The Effect of Pore Directionality of Collagen Scaffolds on Cell Differentiation and In Vivo Osteogenesis.

Although many bone substitutes have been designed and produced, the development of bone tissue engineering products that mimic the microstructural characteristics of native bone remains challenging. It has been shown that pore orientation within collagen scaffolds influences bone matrix formation by the endochondral route. In addition, that the unidirectional orientation of the scaffolds can limit the growth of blood vessels. However, a comparison between the amount of bone that can be formed in scaffolds with different pore orientations in addition to analyzing the effect of loading osteogenic and proangiogenic factors is still required. In this work we fabricated uni- and multidirectional collagen sponges and evaluated their microstructural, physicochemical, mechanical and biological characteristics. Although the porosity and average pore size of the uni- and multidirectional scaffolds was similar (94.5% vs. 97.1% and 260 µm vs. 269 µm, respectively) the unidirectional sponges had a higher tensile strength, Young's modulus and capacity to uptake liquids than the multidirectional ones (0.271 MPa vs. 0.478 MPa, 9.623 MPa vs. 3.426 MPa and 8000% mass gain vs. 4000%, respectively). Culturing of rat bone marrow mesenchymal stem cells demonstrated that these scaffolds support cell growth and osteoblastic differentiation in the presence of BMP-2 in vitro, although the pore orientation somehow affected cell attachment and differentiation. The evaluation of the ability of the scaffolds to support bone growth when loaded with BMP-2 or BMP-2 + VEGF in an ectopic rat model showed that they both supported bone formation. Histological analysis and quantification of mineralized matrix revealed that the pore orientation of the collagen scaffolds influenced the osteogenic process.

Adjustable conduits for guided peripheral nerve regeneration prepared from bi-zonal unidirectional and multidirectional laminar scaffold of type I collagen.

Shortness of donor nerves has led to the development of nerve conduits that connect sectioned peripheral nerve stumps and help to prevent the formation of neuromas. Often, the standard diameters of these devices cannot be adapted at the time of surgery to the diameter of the nerve injured. In this work, scaffolds were developed to form filled nerve conduits with an inner matrix with unidirectional channels covered by a multidirectional pore zone. Collagen type I dispersions (5 mg/g and 8 mg/g) were sequentially frozen using different methods to obtain six laminar scaffolds (P1 to P5) formed by a unidirectional (U) pore/channel zone adjacent to a multidirectional (M) pore zone. The physicochemical and microstructural properties of the scaffolds were determined and compared, as well as their biodegradability, residual glutaraldehyde and cytocompatibility. Also, the Young's modulus of the conduits made by rolling up the bizonal scaffolds from the unidirectional to the multidirectional zone was determined. Based on these comparisons, the proliferation and differentiation of hASC were assessed only in the P3 scaffolds. The cells adhered, aligned in the same direction as the unidirectional porous fibers, proliferated, and differentiated into Schwann-like cells. Adjustable conduits made with the P3 scaffold were implanted in rats 10 mm sciatic nerve lesions to compare their performance with that of autologous sciatic nerve grafted lesions. The in vivo results demonstrated that the tested conduit can be adapted to the diameter of the nerve stumps to guide their growth and promote their regeneration.

Differential responses of endothelial cells on three-dimensional scaffolds to lipopolysaccharides from periodontopathogens.

Studies have been conducted on the pathogenicity of periodontopathogens in cultures of endothelial cells on two-dimensional (2D) polystyrene surfaces, where the monolayer formed is not exposed to proteins of the subendothelial matrix. In this work, we developed a culture system by seeding human coronary artery endothelial cells (HCAECs) onto three-dimensional (3D) scaffolds of collagen type I, a subendothelial protein. The inflammatory responses of the HCAEC monolayers, formed either on 3D scaffolds or directly on a 2D polystyrene plate, to lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) were evaluated. The transcription of 3 genes, the secretion of 40 cytokines and 2 prostanoids, and the adhesion of monocytes to 2D and 3D cultures with or without exposure to lipopolysaccharides (control) were assessed. HCAECs exhibited differences in transcriptional and secretory profiles between the 3D and 2D models. In addition, the inflammatory responses of HCAEC to Aa-LPS and Pg-LPS differed between the two models. In 3D cultures treated with Aa-LPS, the levels of IL-8, RANTES, G-CSF, ICAM-1, IL-6, and TXA2 were significantly higher than those in the controls. In 2D cultures treated with Aa-LPS, IL-8, RANTES, G-CSF, ICAM-1, TNF-RI, PGI2, and TXA2 levels were significantly higher than those in their controls. In the presence of Aa-LPS, monocyte adhesion did not differ between treated and control 3D cultures but was significantly higher in treated 2D cultures than in the controls. In response to Pg-LPS, cytokine-prostaglandin secretion and monocyte adhesion did not differ between 3D and 2D cultures. These data indicate that HCAECs respond differently to these two types of LPS.

Evaluation of collagen type I scaffolds including gelatin-collagen microparticles and Aloe vera in a model of full-thickness skin wound.

Research on collagen type I scaffolds with Aloe vera is sparse. The aim of this work was to develop collagen type I scaffolds with gelatin-collagen microparticles and loaded with a dispersion of A. vera, to assess their performance as grafting material for healing of skin wounds. Scaffolds were evaluated in a Cavia porcellus model with full-thickness skin wound and compared with wounds healed by secondary intention (controls). Animals grafted with scaffolds without A. vera and their control wounds were also included in the study. Evaluation of enzymatic degradation and percentage of the scaffolds' free amino groups-as an indirect assessment of their cross-linking-were also carried out because A. vera contains compounds which affect their stability. We found that dispersions of lyophilized A. vera extract loaded on scaffolds do not have cytotoxic potential, and they decrease collagenase degradation of scaffolds in the range of 0.1 to 0.3% w/v in a dose-dependent manner. Only the A. vera dispersion with the highest concentration (0.3% w/v) decreased the percentage of free amino groups, which are the ones involved in the cross-link of collagen fibers. This finding suggests that cross-linking is not the mechanism by which the tested dispersions stabilize the scaffolds. Preclinical, histochemical, and histomorphometric analyses of repaired wound tissue indicate that loading collagen type I scaffolds, including microparticles of gelatin-collagen, with A. vera in the concentrations tested does not improve wound healing. Low biodegradability of the tested scaffolds caused by the inhibition of collagenase activity might account for these results.

Comparative evaluation of healing biomarkers in skin wound exudates using a nanobiosensor and histological analysis of full-thickness skin wounds grafted with multidirectional or unidirectional artificial dermis.

Analysis of factors that play a role on the healing process in exudates from skin wounds might shed light on the effect that grafted artificial tissue has in wound regeneration and repair. The first objective of this work was to standardize an optic surface plasmon resonance method based on self-assembled monolayers to quantify healing mediator factors (angiopoietin-2, epidermal growth factor, tumour necrosis factor-α, transforming growth factor-ß1, and vascular endothelial growth factor) in wound exudates. Optimal conditions for self-assembling of alkanethiol monolayers, immobilization of antibodies antifactors, and regeneration of sensor surfaces were established. A second objective was to compare healing of wounds grafted with artificial dermis with wounds left to heal by secondary intention (control) in a lagomorph model of full-thickness skin wound. Each animal included in this study had a control wound and an identical contralateral wound grafted with artificial dermis that was made by seeding autologous skin fibroblasts into unidirectional or multidirectional collagen type I scaffolds. Histological and histomorphometric analyses were carried out when animals were sacrificed, in addition to quantifying the factors in the exudates of wounds sampled 3 days after surgery. There were significant differences between the concentrations of evaluated factors in the exudates from grafted and control wounds. This finding coincides with differences observed in the histological and histomorphometric analyses of repaired tissue formed in treated and control wounds.

Terapias Celulares y Productos de Ingeniería de Tejidos para el Tratamiento de Lesiones Condrales de Rodilla / Cell Therapy and Tissue Engineering Products for Chondral Knee Injuries

RESUMEN El cartílago articular es un tejido vulnerable a las lesiones de diferente etiología; siendo uno de los más afectados, el cartílago de la rodilla. Aunque la mayoría de los tratamientos convencionales reducen los síntomas, generalmente conducen a la formación de fibrocartílago; el cual, posee características diferentes a las del cartílago hialino de las articulaciones. Son pocas las aproximaciones terapéuticas que promueven el reemplazo del tejido dañado por cartílago hialino funcional; las más exitosas son las denominadas terapias avanzadas, que aplican células y productos de ingeniería de tejidos con el fin de estimular la regeneración del cartílago. La mayoría de ellas se basan en colocar soportes hechos con biomateriales de diferente origen, que sembrados o no con células exógenas o endógenas, reemplazan al cartílago dañado y promueven su regeneración. Este trabajo revisa algunas de las aproximaciones terapéuticas enfocadas en la regeneración del cartílago articular de rodilla; así como, los biomateriales más empleados en la elaboración de soportes para terapia celular e ingeniería de tejido cartilaginoso.

ABSTRACT The articular cartilage is prone to suffer lesions of different etiology, being the articular cartilage lesions of the knee the most common. Although most conventional treatments reduce symptoms they lead to the production of fibrocartilage, which has different characteristics than the hyaline cartilage of the joint. There are few therapeutic approaches that promote the replacement of damaged tissue by functional hyaline cartilage. Among them are the so-called advanced therapies, which use cells and tissue engineering products to promote cartilage regeneration. Most of them are based on scaffolds made of different biomaterials, which seeded or not with endogenous or exogenous cells, can be used as cartilage artificial replacement to improve joint function. This paper reviews some therapeutic approaches focused on the regeneration of articular cartilage of the knee and the biomaterials used to develop scaffolds for cell therapy and tissue engineering of cartilage.

Controlled release of an extract of Calendula officinalis flowers from a system based on the incorporation of gelatin-collagen microparticles into collagen I scaffolds: design and in vitro performance.

Aiming to develop biological skin dresses with improved performance in the treatment of skin wounds, acellular collagen I scaffolds were modified with polymeric microparticles and the subsequent loading of a hydroglycolic extract of Calendula officinalis flowers. Microparticles made of gelatin-collagen were produced by a water-in-oil emulsion/cross-linking method. Thereafter, these microparticles were mixed with collagen suspensions at three increasing concentrations and the resulting mixtures lyophilized to make microparticle-loaded porous collagen scaffolds. Resistance to enzymatic degradation, ability to associate with the C. officinalis extract, and the extract release profile of the three gelatin-collagen microparticle-scaffold prototypes were assessed in vitro and compared to collagen scaffolds without microparticles used as control. Data indicated that the incorporation of gelatin-collagen microparticles increased the resistance of the scaffolds to in vitro enzymatic degradation, as well as their association with the C. officinalis flower extract. In addition, a sharp decrease in cytotoxicity, as well as more prolonged release of the extract, was attained. Overall results support the potential of these systems to develop innovative dermal substitutes with improved features. Furthermore, the gelatin-collagen mixture represents a low-cost and scalable alternative with high clinical transferability, especially appealing in developing countries.

Real-time quantification of proteins secreted by artificial connective tissue made from uni- or multidirectional collagen I scaffolds and oral mucosa fibroblasts.

Previously, we found that oral autologous artificial connective tissue (AACT) had a different protein secretion profile to that of clot-embedded AACT. Other oral mucosa substitutes, having different cell types and scaffolds, had dissimilar secretion profiles of proteins (including that for AACT) that influence healing outcome; thus, to ascertain the profiles of factors secreted by artificial tissue and whether they are influenced by their microstructure might help in understanding their bioactivity. An important component of tissue microstructure is the fiber orientation of the scaffold used for manufacturing it. This work developed a surface plasmon resonance (SPR) methodology to quantify factors secreted by oral artificial connective tissue (ACT) in culture medium, and a method to manufacture unidirectional laminar collagen I scaffolds. The SPR methodology was used for assessing differences in the protein secretion profile of ACT made with collagen scaffolds having different fiber orientation (unidirectional vs multidirectional). Oral fibroblasts seeded onto unidirectional scaffolds increased the secretion of six factors involved in modulating healing compared to those seeded onto multidirectional scaffolds. Histological analysis of uni- and multidirectional ACT showed that cells differ in their alignment and morphology. This SPR-methodology led to nanoscale detection of paracrine factors and might be useful to study biomarkers of three-dimensional cell growth, cell differentiation, and wound-healing progression.

Neurotensin-loaded collagen dressings reduce inflammation and improve wound healing in diabetic mice.

Impaired wound healing is an important clinical problem in diabetes mellitus and results in failure to completely heal diabetic foot ulcers (DFUs), which may lead to lower extremity amputations. In the present study, collagen based dressings were prepared to be applied as support for the delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. The performance of NT alone and NT-loaded collagen matrices to treat wounds in streptozotocin (STZ) diabetic induced mice was evaluated. Results showed that the prepared dressings were not-cytotoxic up to 72h after contact with macrophages (Raw 264.7) and human keratinocyte (HaCaT) cell lines. Moreover, those cells were shown to adhere to the collagen matrices without noticeable change in their morphology. NT-loaded collagen dressings induced faster healing (17% wound area reduction) in the early phases of wound healing in diabetic wounded mice. In addition, they also significantly reduced inflammatory cytokine expression namely, TNF-α (p<0.01) and IL-1ß (p<0.01) and decreased the inflammatory infiltrate at day 3 post-wounding (inflammatory phase). After complete healing, metalloproteinase 9 (MMP-9) is reduced in diabetic skin (p<0.05) which significantly increased fibroblast migration and collagen (collagen type I, alpha 2 (COL1A2) and collagen type III, alpha 1 (COL3A1)) expression and deposition. These results suggest that collagen-based dressings can be an effective support for NT release into diabetic wound enhancing the healing process. Nevertheless, a more prominent scar is observed in diabetic wounds treated with collagen when compared to the treatment with NT alone.

Enterococcus faecalis internalization in human umbilical vein endothelial cells (HUVEC).

Initial Enterococcus faecalis-endothelial cell molecular interactions which lead to enterococci associating in the host endothelial tissue, colonizing it and proliferating there can be assessed using in vitro models. Cultured human umbilical vein endothelial cells (HUVEC) have been used to study other Gram-positive bacteria-cell interactions; however, few studies have been aimed at establishing the relationship of E. faecalis with endothelial cells. The aggregation substance (AS) family of adhesins represents an E. faecalis virulence factor which has been implicated in endocarditis severity and bacterial persistence. The Asc10 protein (a member of this family) promotes bacterium-bacterium aggregation and bacterium-host cell binding. Evaluating Asc10 role in bacterial internalization by cultured enterocytes has shown that this adhesin facilitates E. faecalis endocytosis by HT-29 cells. A few eukaryotic cell structural components, such as cytoskeletal proteins, have been involved in E. faecalis entry into cell-lines; it is thus relevant to determine whether Asc10, as well as microtubules and actin microfilaments, play a role in E. faecalis internalization by cultured endothelial cells. The role of Asc10 and cytoskeleton proteins in E. faecalis ability to enter HUVEC was assessed in the present study, as well as cell apoptosis induction by enterococcal internalization by HUVEC; the data indicated increased cell apoptosis and that cytoskeleton components were partially involved in E. faecalis entry to endothelial cells, thereby suggesting that E. faecalis Asc10 protein would not be a critical factor for bacterial entry to cultured HUVEC.

Development and preclinical evaluation of acellular collagen scaffolding and autologous artificial connective tissue in the regeneration of oral mucosa wounds.

This work assessed wound healing response in rabbit oral lesions grafted with autologous artificial connective tissue or acellular collagen scaffolds. Autologous artificial oral connective tissue (AACT) was produced using rabbit fibroblasts and collagen I scaffolds. Before implantation, AACT grafts were assayed to demonstrate the presence of fibroblasts and extracellular matrix components, as well as the expression of characteristic genes and secretion of chemokines, cytokines, and growth factors. AACT grafts were tested in the rabbits from which the fibroblasts were obtained, whereas acellular collagen type I scaffolds (CS) were evaluated in a separate group of rabbits. In both cases, contralateral wounds closed by secondary intention were used as controls. In a separate experiment, AACT-grafted wounds were directly compared with contralateral CS-grafted wounds in the same animals. Wound contraction and histological parameters were examined to evaluate closure differences between the treatments in the three animal experiments performed. Contraction of wounds grafted with AACT and CS was significantly lower than in their controls (p < 0.05). Additionally, AACT significantly lowered wound contraction when compared with CS (p < 0.05). Intriguingly, it was observed that AACT-grafted wounds initially displayed a significantly higher (p < 0.05)-albeit transient-inflammatory response than seen in CS-grafted wounds and secondary healed wounds. This suggests that an early inflammatory component may contribute to tissue regeneration. Altogether, the results suggest that AACT- and CS-grafted wounds favor regeneration of oral mucosa.

Estandarización de un modelo basado en monocapas de células Caco-2 con aplicación en estudios de absorción de fármacos / Standardization of a model based on Caco-2 cell monolayers with application on in vitro drug absorption tests

La Biodisponibilidad de los fármacos administrados oralmente depende de su absorción y de su metabolismo. Dentro de los factores que determinan la absorción de un compuesto se encuentran la permeabilidad y las características de su transporte a través de la mucosa intestinal. La administración de alimentos y drogas norteamericana (Food and Drug Administration / FDA) reconoce que la determinación in vitro de la permeabilidad usando modelos basados en monocapas de células Caco-2, puede ayudar a predecir la absorción in vivo de un fármaco. A pesar de su utilidad, en Colombia no se ha reportado la utilización de este modelo en los análisis de clasificación biofarmacéutica y de predicción de la biodisponibilidad oral. El objetivo de este trabajo fue estandarizar un modelo para el estudio de absorción basado en células Caco-2. Los coeficientes unidireccionales de permeabilidad aparente (Papp) de ranitidina y teofilina fueron establecidos en monocapas celulares en dirección apical-basolateral. Las muestras provenientes de la cámara basolateral, fueron analizadas mediante cromatografía líquida de alta eficiencia y los valores de Papp calculados con las concentraciones halladas. Los coeficientes de variación intra e interdías encontrados permitieron establecer la precisión del sistema estandarizado. Con los valores de Papp obtenidos se hizo la asignación de los compuestos a la clase de permeabilidad que concuerda con la reportada para cada uno. Para que el modelo sea utilizado en la determinación de permeabilidad de compuestos de interés, es necesaria su validación con moléculas de referencia