Bioprinting of Collagen: Considerations,Potentials, and Applications

Collagen is the most abundant extracellular matrix protein that is widely used in tissue engineering (TE). There is little research done on printing pure collagen. To understand the bottlenecks in printing pure collagen, it is imperative to understand collagen from a bottom‐up approach. Here it is aimed to provide a comprehensive overview of collagen printing, where collagen assembly in vivo and the various sources of collagen available for TE application are first understood. Next, the current printing technologies and strategy for printing collagen‐based materials are highlighted. Considerations and key challenges faced in collagen printing are identified. Finally, the key research areas that would enhance the functionality of printed collagen are presented.     

Collagen-like peptides and peptide–polymer conjugates in the design of assembled materials

Collagen is the most abundant protein in mammals, and there has been long-standing interest in understanding and controlling collagen assembly in the design of new materials. Collagen-like peptides (CLPs), also known as collagen-mimetic peptides (CMPs) or collagen-related peptides (CRPs), have thus been widely used to elucidate collagen triple helix structure as well as to produce higher-order structures that mimic natural collagen fibers. This mini-review provides an overview of recent progress on these topics, in three broad topical areas. The first focuses on reported developments in deciphering the chemical basis for collagen triple helix stabilization, which we review not with the intent of describing the basic structure and biological function of collagen, but to summarize different pathways for designing collagen-like peptides with high thermostability. Various approaches for producing higher-order structures via CLP self-assembly, via various types of intermolecular interaction, are then discussed. Finally, recent developments in a new area, the production of polymer–CLP bioconjugates, are summarized. Biological applications of collagen contained hydrogels are also included in this section. The topics may serve as a guide for the design of collagen-like peptides and their bioconjugates for targeted application in the biomedical arena.     

Characterization of keratin–collagen 3D scaffold for biomedical applications

Fabrication of keratin–collagen (KC) 3D scaffold with improved thermal denaturation rate is reported. In vitro application of (KC) scaffold stimulates basic extra cellular matrix constituents. KC Scaffold considerably reduced undesirable properties of both collagen and keratin while collagen incorporation reduces the fragility with increases of strength and flexibility in the scaffold. In addition to this, the scaffold showed homogenous well‐interconnected pores in the range of 10–100 µm when observed in scanning electron microscope. Usage of keratin in KC scaffold offers increased biodegradation rate and higher denaturation rate in addition to its rapid cell growth with normal morphology ultimately reaching cell population of 3.9–9.7 million per cm³ after 48 hr in KC scaffold. Circular dichroism (CD) and Fourier transform spectroscopy (FT‐IR) of KC showed presence of helical structure of collagen and ß‐turns of keratin confirming retention of native structures of both the proteins KC scaffold showed good swelling behavior and water uptake. Our study strongly supports the superidity of KC scaffold over the collagen or keratin when they are independently used for tissue engineering applications. Copyright © 2011 John Wiley & Sons, Ltd.     

Raman spectroscopy monitors adverse bone sequelae of cancer radiotherapy

Raman spectroscopy provides information on bone chemical composition and structure via widely used metrics including mineral to matrix ratio, mineral crystallinity and carbonate content, collagen crosslinking ratio and depolarization ratios. These metrics are correlated with bone material properties, such as hardness, plasticity and Young''s modulus. We review application of Raman spectroscopy to two important irradiated animalmodels: the mouse tibia, amodel for damage to cortical bone sites including the rib (breast cancer) and to healthy tissue adjacent to extremity sarcomas, and the rat mandible, a model for radiation damage in head and neck cancer radiotherapy. Longitudinal studies of irradiated mouse tibia demonstrate that radiation-induced matrix abnormalities can persist even 26 weeks postradiation. Polarized Raman spectroscopy shows formation of more ordered orientation of both mineral and collagen. At 8 weeks post-radiation, irradiated rat hemimandible exhibits transient hypermineralization, increased collagen cross-linking and decreased depolarization ratios of mineral and collagen. A standard radioprotectant, amifostine, mitigates rat mandible radiation damage, with none remaining detectable 18 weeks post-radiation. Already a powerful tool to monitor radiation damage, Raman spectroscopy may be important in development of new radiotherapy protocols and radioprotective agents. Further in vivo studies of radiation effects on the rodent models are underway, as are development of methodologies for eventual use in human subjects.     

Structure and conformation of intramolecularly cross-linked collagen

This work reports the effects of cross-linking agent like formaldehyde (HCHO) and glutaraldehyde (GA) on monomeric collagen and its conformational stability using circular dichroism. The nature of the bonds formed and the stability of the cross-links introduced vary with the aldehyde. The amount of HCHO required is four times greater than GA to induce similar changes in the molar ellipticity. This is due to the structural changes of collagen-aldehyde reaction. The change in the molar ellipticity and Rpn ratio for HCHO and GA treated collagen suggest possible aggregation of collagen molecule. The relative viscosity of native and aldehyde treated collagen was measured and correlated to the changes seen in the CD spectra. The small amount of aldehydes are needed to induce changes in the conformational stability of collagen. This can be applied to the biomaterial and biomedical application.     

Effects of botulinum toxin type a on collagen deposition in hypertrophic scars

A recent study reported that Botulinum toxin type A (BTXA) could inhibit the growth of hypertrophic scars and improve their appearance. However, the mechanism of BTXA's action on hypertrophic scars is still unknown. Some in vitro studies had shown BTXA could alleviate hypertrophic scars by acting on the biological behavior of fibroblasts, but there are few in vivo experiments, especially animal model experiments, supporting these findings. The aim of the study reported herein was to investigate the effect of BTXA on collagen deposition on hypertrophic scars in a rabbit ear model and partially clarify the mechanism of BTXA on the hypertrophy of scars. The rabbit hypertrophic scar model was used and eight rabbits were employed. BTXA was injected into the hypertrophic scar tissue of one ear; and the other ear in the same rabbit was the control without BTXA injection. The scar thickness and deposition of collagen was examined through immune histochemistry including haematoxylin and eosin (H&E) and Masson trichrome staining. The thicknesses of hypertrophic scars in the BTXA treatment group were obviously lower than in the control groups (P < 0.01). H&E and Masson staining showed that collagen fibers were stained blue. Compared with the treatment group, the collagen fibers were thicker and the arrangement of collagen fibers were disordered in the control group. This study used the rabbit ear model of hypertrophic scars to assess the effects of BTXA on scar hypertrophy. The application of BTXA may be useful for inhibiting hypertrophic scars.     

皮革固体废弃物的高值转化

传统的制革工业在生产过程中要产生原皮重60%以上的固体废弃物，由于没有充分利用，成为造成制革工业污染严重的重要原因之一，随着资源，环境等全球性生态问题的日益严峻，制革废弃物的资源化，特别是高值转化，已成为国内外关注的重要课题，本文对作为天然的生物质资源的胶原适用于食品，医药和人妆品的属性及其应用以及从制革皮边角废弃物中提取胶原的方法作了简要论述。     

Collagen-Derived Biomaterials in Bone and Cartilage Repair

Summary: We have analyzed a number of collagen-derived biomaterials for the matrix- induced and assisted bone and cartilage tissue regeneration. These include the Small intestine submuosa (SIS) Restor ™, ACI-Maix collagen membrane, Chondro- Gide collagen membrane, Permacol collagen Ossix and lycoll collagen membrane and five types of collagen-based marine sponge skeletons. Certain characteristics of different scaffold materials with comparable chemical composition may vary significantly. This variation may have a relevant impact on the suitability of the scaffolds for bone and cartilage regeneration. It suggests that the ACI-Maix® membrane is the best available collagen-derived material for an MACI®/MACT® application. In addition, the study of marine sponge indicates that the collagenous fibre skeleton of marine sponges provides a suitable bioscaffold for bone regeneration, as it supports the adhesion, migration and proliferation of osteoblasts in vitro.     

Conformational dynamics accompanying the proteolytic degradation of trimeric collagen I by collagenases

Collagenases are the principal enzymes responsible for the degradation of collagens during embryonic development, wound healing, and cancer metastasis. However, the mechanism by which these enzymes disrupt the highly chemically and structurally stable collagen triple helix remains incompletely understood. We used a single-molecule magnetic tweezers assay to characterize the cleavage of heterotrimeric collagen I by both the human collagenase matrix metalloproteinase-1 (MMP-1) and collagenase from Clostridium histolyticum. We observe that the application of 16 pN of force causes an 8-fold increase in collagen proteolysis rates by MMP-1 but does not affect cleavage rates by Clostridium collagenase. Quantitative analysis of these data allows us to infer the structural changes in collagen associated with proteolytic cleavage by both enzymes. Our data support a model in which MMP-1 cuts a transient, stretched conformation of its recognition site. In contrast, our findings suggest that Clostridium collagenase is able to cleave the fully wound collagen triple helix, accounting for its lack of force sensitivity and low sequence specificity. We observe that the cleavage of heterotrimeric collagen is less force sensitive than the proteolysis of a homotrimeric collagen model peptide, consistent with studies suggesting that the MMP-1 recognition site in heterotrimeric collagen I is partially unwound at equilibrium.     

Use of biospecific interactions of collagen, fibronectin and their fragments in affinity chromatography.

Various aspects of the application of fibronectin-collagen biospecific interactions in affinity chromatography are described. A new biospecific method for one-stage isolation of collagen peptides containing fibronectin-binding sites is proposed. The alpha 1 CB7-peptide of type-I collagen cyanogen bromide cleavage was isolated by means of affinity chromatography on adsorbents containing an immobilized gelatin-binding domain (45,000 relative molecular mass) of fibronectin. The method gives highly purified preparations of alpha 1 CB7-peptide. This peptide, as well as some other collagen molecular fragments (alpha-chains, beta-components, alpha 1 CB8-peptide), were immobilized on Sepharose, and the properties of such affinity adsorbents obtained were studied. Adsorbents with immobilized alpha-chains and alpha 1 CB7-peptide had a fibronectin-binding capacity 1.5-2.0 times higher than commercial gelatin-Sepharose. Large-scale production of highly purified fibronectin from human plasma, using affinity chromatography on immobilized individual alpha-chains of collagen, was developed.     

Systematically engineered electrospun conduit based on PGA/collagen/bioglass nanocomposites: The evaluation of morphological,mechanical, and bio‐properties

Peripheral nerve injury can considerably affect the daily life of affected people through reduced function and permanent deformation of the nerve. One of the conventional treatments used for the management of the disease is the application of autograft, which is recognized as a golden standard method; however, the process of gaining access to autograft has posed a significant challenge to its use. Nerve guidance channels (conduits), which are made in different methods, can act as an alternative therapy for patients that have undergone nerve injury; but, achieving these conduits has always been a major dilemma to be applied for patients with nerve injury. In this study, a novel conduit based on polymer blend nanocomposites of polyglycolic acid (PGA), collagen, and nanobioglass (NBG) were prepared by electrospinning technique and then compared with PGA/collagen and PGA conduits that were made in previous studies. Additionally, their various properties were characterized by scanning electron microscopy (SEM), X‐ray diffraction (XRD), contact angle, dynamic mechanical thermal analysis (DMTA), tensile strength, Fourier‐transform infrared (FTIR), and the porosity and degradation. The results showed that the mechanical, chemical, biocompatibility, and biodegradability properties of PGA/collagen/NBG conduits were more favorable in comparison with other materials. According to 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and 4′,6‐diamidino‐2‐phenylindole (DAPI) staining technique, nanofibrous electrospun PGA/collagen/NBG conduits are more suitable for cell adhesion and proliferation in comparison with either PGA or PGA/collagen conduits and can have potential for nerve regeneration.     

Decomposition of Insoluble and Hard-to-Degrade Animal Proteins by Enzyme E77 and Its Potential Applications

Insoluble and hard-to-degrade animal proteins are group of troublesome proteins, such as collagen, elastin, keratin, and prion proteins that are largely generated by the meat industry and ultimately converted to industrial wastes. We analyzed the ability of the abnormal prion protein-degrading enzyme E77 to degrade insoluble and hard-to-degrade animal proteins including keratin, collagen, and elastin. The results indicate that E77 has a much higher keratinolytic activity than proteinase K and subtilisin. Maximal E77 keratinolytic activity was observed at pH 12.0 and 65 °C. E77 was also adsorbed by keratin in a pH-independent manner. E77 showed lower collagenolytic and elastinolytic specificities than proteinase K and subtilisin. Moreover, E77 treatment did not damage collagens in ovine small intestines but did almost completely remove the muscles. We consider that E77 has the potential ability for application in the processing of animal feedstuffs and sausages.     

胶原接枝改性用于制备红外低发射率涂层的研究

用甲基丙烯酸甲酯在硝酸铈铵和偶氮二异丁腈的联合引发下对胶原进行接枝共聚改性,并用制得的胶原接枝共聚物颗粒与氧化铟纳米粒子复合制成涂层.研究了接枝反应温度及萃取剂对胶原接枝共聚物及其复合物涂层的红外发射率的影响,同时对复合物涂层红外发射率的降低机理进行了初步探讨.结果表明,在反应温度为50～55℃时,先后用丙酮和水作为萃取剂,可制得粒径为40～80nm的胶原接枝共聚物颗粒,该颗粒与氧化铟纳米粒子复合后,涂层的红外发射率(8～14μm)较单一的胶原接枝共聚物和氧化铟纳米粒子的红外发射率明显降低,胶原接枝共聚物纳米颗粒和氧化铟纳米粒子之间显示出较强的复合协同效应.     

Role of solvents in stability of collagen

The influence of hydroxymethyl chain length of the solvents on collagen was established with conformational stability and thermal stability. Thermal stability of monomeric collagen and RTT fibres (rat tail tendon) treated with methanol, ethylene glycol (EG) and glycerol were reported using the melting temperature for helix-coil transition and the peak temperature for collagen-gelatin transition. Both melting temperature and peak temperature increases as the hydroxymethyl chain length increases. Conformational stability of collagen solution treated with lower and higher concentrations of methanol, ethylene glycol and glycerol indicates that aggregation of collagen molecule is more at higher concentrations of these solvents. The concentration dependence is greater for the increased number of OH groups. Since protein aggregation is associated with neuro degenerative diseases, aggregation of collagen molecule in the presence of solvents is of great importance for biomedical application.     

Photoactivated coumaryl-diazopyruvate fluorescent label for amine functional groups of tissues containing type-I collagen

The design, synthesis and application of a new fluorescent-labeling reagent for collagen has been developed as a prerequisite for the design of a photoactivated collagen-crosslinking compound for surgical wound closure. The amine groups in collagen are the targets of a rational design for a new fluorophore because natural collagen crosslinks are formed between primary (1(o)) amine groups of lysine and hydroxylysine. The availability of 1(o) amines for crosslinking in native collagenous tissues was evaluated by reacting tendon and corneal samples with o-phthalaldehyde and dansyl chloride, fluorophores commonly used for the detection of 1(o) and 2(o) amines. The resulting fluorescent collagen fibrils indicated the presence of amines in native tissue. Subsequently, a photoactivated fluorescent label for 1(o) and 2(o) amines, coumaryl gamma-amino-butyric acid diazopyruvate (CGDP), was designed and synthesized. CGDP was first used to photolabel poly-L-lysine, forming a fluorescent, covalent bond to the 1(o) amine. CGDP was then photoreacted with corneal and tendon tissue samples to produce CGDP fluorescent-labeled samples that were statistically significantly more fluorescent than were the controls. These experiments support the postulate that 1(o) or 2(o) (or both) amines in native collagenous tissues are available to serve as targets for photoactivated collagen crosslinkers for wound closure.     

Effect of the self-assembly of collagen on crystallisation of calcium carbonate in aqueous solution

In order to investigate how the self-assembly of organic matrix influences crystallisation and growth of inorganic minerals, we selected collagen as the matrix and conducted three experiments of crystallisation of CaCO₃ in different reaction systems: H₂O system, as-assembled collagen fibrils system and self-assembling of collagen system. It is found that (i) the self-assembly process of organic matrix had a remarkable effect on the morphology of inorganic minerals: CaCO₃ crystals formed in the as-assembled collagen fibrils system were global clusters and those formed in the self-assembling of collagen system appeared as interlaced networks and (ii) the organic matrix decided the polymorph of crystals: CaCO₃ crystals were calcite in the H₂O system and appeared vaterite in the collagen system. From this study, we can conclude that the self-assembly of collagen fibrils greatly affect the crystallisation and growth of CaCO₃. Such results are significant in understanding the mechanism of biomineralisation in calcified tissues in general, and useful in the synthesis of biominerals.

(a)?CaCO₃ formed in the as-assembled collagen fibrils system. (b)?CaCO₃ formed in the self-assembling of collagen monomer system.The TEM images of samples obtained in the as-assembled collagen fibrils and self-assembling of collagen monomer system, were observed, respectively. The result shows that crystals CaCO₃ formed in the as-assembled collagen fibrils system were global clusters; crystals CaCO₃ formed in the self-assembling of collagen monomer system appeared interlaced networks.     

Properties of Spreaded Collagen I Monolayers on the Surface of Aqueous tert-Butanol and n-Hexanol Solutions

Properties of the monolayers of collagen isolated from the sclera of pig's eye are studied at the air–water interface with increasing tert-butanol or n-hexanol concentrations in a subphase. In the case of aqueous n-hexanol solutions, its adsorption on the subphase surface results in the formation of mixed monolayer whose properties depend on n-hexanol concentration in the subphase and the ratio between the number of alcohol and collagen molecules in the monolayer. At higher n-hexanol surface concentration, the phase separation of the monolayer into the domains of the condensed phase of alcohol and fibrous collagen occurs. A decrease in water activity in the presence of tert-butanol leads to a drastic reduction of collagen surface activity. This effect can be explained by both the constrained collagen spreading on the surface of tert-BuOH solutions and adsorption of alcohol molecules on collagen resulting in macromolecule hydrophilization. Alcohol critical concentrations are disclosed above which collagen monolayers are not formed.     

Fluorescence characterization of the thermal stability of collagen mimic peptides

The thermal stability of triple helical structure plays a critical role in collagen biosynthesis, function and degradation. CD technique was utilized to characterize the thermal stability of synthetic collagen mimic peptides. Fluorescence spectroscopy is widely used with easy access all around the world because of its inexpensive instrumentation, low operation cost, easy operation, and high sensitivity. Here we have developed an alternative fluorescence method to detect the thermal stability of collagen mimic peptides. We have demonstrated that fluorescence spectroscopy could measure the thermal stability of collagen mimic peptides with low concentrations under different circumstances. This highly sensitive fluorescence self-quenching assay will greatly expedite the studies of sequence-dependent properties of collagen mimic peptides, and it has great potential in the application of determining the thermal stability of triple helix systems such as collagens, collectins, adiponectin, macrophage scavenger and C1q.     

Redox Potential and Antioxidant Capacity of Bovine Bone Collagen Peptides towards Stable Free Radicals,and Bovine Meat Lipids and Proteins. Effect of Animal Age,Bone Anatomy and Proteases—A Step Forward towards Collagen-Rich Tissue Valorisation

Collagen antioxidant peptides are being widely studied. However, no research has paid attention to biological parameters such as the age and anatomy of collagen-rich tissues, which can determine a change in tissue structure and composition, and then in bioactivity. Moreover, only few research works have studied and assessed peptides antioxidant activity on the food matrix. This work aimed to investigate the effect of bovine’s bone age and anatomy, and of six different enzymes, on the antioxidant activity of collagen peptides. Collagen was extracted from young and old bovine femur and tibia; six different enzymes were used for peptides’ release. The redox potential, the quenching of stable free radicals, and the antioxidant capacity on bovine meat lipids and proteins was evaluated, under heating from ambient temperature to 80 °C. Age and anatomy showed a significant effect; the influence of anatomy becomes most important with age. Each enzyme’s effectiveness toward age and anatomy was not the same. The greatest amount of peptides was released from young bones’ collagen hydrolysed with papain. The antioxidant activity was higher at higher temperatures, except for meat proteins. Assessing the effect of age and anatomy of collagen-rich tissues can promote a better application of collagen bioactive peptides.     

Reactivity of alkaline protease to keratin and collagen containing substances

The reactivity of partially purified alkaline protease fromBacillus subtilis, to keratin and collagen containing substances has been investigated. The experimentally obtained apparent values of the Michaelis-Menten constant (K_m), the maximum reaction rate (Kmax, and the energy of activation (Ea), lead to the conclusion that: