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Hypertrophic scar and keloids have affected patients and frustrated physicians for centuries. Hypertrophic scar (HSc) and keloids are a major problem for patients who survive extensive thermal and traumatic skin injuries. HSc and other fibroproliferative disorders are associated with excessive accumulation of collagen and extracellular matrix proteins due to an imbalance between synthesis and degradation. The therapeutic management of hypertrophic scars and keloids include occlusive dressings, compression therapy, intralesional corticosteroid injections, cryosurgery, excision, radiation therapy, laser therapy, interferon therapy and other promising lesser known therapies directed at collagen synthesis. In this study we investigated the effect of phenergan (promethazine hydrochloride) as one of the most potent histamine antagonists on cell proliferation, DNA synthesis and collagen production in fibroblast isolated from human post burn hypertrophic scar, keloids and normal skin. The proliferation of normal skin fibroblast was slightly decreased but hypertrophic scar and keloids showed significant (p<0.001) level of decrease after 72 hours of phenergan (750 μM) treatment. The results of DNA synthesis also significantly (p<0.001) decreased in hypertrophic scar and keloid fibroblasts. Phenergan (1.5 mM) decreased the collagen synthesis upto 61% and 66% in HSc and keloids in comparison to normal skin fibroblast, which showed reduction of 38% after 72 hours. Improved understanding of such regulatory mechanisms may eventually be of therapeutic significance in the control of hypertrophic scar and keloids.

The development of shape and form is intrinsic to the structure and function of many biological macromolecules including tubulin, actin and collagen. Type I collagen is a major structural protein in the body, providing mechanical strength for tissues such as bone and skin. It is present in the form of fibrils which display a regular banding pattern known as D-periodicity (where D = 67 nm). Type I collagen is a long rod-like molecule (300 nm ×1.5 nm) consisting of a triple helix formed from three polypeptide chains. In vivo and in vitro studies have shown that collagen molecules self-assemble in a regular D-staggered array to form striated fibrils. Further studies have shown that the process, termed fibrillogenesis, is entropy driven. A model based on diffusion limited aggregation was used to investigate the properties of rod self-assembly. This simple model reproduced several experimentally observed features of collagen fibril morphology including a linear mass/unit length profile and a preference for tip growth.

This discussion about diagnostic tests for cancer incorporates a powerful branch of Physics namely X-ray diffraction. Although this technique was used to solve the DNA structure using the X-ray diffraction pictures of Rosalind Franklin,¹ and the structure of vitamin B12 by Dorothy Hodgkin² and hosts of other medical related structures, it is poorly understood by the general medical profession and the community at large. To the nonphysicist the patterns appear to have no relation to the results produced. It might as well be written in Greek. The well-known quote of Poincaré, the famous French mathematician and scientist, in 1885 comes to mind: "Science is built up with facts as a house is with stones. But a collection of facts is no more a science than a heap of stones is a house."

In order therefore to build a true understanding of this powerful technique it is necessary to build a firm understanding of the basic facts about this technique, so that the final results will be clear to all, as they will be held up by a firm house of knowledge. So let us take up the first stone.

Type I collagen fibrils have circular cross sections with radii mostly distributed in between 50 and 100 nm and are characterized by an axial banding pattern with a period of 67 nm. The constituent long molecules of those fibrils, the so-called triple helices, are densely packed but their nature is such that their assembly must conciliate two conflicting requirements. One is a double-twist around the axis of the fibril induced by their chirality and the other is a periodic layered organization, corresponding to the axial banding, built by specific lateral interactions. We examine here how such a conflict could contribute to the control of the radius of a fibril. We develop our analysis with the help of two geometrical archetypes: the Hopf fibration and the algorithm of phyllotaxis. The first one provides an ideal template for a twisted bundle of fibres and the second ensures the best homogeneity and local isotropy possible for a twisted dense packing with circular symmetry. This approach shows that, as the radius of a fibril with constant double-twist increases, the periodic layered organization can not be preserved without moving from planar to helicoidal configurations. Such changes of configurations are indeed made possible by the edge dislocations naturally present in the phyllotactic pattern. The distribution of those defects is such that the lateral growth of a fibril should stay limited in the observed range. Because of our limited knowledge about the elastic constants involved, this purely geometrical development stays at a quite conjectural level.

Tumor invasion, the process by which tumor cells break away from their primary tumor and gain access to vascular systems, is an important step in cancer metastasis. Most current 3D tumor invasion assays consisted of a single tumor cell embedded within an extracellular matrix (ECM). These assays taught us much of what we know today on how key biophysical (e.g., ECM stiffness) and biochemical (e.g., cytokine gradients) parameters within the tumor microenvironment guided and regulated tumor invasion. One limitation of the single tumor cell invasion assays was that it did not account for cell–cell adhesion within the tumor. In this paper, we developed a micrometer scale 3D co-culture spheroid invasion assay that recapitulated physiologically realistic tumor microenvironment and was compatible with microscopic imaging. Micrometer scale co-culture spheroids (1:1 ratio of metastatic breast cancer MDA-MB-231 and non-tumorigenic epithelial MCF-10A cells) were made using an array of microwells, and then were embedded within a collagen matrix in a microfluidic platform. Real time imaging of tumor spheroid invasion revealed that the spatial distribution of the two cell types within the tumor spheroid critically regulated tumor invasion. This work linked tumor architecture with tumor invasion and highlighted the importance of the biophysical cues within the bulk of the tumor in tumor invasion.

Collagen is an endogenous fluorophore that accounts for about 70% of all proteins of human skin, so it can be an optical marker for structural abnormalities in tissues registered by laser fluorescent diagnostics in vivo. Using the examples of such abnormalities as scars, scleroderma and basal cell carcinoma, this study shows the differences between coefficients of fluorescent contrast k_f(λ) of abnormalities from the ones for healthy tissues at fluorescent excitation wavelength 360–380 nm. It is shown that scars and dysplasia are characterized by reduced values of k_f(λ) for collagen. Due to high turbidity and phase heterogeneousness as well as variation of parameters of blood microcirculation and concentrations of other related chromophores, there is no mathematical model that precisely calculates the concentration of collagen in tissues only with the use of the value of fluorescent signal intensity. So, probably, the best marker of the pathological process is a comprehensive representation of k_f(λ) for all endogenous fluorophores, i.e., for all used visible wavelengths. In this case identification of abnormal tissues is quite possible by detecting some deviations of coefficients k_f(λ) for the optically identical and symmetrical regions of the human body.

Changes of the blood vessels and collagen are associated with the development of abnormal cervical cells. Recently, optical coherence tomography and Mueller polarization images were used to provide information regarding the presence of collagen fibers in the cervical tissue. However, most of these methods need a lot of time for image recording and are expensive. In addition, the general survey on the absorption and distribution characteristics of collagen and blood in the cervical is still lacking. In this study, we developed a colposcopy combining cross-polarized image and image processing algorithm with an efficient analytical model to map the distribution of blood and collagen in the uterine. For this system’s proof of concept, we captured and processed the case of cervical ectopy and Nabothian cyst. The results show that the distribution of blood and collagen maps matched with anatomical and physiological when compared with Lugol’s iodine images. This technology has some advantages, such as low cost, real time, and can replace the use of acetic acid or Lugol’s iodine in the future.

Tissue engineering is an innovative field of research applied to treat intestinal diseases. Engineered smooth muscle requires dense smooth muscle tissue and robust vascularization to support contraction. The purpose of this study was to use heparan sulfate (HS) and collagen coatings to increase the attachment of smooth muscle cells (SMCs) to scaffolds and improve their survival after implantation. SMCs grown on biologically coated scaffolds were evaluated for maturity and cell numbers after 2, 4 and 6 weeks in vitro and both 2 and 6 weeks in vivo. Implants were also assessed for vascularization. Collagen-coated scaffolds increased attachment, growth and maturity of SMCs in culture. HS-coated implants increased angiogenesis after 2 weeks, contributing to an increase in SMC survival and growth compared to HS-coated scaffolds grown in vitro. The angiogenic effects of HS may be useful for engineering intestinal smooth muscle.

Collagen is the most abundant natural protein found in living systems. While there is a whole family of different collagen types, each differing in sequence, the properties that make this protein so attractive as the building blocks for medical devices, are reflected largely by the unique fibrillar structure of the molecule, as well as defined functional regions that interact with the surrounding cells and other matrix components. As a commercial medical product, collagen can be part of the natural tissue used in the device, or it can be fabricated as a reconstituted product from animal or recombinant sources. Both types of uses have distinct properties that convey advantages and disadvantages to the end product. This review examines the chemistry and biology of collagen and describes some well-documented examples of collagen-based medical devices produced in one or other of these formats.

The interaction between cells and biomaterials strongly depends on the assembled structure of collagen adsorption upon the solid surface. Due to its self-assembling property, Type I collagen may aggregate and form fibrils in vivo and in vitro. This study utilizes an atomic force microscope to investigate nanometer-scale organization of adsorbed Type I collagen layers on mica and on poly(methyl methacrylate) (PMMA). We have observed various film morphologies, depending on substrate hydrophobicity and the state of collagen solution used. On mica, the atomic force microscopy (AFM) study obtains dense felt-like structures of randomly distributed assemblies. Images of network-like assemblies composed of interwoven fibrils appear on PMMA. According to the above results, we believe that these assemblies are associated at the interface rather than aggregated in the solution. This work also investigates the adsorbed collagen structure on PMMA after collagen aggregation in solution, to realize the relation between adsorption and aggregation. Consequently, the result exhibits a dendritic fibrillar structure adsorbed on PMMA, following collagen molecule aggregation, to form a fibrillar structure in the solution. This result suggests that the adsorption of aggregates preformed in the solution is preferable to collagen molecules adsorption. This research created all assembled structures of adsorbed collagen layers in nanometer-scale thickness.

Developments on tissue engineering, especially on tissue regeneration and drug delivery, demand also developments on biomaterials. Research on the preparation methods of biomaterials has exhibited remarkable advances in the recent years. Natural biomaterials, such as chitosan and collagen, or synthetic materials like poly(lactic acid) can be shaped in various forms. The parameters involved in the fabrication processes provide methodologies for control of the materials' properties, such as morphology, biodegradability, mechanical strength, and adhesion. As new applications develop for these materials, the preparation methods have to be optimized to achieve the desired material properties. These properties mostly not only mimic the conditions in the human body, but also may divert the microenvironment of cells in the diseased area in order to promote faster or guided healing and tissue regeneration. This review pays attention on some of the fabrication methods for biomaterial particulates of sizes in the micro- and nanoscale. The views expressed here focus on the many years of experience of the authors with electrostatic and ultrasonic fabrication methods. These methods are still under development and up to now can produce particulates of various sizes down to the nanometer scale with narrow size distributions. Such biomaterials that have extraordinary properties may provide ways for the development of remarkable biomedical applications.

Injectable filler, which is often applied in minimally invasive surgery, has been widely-used in facial rejuvenation. Because of its convenience, effective usage and less downtime after treatment for the patients, it becomes one of the most popular treatment methods at present. The currently available products containing collagen base have been proven to have much satisfactory safety and effectiveness, but one of its disadvantages is the lack of long-term volume persistence. We have previously prepared the reconstituted collagen fibrils with hyaluronic acid (HA) by modifying the fibril surface. This study is to evaluate the potential of these materials as injectable filler in vitro. A preparation of collagen fibrils with a diameter of 100–150 nm was used. The modification rate of HA on the fibril surface was 20%. In assessment of the biocompatibility, it was proven that the collagen fibril and HA-collagen fibril treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) had no cytotoxicity or inhibitory actions. In interactions between these materials and cells, it was found that the existence of HA could improve the migration ability of L929 fibroblasts and breast cancer cells (MDA-MB 435s). In conclusion, collagen fibril and HA-collagen fibril have the potential to be developed into an injection as a soft tissue filler for biomedical applications.

Hydroxyapatite (HAp) is the major inorganic component in human bones and well known for its biocompatibility. It has been widely prepared in many forms for orthopedic and dental applications. Color-center-doped HAps are also prevalently used as fluorescent materials. Further, HAp based photoluminescent material might be an ideal agent for bioimaging. Moreover, self-assembly of HAp in calcium- and phosphate-containing solutions gains a lot of attention because organic–inorganic HAp hybrids can be tailor-made. In this study, the author used a biomimetic process to synthesize organic–inorganic HAp nano hybrid at room temperature. The natural and synthesized polymeric templates used to prepare the organic–inorganic HAp hybrids were collagen, PEG–PLGA (di block), and PEG–PLGA–PEG (tri block), respectively. X-ray diffraction (XRD) diagrams revealed that the synthesized powders had crystalline HAp phase, and transmission electron microscope (TEM) photographs showed their nano grain structure. Characterization using XRD, TEM, and Fourier transform infrared spectrometer (FTR) indicated the existence of crystalline HAp phase and the variation in HAp shape versus different polymers in the composite powders. The measurement of photoluminescent (PL) spectra used a 325 nm He–Cd laser as source. The di HAp and tri HAp emitted light with a wide range of wavelength from 350 to 550 nm, much stronger than pure HAp. Collagen alone emitted brighter fluorescence peaked around 410 nm but was quickly quenched, while collagen–HAp composite powders emitted sustaining PL light peaking around 415 nm. The developed PL bioceramics are of great potential in bio sensing and bio optoelectronics.

In this work, we used multiphoton microscopic system for characterizing three-dimensional microstructure of collagen/chitosan polymeric scaffolds in a noninvasive fashion. Nonlinear optical signals including multiphoton autofluorescence (MAF) and second harmonic generation (SHG) derived from collagen/chitosan scaffolds were collected and analyzed. The three-dimensional porous microstructures of collagen/chitosan scaffolds were visualized by co-localized and evenly distributed MAF and SHG signals. The distribution of collagen and chitosan compositions within miscible collagen/chitosan blends cannot be either localized or differentiated simply using these nonlinear optical signals. However, the intensity of MAF signals in scaffolds was found to be markedly decreased in correlation to the supplementation of chitosan within blends, regardless of collagen/chitosan weight ratios. It therefore implied that the MAF-generating molecules within collagen being altered in miscible collagen/chitosan blends. And the SHG signals also decreased significantly in collagen/chitosan scaffolds with the supplementation of chitosan, regardless of different weight ratios. This finding supported the hypothesis regarding the miscibility of collagen/chitosan blends that triple helix structure of collagen, a proven SHG-generating microstructure, was altered in miscible collagen/chitosan blends. In conclusion, our work demonstrated that multiphoton imaging modality can be versatile for investigating three-dimensional microstructure of miscible polymeric scaffolds in a minimal invasive fashion, and may potentially be applicable in the field of tissue engineering.

Purpose: Plasmid loading into scaffolds to enhance sustained release of growth factors is an important focus of regenerative medicine. The aim of this study was to build gene-activated matrices (GAMs) and examine the bone augmentation properties. Methods: Generation 5 polyamidoamine dendrimers (G5 dPAMAM)/plasmid recombinant human bone morphogenetic protein-2 (rhBMP-2) complexes were immobilized into beta-tricalcium phosphate (β-TCP)/type I collagen porous scaffolds. After cultured with rat mesenchymal stem cells (rMSCs), transfection efficiencies were examined. The secretion of rhBMP-2 and alkaline phosphatase (ALP) were detected to evaluate the osteogenic properties. Scanning electron microscopy (SEM) was used to observe attachment and proliferation. Moreover, we applied these GAMs directly into freshly created segmental bone defects in rat femurs, and their osteogenic efficiencies were evaluated. Results: Released plasmid complexes were transfected into stem cells and were expressed, which caused osteogenic differentiations of rat mesenchymal stem cells (rMSCs). SEM analysis showed excellent cell attachment. Bioactivity of plasmid rhBMP-2 was maintained in vivo, and the X-ray observation, histological analysis and immunohistochemistry (IHC) of bone tissue demonstrated that the bone healing in segmental femoral defects was enhanced by implantation of GAMs. Conclusions: Such biomaterials offer therapeutic opportunities in critical-sized bone defects.

Collagen and noncollagenous proteins have an important role in the formation of mineral constituent of bone matrix. In this research, the morphology and phase characteristics of calcium phosphate nanoparticles in presence of collagen were investigated. The synthesis reaction was initiated by mixing H₃PO₄ as phosphorous source and CaCl₂ as calcium source and type I collagen. Collagen concentration in suspension and Ca to P ratio was 1% and 1.67, respectively. The samples (with collagen and without collagen), were heat treated at 600°C and characterized by X-Ray diffraction (XRD), Fourier transformation infrared (FTIR) and scanning electron microscopy (SEM). More smaller and flake-like shape particles were observed in the SEM images of sample synthesized in the presence of collagen compared to the control sample which was constituted of larger granular particles. The XRD results revealed that the synthesized mineral powders with collagen were composed of hydroxyapatite and octacalcium phosphate. P–O and OH characteristic peaks were identified in FTIR spectra. In hybrid sample, the shift of amides band, revealed the electrostatic interactions between calcium phosphate ions and carboxyl or amino groups of collagen fibrils. The Ca to P molar ratio for sample with collagen was 1.9. It was found that the sample synthesized in the presence of collagen has a similar microstructure to natural bone.

Background and aim: Healing of fire-induced wounds has been still a challenge in clinical issues. The aim of this study was to fabricate a nanofibrous poly (L-lactic acid)/collagen (PLLA/COL) scaffold with sustained release of aloe vera (AV) gel using a chitosan (CT)-coated layer for skin tissue engineering applications. Material and methods: Morphology, porosity, tensile strength, hydrophilicity, degradation rate, water vapor permeability and water uptake ratio of the scaffold were characterized. The behaviors of mouse fibroblasts (L929) were evaluated on the scaffold. Results: We observed that although the porosity of the scaffold was decreased, other characteristics were enhanced by coating a CT layer. The scaffold supports attachment, viability and proliferation of mouse fibroblasts. Conclusion: Consequently, the PLLA/COL scaffold coated with CT for sustained release of AV gel can be considered as a desirable scaffold for skin tissue engineering.

In this chapter we discuss how to engineer 3D pulmonary tissue constructs in vitro using primary isolates of foetal mouse distal lung cells. When cultured in hydrogel-based 3D constructs, the mixed cell population, comprised epithelial, mesenchymal and endothelial cells, organised into alveolar forming unit (AFU)-like sacculated structures, which, in terms of morphology and cytodifferentiation, were reminiscent of native distal lung. By using a unique, serum-free medium supplemented with a cocktail of tissue-specific growth factors, we were able to induce concomitant alveolisation and neovascularisation when culturing the cells in the hydrogels, but not in scaffolds composed of synthetic polymers. Our data suggest that our in vitro model is capable of recapitulating the parallel morphogenesis of epithelial and endothelial pulmonary tissue components, which may occur through dynamic paracrine interactions. These results also stress the importance of the complex input from co-cultures, tissue-specific growth factors and integrin signalling for successful tissue engineering in vitro. In a mouse model in vivo, incorporation of the primary lung cell isolates into Matrigel plugs, implanted either subcutaneously (s.c.), or under the kidney capsule, leads to the formation of sacculated AFUs in close proximity to patent capillaries. Effective functional vascularisation, however, was only observed upon addition of angiogenic growth factors to the scaffolds and their controlled release over time. Use of a fluorescent cell tracker confirmed that the neovessels in the constructs comprised endothelial cells from both the host and the grafts. These data demonstrate that it is feasible to generate vascularised pulmonary tissue constructs in vivo with proper epithelial differentiation, and that the degree of vascularisation may be manipulated by incorporating the release of an angiogenic factor within the construct.

Tumor invasion, the process by which tumor cells break away from their primary tumor and gain access to vascular systems, is an important step in cancer metastasis. Most current 3D tumor invasion assays consisted of a single tumor cell embedded within an extracellular matrix (ECM). These assays taught us much of what we know today on how key biophysical (e.g., ECM stiffiness) and biochemical (e.g., cytokine gradients) parameters within the tumor microenvironment guided and regulated tumor invasion. One limitation of the single tumor cell invasion assays was that it did not account for cell–cell adhesion within the tumor. In this chapter, we developed a micrometer scale 3D co-culture spheroid invasion assay that recapitulated physiologically realistic tumor microenvironment and was compatible with microscopic imaging. Micrometer scale co-culture spheroids (1:1 ratio of metastatic breast cancer MDA-MB-231 and non-tumorigenic epithelial MCF-10A cells) were made using an array of microwells, and then were embedded within a collagen matrix in a microfluidic platform. Real time imaging of tumor spheroid invasion revealed that the spatial distribution of the two cell types within the tumor spheroid critically regulated tumor invasion. This work linked tumor architecture with tumor invasion and highlighted the importance of the biophysical cues within the bulk of the tumor in tumor invasion.