydrogels significantly reduced the infarct size and increased the wall thickness, and these were further improved with the BMSC-encapsulating hybrid hydrogel treatment, probably related to the enhanced engraftment and persistence of the BMSCs when delivered within the hybrid hydrogel. Thus, these injectable hybrid hydrogels combining intrinsic bioactivity of collagen, controlled mechanical properties and enhanced stability provide a versatile platform for functional cardiac regeneration.

Effects of material and surface functional group on collagen self-assembly and subsequent cell adhesion behaviors.

Collagen fibrous network not only provides structural support for cells but also serves as critical environment modulating various cell functions. Various factors would influence the collagen self-assembly but the effect of substrate surface on such process has been rarely studied. Here we examined the effects of materials (Ti and hydroxyapatite) and their surface characteristics (with and without the enrichment of hydroxyl group) on collagen self-reconstitution and fibrous network formation, and on subsequent cell adhesion and cytoskeleton organization of mesenchymal stem cells (MSCs). For both Ti and hydroxyapatite (HA) substrates, the enrichment of hydroxyl group (OH) on substrate surfaces promoted the collagen self-reconstitution and facilitated the formation of the fibrous network after 4h immersion in phosphate buffer solution (PBS), while all samples showed clear fibrous network formation after 2 day soaking in PBS. Compared with the Ti surfaces, the HA surfaces facilitated the self-reconstitution of collagen, leading to a more mature fibrous network with a twisted structure and enhanced lateral aggregation of fibrils. The fibrous network difference resulted in different behaviors of the subsequent MSC adhesion and spreading. The MSCs had the best adhesion and cytoskeleton organization on the OH enriched HA surface with collagen modification. Our results suggested that both the material selection and the hydroxyl group significantly influenced the collagen self-assembly and fibrous network formation and, as a result, the subsequent cell adhesion behaviors.

Chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines formed by cross-link of bone and synovium collagen.

This review focuses on the chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines (Pyds), fluorescent collagen cross-links, with a pyridinium salt structure. Pyds derive from the degradation of bone collagen and have attracted attention for their use as biochemical markers of bone resorption and to assess fracture risk prediction in persons suffering from osteoporosis, bone cancer and other bone or collagen diseases. We consider and critically discuss all reported syntheses of free and glycosylated Pyds evidencing an unrevised chemistry, original and of general utility, analysis of which allows us to also support a previously suggested non-enzymatic formation of Pyds in collagen better rationalizing and justifying the chemical events.

Development of a salmon-derived crosslinked atelocollagen sponge disc containing osteogenic protein-1 for articular cartilage regeneration: in vivo evaluations with rabbits.

BACKGROUND: We have developed crosslinked salmon-derived atelocollagen sponge, which has a denaturation temperature of 47 degrees Celsius. The purpose of this study is to evaluate the fundamental in vivo efficacy of the osteogenic protein (OP) -1 containing salmon-derived collagen sponge disc (SCS) on cartilage regeneration, using a rabbit model. METHODS: A total of 24 rabbits were used in this study. In each animal, a full-thickness osteochondral defect was created in each femoral trochlea. Then, each 12 rabbits were randomly divided into the two groups. In Group I, an OP1-SCS disc was implanted into the defect in the right knee. In Group II, a SCS disc without OP-1 was implanted into the defect in the right knee. A control group of 12 rabbits was assembled from randomly-selected left knees from among the first two groups. In Group-III, we applied no treatment for a defect in the left knee to obtain the untreated control. All rabbits were sacrificed at 12 weeks after surgery. In each group, 10 animals were used for histological and immunohistological evaluations, and the remaining 2 were used for real-time polymerase chain reaction (PCR) analyses. RESULTS: In Group I, a regenerated cartilage tissue rich in proteoglycan and type-2 collagen was found at 12 weeks, although the width was thicker than that of Group II. In Group II, the defect was filled with thick inhomogeneous tissues, including cartilage, fibrous, and bone tissues at 12 weeks. Concerning the gross observation and histological scores at 12 weeks, the ANOVA showed significant differences (p < 0.0001, and p < 0.0001, respectively). The post-hoc test indicated that the gross observation and histological scores of Group I was significantly greater than those of Groups II (p = 0.035, and p = 0.0104, respectively) and III (p < 0.0001, and p < 0.0001, respectively), while Group II was significantly greater than Group III (p = 0.0069, and p = 0.005, respectively). The real time PCR analysis showed that gene expression of type-2 collagen and aggrecan of Group I was greater than that of Group II. CONCLUSIONS: The present study clearly demonstrated that the implantation of the OP1-SCS disc without any cultured cells may induce spontaneous hyaline-like cartilage regeneration to greater degrees than implantation of only the salmon-derived collagen sponge disc.