Type I collagen hydrogels as a delivery matrix for royal jelly derived extracellular vesicles.

Throughout the last decade, extracellular vesicles (EVs) have become increasingly popular in several areas of regenerative medicine. Recently, Apis mellifera royal jelly EVs (RJ EVs) were shown to display favorable wound healing properties such as stimulation of mesenchymal stem cell migration and inhibition of staphylococcal biofilms. However, the sustained and effective local delivery of EVs in non-systemic approaches - such as patches for chronic cutaneous wounds - remains an important challenge for the development of novel EV-based wound healing therapies. Therefore, the present study aimed to assess the suitability of type I collagen -a well-established biomaterial for wound healing - as a continuous delivery matrix. RJ EVs were integrated into collagen gels at different concentrations, where gels containing 2 mg/ml collagen were found to display the most stable release kinetics. Functionality of released RJ EVs was confirmed by assessing fibroblast EV uptake and migration in a wound healing assay. We could demonstrate reliable EV uptake into fibroblasts with a sustained pro-migratory effect for up to 7 d. Integrating fibroblasts into the RJ EV-containing collagen gel increased the contractile capacity of these cells, confirming availability of RJ EVs to fibroblasts within the collagen gel. Furthermore, EVs released from collagen gels were found to inhibit Staphylococcus aureus ATCC 29213 biofilm formation. Overall, our results suggest that type I collagen could be utilized as a reliable, reproducible release system to deliver functional RJ EVs for wound healing therapies.

Insight on collagen self-assembly mechanisms by coupling molecular dynamics and UV spectroscopy techniques.

Self-assembly of rat tail collagen type I was investigated by means of turbidity measurements and molecular dynamics simulations. Turbidity curves collected at different pH values show that the rate of aggregation was not linear in dependence from pH, with the fastest kinetics at pH 5.0 and the lowest at neutral pH. MD simulations were carried out on two regions with different hydropathicity, monitoring the aggregation of up to four staggered tropocollagen fragments at different ionic strength. At physiological conditions, association of lowly charged regions occurs more easily than for highly charged ones, the latter seeming to aggregate in a sequential way. The first contacts indicate for both regions that the driving force is hydrophobic, the electrostatic contribution becoming relevant at short distance. The direct inter-tropocollagen H-bonds confirm that fibrillogenesis is driven by loss of surface water from the monomers and involves in large percentage hydroxyproline residues. Low ionic strength dynamics leads to the formation of incorrect assemblies, driven by not shielded pairwise charge interactions.

Estudo da resistência cicatricial cutânea de ratos tratados com óleo de pequi (Caryocar brasiliense) / Study of skin scarring resistance of rats treated with pequi oil (Caryocar brasiliense)

Introdução: Existem vários estudos sobre a utilização do pequi (Caryocar brasiliense) no processo cicatricial; contudo, em poucos trabalhos desenvolvidos, avaliou-se resistência dos tecidos à tensão pós-tratamento. Objetivo: Analisar a tensão cicatricial em incisões cutâneas de ratos, após terapia com Caryocar brasiliense. Método: Vinte ratos Wistar, divididos em dois grupos (placebo/tratado), sofreram incisão cutânea no dorso. O grupo tratado recebeu doses diárias de óleo de Caryocar brasiliense, e o placebo aplicação de óleo mineral. Após sacrifício, em sete e quatorze dias pós-cirurgia, amostras de pele foram submetidas à análise tênsil-histológica. Resultados: Observou-se diferença significante intergrupos na força máxima de tração, assim como uma elevação da síntese de colágeno na área das lesões no grupo tratado com óleo Caryocar brasiliense. Conclusão: A terapia com óleo de Caryocar brasiliense aumenta a resistência tênsil da pele, melhorando a resposta reparacional, reduzindo riscos de deiscência e complicações pós-cirúrgicas.

Introduction: There are several studies on the use of Caryocar brasiliense in the scarring process; however, few studies have evaluated posttreatment skin tissue resistance to tension. Objective: To analyze the scar tension in skin incisions of rats after therapy Caryocar brasiliense. Method: Twenty Wistar rats were divided into two groups (placebo / rough) and suffered skin incision in dorso. The treatment group received daily dose of Caryocar brasiliense oil and the placebo group with application of mineral oil. After sacrifice, in seven fourteen days after surgery, skin samples were subjected to tensile-histological analysis. Results: There was a significant intergroup difference in the maximum strength of traction, as well as an increase in collagen synthesis in the area of lesions in the treated group. Conclusion: Treatment with oil from Caryocar brasiliense increases the tensile strength of the skin, improving the healing response and reducing the risks of dehiscence and postoperative complications.

Engineering D-Amino Acid Containing Collagen Like Peptide at the Cleavage Site of Clostridium histolyticum Collagenase for Its Inhibition.

Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen) on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD). Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins.

Methacrylation induces rapid, temperature-dependent, reversible self-assembly of type-I collagen.

Type-I collagen self-assembles into a fibrillar gel at physiological temperature and pH to provide a cell-adhesive, supportive, structural network. As such, it is an attractive, popular scaffold for in vitro evaluations of cellular behavior and for tissue engineering applications. In this study, type-I collagen is modified to introduce methacrylate groups on the free amines of the lysine residues to create collagen methacrylamide (CMA). CMA retains the properties of collagen such as self-assembly, biodegradability, and natural bioactivity but is also photoactive and can be rapidly cross-linked or functionalized with acrylated molecules when irradiated with ultraviolet light in the presence of a photoinitiator. CMA also demonstrates unique temperature-dependent behavior. For natural type-I collagen, the overall structure of the fiber network remains largely static over time scales of a few hours upon heating and cooling at temperatures below its denaturation point. CMA, however, is rapidly thermoreversible and will oscillate between a liquid macromer suspension and a semisolid fibrillar hydrogel when the temperature is modulated between 10 and 37 °C. Using a series of mechanical, scattering, and spectroscopic methods, we demonstrate that structural reversibility is manifest across multiple scales from the protein topology of the triple helix up through the rheological properties of the CMA hydrogel. Electron microscopy imaging of CMA after various stages of heating and cooling shows that the canonical collagen-like D-periodic banding ultrastructure of the fibers is preserved. A rapidly thermoreversible collagen-based hydrogel is expected to have wide utility in tissue engineering and drug delivery applications as a biofunctional, biocompatible material. Thermal reversibility also makes CMA a powerful model for studying the complex process of hierarchical collagen self-assembly.

Preparation of differently sized injectable collagen micro-scaffolds.

Collagen scaffolds have been widely used as biomaterials for tissue engineering. In general, application of scaffolds requires surgery. In this study, we describe a new and simple technique to prepare porous micro-scaffolds from type I collagen fibrils which can be injected, thus preventing surgery. The size of the micro-scaffolds could be easily controlled using sieves with varying cut-offs. EDC-NHS crosslinking was essential to stabilize the collagen micro-scaffolds. Micro-scaffolds were highly porous and could be injected through small diameter needles (18-21 gauge). Collagen micro-scaffolds may be used as injectables for the local delivery of effector molecules and/or cells, thus creating specific niches to enhance local tissue regeneration.

Electrospinning of matrigel to deposit a basal lamina-like nanofiber surface.

Schwann cell basal lamina is a nanometer-thin extracellular matrix layer that separates the axon-bound Schwann cells from the endoneurium of the peripheral nerve. It is implicated in the promotion of nerve regeneration after transection injury by allowing Schwann cell colonization and axonal guidance. Hence, it is desired to mimic the native basal lamina for neural tissue engineering applications. In this study, basal lamina proteins from BD Matrigel (growth factor-reduced) were extracted and electrospun to deposit nonwoven nanofiber mats. Adjustment of solute protein concentration, potential difference, air gap distance and flow rate produced a basal lamina-like construct with an average surface roughness of 23 nm and composed of 100-nm-thick irregular and relatively discontinuous fibers. Culture of embryonic chick dorsal root ganglion explants demonstrated that the fabricated nanofiber layer supported explant attachment, elongation of neurites, and migration of satellite Schwann cells in a similar fashion compared to electrospun collagen type-I fibers. Furthermore, the presence of nanorough surface features significantly increased the neurite spreading and Schwann cell growth. Sciatic nerve segment incubation also showed that the construct is promigratory to nerve Schwann cells. Results, therefore, suggest that the synthetic basal lamina fibers can be utilized as a biomaterial for induction of peripheral nerve repair.

Engineering of implantable cartilaginous structures from bone marrow-derived mesenchymal stem cells.

Fabrication of implantable cartilaginous structures that could be secured in the joint defect could provide an alternative therapeutic approach to prosthetic joint replacement. Herein we explored the possibility of using biodegradable hydrogels in combination with a polyglycolic acid (PGA) scaffold to provide an environment propitious to mesenchymal stem cells (MSCs) chondrogenic differentiation. We examined the influence of type I collagen gel and alginate combined with PGA meshes on the extracellular matrix composition of tissue-engineered transplants. MSCs were isolated from young rabbits, expanded in monolayers, suspended in each hydrogel, and loaded on PGA scaffolds. All constructs (n=48) were cultured in serum-free medium containing transforming growth factor beta-1, under dynamic conditions in specially designed bioreactors for 3-6 weeks. All cell-polymer constructs had a white, shiny aspect, and retained their initial size and shape over the culture period. Their thickness increased substantially over time, and no shrinkage was observed. All specimens developed a hyalin-like extracellular matrix containing glycosaminoglycans (GAGs) and type II collagen, but significant differences were observed among the three different groups. In PGA/MSCs and collagen-PGA/MSCs constructs, the cell growth phase and the chondrogenic differentiation phase of MSCs occurred during the first 3 weeks. In alginate-PGA/MSCs constructs, cells remained round in the hydrogel and cartilage extracellular matrix deposition was delayed. However, at 6 weeks, alginate-PGA/MSCs constructs exhibited higher contents of GAGs and lower contents of type I collagen. These results suggest that the implied time for the transplantation of in vitro engineered constructs depends, among other factors, on the nature of the scaffold envisioned. In this study, we demonstrated that the use of a composite hydrogel-PGA scaffold supported the in vitro growth of implantable cartilaginous structures cultured in a bioreactor system.

Rapid synthesis of a register-specific heterotrimeric type I collagen helix encompassing the integrin alpha2beta1 binding site.

We report a rapid method to synthesize cystine cross-linked heterotrimeric collagenous peptides. They can be engineered to favour one particular axial alignment of the strands, called the register of the helix. Here, the sequence of the constituent peptides contains 18 residues of "guest" collagen type I sequence flanked by N and C-terminal (Gly-Pro-Pro)5 "host" modules which ensure helicity. Further C-terminal residues include appropriately spaced cysteine residues and alanine to provide the necessary flexibility for helix formation. The cross-linking reaction and subsequent separation protocols have been designed for any inserted collagen sequence that does not contain a cysteine residue. Mass spectrometry and ion-exchange chromatography allow us to distinguish between different disulphide-bonded species and to monitor the formation of side-products. Starting peptide can be recovered simply from the reaction mixture by reduction and separation. Yields are typically 30%, working on a 10 mg scale. 15N-1H NMR and platelet adhesion studies show that the peptide heterotrimers presented here can reshuffle to cover all three axial registers. Less flexible spacers between the disulphide linkages and the helix will restrict each heterotrimer to one register only.

Intact glycation end products containing carboxymethyl-lysine and glyoxal lysine dimer obtained from synthetic collagen model peptide.

Advanced glycation end products (AGE) are accumulated in human tissues when long-lived proteins are glycated due to hyperglycemia and/or aging. In this study, we synthesized a collagen model peptide, Ac-(Pro-Hyp-Gly)(5)-Pro-Lys-Gly-(Pro-Hyp-Gly)(5)-Ala-NH(2) to investigate intact AGEs in peptides. The peptide formed a stable triple helix structure, and was subjected to glycation reactions with glucose, ribose and glyoxal. Besides carboxymethyl-lysine in the peptide, a conjugated form linked with glyoxal lysine dimer (GOLD) was detected upon treatment with glyoxal. This is the first example of intact glycation-derived dimers of peptides retaining intrinsic protein structures.

Type I collagen can function as a reservoir of basic fibroblast growth factor.

Storage and release of endogenous growth factors by the extracellular matrix (ECM) are important biological events that control tissue homeostasis and regeneration. The interaction between basic fibroblast growth factor (bFGF) and heparan sulfate proteoglycans has been extensively studied and used as a prototype model of such a system, while the lower affinity of fibrillar type I collagen for bFGF has generally been considered biologically insignificant. However, our present investigation revealed that bFGF spontaneously interacts with type I collagen solution and sponges under in vitro and in vivo physiological conditions, and is protected from the proteolytic environment by the collagen. bFGF incorporated in a collagen sponge sheet was sustainedly released in the mouse subcutis according to the biodegradation of the sponge matrix, and exhibited local angiogenic activity in a dose-dependent manner. Intramuscular injection of collagen microsponges incorporating bFGF induced a significant increase in the blood flow in the murine ischemic hindlimb, which could never have been attained by bolus injection of bFGF. These results suggest the significance and therapeutic utility of type I collagen as a reservoir of bFGF.

Characterization of the nucleation step and folding of a collagen triple-helix peptide.

Peptide T1-892 is a triple-helical peptide designed to include two distinct domains: a C-terminal (Gly-Pro-Hyp)(4) sequence, together with an N-terminal 18-residue sequence from the alpha1(I) chain of type I collagen. Folding experiments of T1-892 using CD spectroscopy were carried out at varying concentrations and temperatures, and fitting of kinetic models to the data was used to obtain information about the folding mechanism and to derive rate constants. Proposed models include a heterogeneous population of monomers with respect to cis-trans isomerization and a third-order folding reaction from competent monomer to the triple helix. Fitting results support a nucleation domain composed of all or most of the (Gly-Pro-Hyp)(4) sequence, which must be in trans form before the monomer is competent to initiate triple-helix formation. The folding of competent monomer to a triple helix is best described by an all-or-none third-order reaction. The temperature dependence of the third-order rate constant indicates a negative activation energy and provides information about the thermodynamics of the trimerization step. These CD studies complement NMR studies carried out on the same peptide at high concentrations, illustrating how the rate-limiting folding step is affected by changes in concentration. This sequence preference of repeating Gly-Pro-Hyp units for the initiation of triple-helix formation in peptide T1-892 may be related to features in the triple-helix folding of collagens.

Structural properties of a collagenous heterotrimer that mimics the collagenase cleavage site of collagen type I.

Collagens contain sequence- and conformation-dependent epitopes responsible for their digestion by collagenases at specific loci. A synthetic heterotrimer construct containing the collagenase cleavage site of collagen type I was found to mimic perfectly native collagen in terms of selectivity and mode of enzymatic degradation. The NMR conformational analysis of this molecule clearly revealed the presence of two structural domains, i.e. a triple helix spanning the Gly-Pro-Hyp repeats and a less ordered portion corresponding to the collagenase cleavage site where the three chains are aligned in extended conformation with loose interchain contacts. These structural properties allow for additional insights into the very particular mechanism of collagen digestion by collagenases.

Folding and conformational consequences of glycine to alanine replacements at different positions in a collagen model peptide.

The collagen model peptide T1-892 includes a C-terminal nucleation domain, (Gly-Pro-Hyp)(4), and an N-terminal (Gly-X-Y)(6) sequence taken from type I collagen. In osteogenesis imperfecta (OI) and other collagen diseases, single base mutations often convert one Gly to a larger residue, and T1-892 homologues modeling such mutations were synthesized with Gly to Ala substitutions in either the (Gly-Pro-Hyp)(4) domain, Gly25Ala, or the (Gly-X-Y)(6) domain, Gly10Ala. CD and NMR studies show the Gly10Ala peptide forms a normal triple-helix at the C-terminal end and propagates from the C- to the N-terminus until the Gly --> Ala substitution is encountered. At this point, triple-helix folding is terminated and cannot be reinitiated, leaving a nonhelical N-terminus. A decreased thermal stability is observed as a result of the shorter length of the triple-helix. In contrast, introduction of the Gly to Ala replacement at position 25, in the nucleation domain, shifts the monomer/trimer equilibrium toward the monomer form. The increased monomer and lower trimer populations are reflected in the dramatic decrease in triple-helix content and stability. Unlike the Ala replacement at position 10, the Ala substitution in the (Gly-Pro-Hyp)(4) region can still be incorporated into a triple-helix, but at a greatly decreased rate of folding, since the original efficient nucleation site is no longer operative. The specific consequences of Gly to Ala replacements in two distinctive sequences in this triple-helical peptide may help clarify the variability in OI clinical severity resulting from mutations at different sites along type I collagen chains.