Narrow Results

Rotator cuff repair (RCR) is a crucial surgical procedure, but has unacceptable mechanical failure rates between 25–60%. Examining supplemental synergistic interventions, such as biological augmentations (ex: growth factors) to improve fibrocartilage formation rather than scar tissue formation, would make tears more amenable to surgical repair. Due to the large number of agents and application methods (and times), improved techniques are needed for assessing RCR in animals. In particular, high-resolution real-time imaging is needed to guide tissue engineering in animal models. Optical coherence tomography (OCT) is well suited for this role, with resolutions 25 × greater than any clinical imaging modality and an ability to identify organized collagen with polarization sensitive techniques. For example, it can determine severe collagen depletion in visually normal tendons. The images here show the first OCT and PS-OCT of the rotator cuff in male Wistar rats. The structure of the supraspinatus tendon, enthesis, and humerus are well defined. For histological comparison, this sample was stained with both Masson's Trichrome, to expose any structural abnormalities, and Picrosirius Red, to determine collagen content using a polarization filter. OCT studies offer the potential of understanding RCR failure mechanisms and potential tissue altering agents, substantially impacting outcomes.

Cartilage can redistribute human body’s daily loads and decrease the friction force in the diarthrodial joints. However, it may be injured due to trauma, sports injury, biomechanical imbalance, and genetic disease. Microfracture (MF), osteochondral autograft transplantation (OAT), and autologous chondrocyte implantation (ACI) are the most common treatment procedures in the hospital. Recently, the concept of tissue engineering involving the combination of cells, scaffolds, and bioactive signals has inspired researchers. Our team of researchers synthesized a tri-copolymer from biological polymer by using gelatin, chondroitin-6-sulfate, and hyaluronic acid through cross-linking reaction. Lacuna formation could be seen in the tri-copolymer surrounding the chondrocytes, and some newly formed glycosaminoglycan was found in the engineered cartilage. Considering the dedifferentiation possibility of chondrocyte, bone marrow mesenchymal stem cells (BMSCs) become an ideal cell source for cartilage tissue regeneration, since they can be easily harvested from adult tissue, and be expanded in vitro. In an in-vivo porcine pilot study, the results showed that the defect site could be regenerated by BMSCs/collagen gel, and is formed with fibro/hyaline mixed cartilage tissue after implantation for six months. Several clinical studies using BMSCs for cartilage defect treatment were also conducted recently; clinical outcomes such as IKDC, Lysholm, and Tegner scores improved when the cartilage defects were repaired by several millions of mesenchymal stem cells, and there is no tumor formation after being treated with BMSCs during the 10-year follow-up. Moreover, recently a commercial BMSCs/collagen gel composite for cartilage repair was developed in Taiwan and clinical trial was conducted in 2008; the results showed that there is an improvement in IKDC and MRI scores during the nine-year follow-up. It seems that using an engineered cartilage made from BMSCs/collagen gel for cartilage defect treatment is a promising method.

The chemical reactions and physical effects involved in the cessation of bone formation with age, the formation of blood and other cells in bone marrow plus the development of osteoporosis and the link of the latter to anaemia and diabetes are reconsidered with respect to the physical and biochemical conditions present in the human body.

The present study was performed to establish whether intact human tendons exhibit time-dependent tensile properties, as they do in the in vitro state. Measurements were taken in seven men and involved ultrasound-based recording of the gastrocnemius tendon elongation during three sets of five repeated isometric plantarflexion contractions elicited by tetanic electrical stimulation. The plantarflexion moment corresponding to the tendon elongation in the fifth contraction presented a pattern dependent on the voltage applied: it was approximately constant when applying 50% of maximal voltage, but it decreased curvilinearly as a function of contraction number when applying 70 and 100% of maximal voltage, reaching in the fifth contraction 84% of the plantarflexion moment corresponding to the elongation examined in the first contraction. These results suggest that, once a threshold tendon elongation is undergone, in vivo tendons may exhibit substantial viscoelasticity. The present findings have implications for muscle and joint function and need to be accounted for by musculoskeletal models.

There have been no previous reports of tendon tissue engineering using mesenchymal stem cells (MSCs) with regard to quantitative evaluation of protein expression levels and observation of derived extracellular matrix (ECM) state. Therefore, we approached tendon tissue engineering from both perspectives. Human bone marrow MSCs (hBMSCs) were subjected to 8% or 10% cyclic stretching at 1 Hz to promote differentiation into tenocytes and ECM production. The type I collagen (Col I) and Tenascin-C (Tnc) protein expression levels were evaluated quantitatively by enzyme-linked immunosorbent assay (ELISA). Confocal fluorescence microscopy was employed to observe the derived ECM state. Col I state derived from 10%-stretched cells as ECM was elongated like actual tendon ECM, although the quantitative protein expression levels were slightly higher in 8%-stretched cells. The results suggested that the optimal uniaxial stretching ratio was different between protein expression levels and derived ECM state. Therefore, it is important to pay attention to both protein expression levels and ECM state in tendon tissue engineering.

Bone is a multiscale combination of collagen molecules merged with mineral crystals. Its high rigidity and stability stem amply from its polymeric organic matrix and secondly from the connections established between interdifferent and intradifferent scale components through cross-links. Several studies have shown that the cross-links inhibition results in a reduction in strength of bone but they do not quantify the degree to which these connections contribute to the bone rigidity and toughness. This report is classified among the few works that measure the cross-links multiscale impact on the ultrastructure bone mechanical behavior.

This work aims firstly to study the effect of cross-links at the molecule scale and secondly to gather from literature studies results handling with cross-links effects on the other bone ultrastructure scales in order to reveal the multiscale effect of cross-links. This study proves that cross-links increasing number improves the mechanical performance of each scale of bone ultrastructure. On the other hand, cross-links have a multiscale contribution that depends on its rank related to existing cross-links connecting the same geometries and it depends on mechanical characteristics of geometries connected.

Pathological analysis as well as biomechanical methods are powerful approaches for collagen assessment, which plays an important role in understanding the wound healing process and choosing a treatment method in clinical situations. Due to the limitations of preparing and evaluating pathological images, this study was designed to establish a machine learning technique to predict the wound collagen content through its biomechanical parameters. For this purpose, the artificial neural network (ANN) and adaptive neuro-fuzzy inference system (ANFIS) were compared. The wound was created with an incision on the back of 30 male BALB/c mice. On the 7th and 14th days, animals were sacrificed and 60 wound tissue samples were evaluated using histopathological and biomechanical methods to quantify the amount of collagen and wound tensile strength to feed the ANN and ANFIS developed models. Based on the results, both models have appropriate performance to predict the wound collagen content. However, the comparison of coefficient of determination ($R^2$) and root mean square error (RMSE) for testing dataset revealed that ANN ($R^2=0.95$, $\mathrm{\text{RMSE}}=0.29$) had more prediction capability than ANFIS ($R^2=0.84$, $\mathrm{\text{RMSE}}=0.87$). As a decision support system, ANN model could assist in the evaluation of wound healing process with collagen values prediction.

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.