Cristina Velasquillo

Anti-biofilm activity of chitosan gels formulated with silver nanoparticles and their cytotoxic effect on human fibroblasts.

The development of multi-species biofilms in chronic wounds is a serious health problem that primarily generates strong resistance mechanisms to antimicrobial therapy. The use of silver nanoparticles (AgNPs) as a broad-spectrum antimicrobial agent has been studied previously. However, their cytotoxic effects limit its use within the medical area. The purpose of this study was to evaluate the anti-biofilm capacity of chitosan gel formulations loaded with AgNPs, using silver sulfadiazine (SSD) as a standard treatment, on strains of clinical isolates, as well as their cytotoxic effect on human primary fibroblasts. Multi-species biofilm of Staphylococcus aureus oxacillin resistant (MRSA) and Pseudomonas aeruginosa obtained from a patient with chronic wound infection were carried out using a standard Drip Flow Reactor (DFR) under conditions that mimic the flow of nutrients in the human skin. Anti-biofilm activity of chitosan gels and SSD showed a log-reduction of 6.0 for MRSA when chitosan gel with AgNPs at a concentration of 100 ppm was used, however it was necessary to increase the concentration of the chitosan gel with AgNPs to 1000 ppm to get a log-reduction of 3.3, while the SSD showed a total reduction of both bacteria in comparison with the negative control. The biocompatibility evaluation on primary fibroblasts showed better results when the chitosan gels with AgNPs were tested even in the high concentration, in contrast with SSD, which killed all the primary fibroblasts. In conclusion, chitosan gel formulations loaded with AgNPs effectively prevent the formation of biofilm and kill bacteria in established biofilm, which suggest that chitosan gels with AgNPs could be used for prevention and treatment of infections in chronic wounds. The statistic significance of the biocompatibility of chitosan gel formulations loaded with AgNPs represents an advance; however further research and development are necessary to translate this technology into therapeutic and preventive strategies.

Anti-biofilm and cytotoxicity activity of impregnated dressings with silver nanoparticles

Infections arising from bacterial adhesion and colonization on chronic wounds are a significant healthcare problem. Silver nanoparticles (AgNPs) impregnated in dressing have attracted a great deal of attention as a potential solution. The goal of the present study was to evaluate the anti-biofilm activities of AgNPs impregnated in commercial dressings against Pseudomonas aeruginosa, bacteria isolated of chronic wounds from a hospital patient. The antimicrobial activity of AgNPs was tested within biofilms generated under slow fluid shear conditions using a standard bioreactor. A 2-log reduction in the number of colony-forming units of P. aeruginosa was recorded in the reactor on exposure to dressing impregnated with 250ppm of AgNPs, diameter 9.3±1.1nm, and also showed compatibility to mammalian cells (human fibroblasts). Our study suggests that the use of dressings with AgNPs may either prevent or reduce microbial growth in the wound environment, and reducing wound bioburden may improve wound-healing outcomes.

Silver nanoparticles with antimicrobial activities against Streptococcus mutans and their cytotoxic effect

Microbial resistance represents a challenge for the scientific community to develop new bioactive compounds. The goal of this research was to evaluate the antimicrobial activity of silver nanoparticles (AgNPs) against a clinical isolate of Streptococcus mutans, antibiofilm activity against mature S. mutans biofilms and the compatibility with human fibroblasts. The antimicrobial activity of AgNPs against the planktonic clinical isolate was size and concentration dependent, with smaller AgNPs having a lower minimum inhibitory concentration. A reduction of 2.3 log in the number of colony-forming units of S. mutans was observed when biofilms grown in a CDC reactor were exposed to 100 ppm of AgNPs of 9.5±1.1 nm. However, AgNPs at high concentrations (>10 ppm) showed a cytotoxic effect upon human dermal fibroblasts. AgNPs effectively inhibited the growth of a planktonic S. mutans clinical isolate and killed established S. mutans biofilms, which suggests that AgNPs could be used for prevention and treatment of dental caries. Further research and development are necessary to translate this technology into therapeutic and preventive strategies.

Cartilage Tissue Engineering: The Role of Extracellular Matrix (ECM) and Novel Strategies

Articular cartilage is a hyaline cartilage that consists primarily of extracellular matrix with a sparse population of cells, lacking blood vessels, lymphatic vessels and nerves. The only cell type within cartilage is the chondrocyte and has a low level of metabolic activity with little or no cell division and is the responsible for maintaining in a low-turnover state the unique composition and organization of the matrix that was determined during embryonic and postnatal development. The biological and mechanical properties of articular cartilage depend on the interactions between the chondrocytes and the matrix that maintain the tissue. Chon‐ drocytes form the macromolecular framework of the tissue matrix from three classes of molecules: collagens, proteoglycans, and non-collagenous proteins and maintain the extrac‐ ellular matrix (ECM) by low-turnover replacement of certain matrix proteins [1, 2].

Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis.

The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy-pertrophy.

The Holy Grail of Orthopedic Surgery: Mesenchymal Stem Cells—Their Current Uses and Potential Applications

Only select tissues and organs are able to spontaneously regenerate after disease or trauma, and this regenerative capacity diminishes over time. Human stem cell research explores therapeutic regenerative approaches to treat various conditions. Mesenchymal stem cells (MSCs) are derived from adult stem cells; they are multipotent and exert anti-inflammatory and immunomodulatory effects. They can differentiate into multiple cell types of the mesenchyme, for example, endothelial cells, osteoblasts, chondrocytes, fibroblasts, tenocytes, vascular smooth muscle cells, and sarcomere muscular cells. MSCs are easily obtained and can be cultivated and expanded in vitro; thus, they represent a promising and encouraging treatment approach in orthopedic surgery. Here, we review the application of MSCs to various orthopedic conditions, namely, orthopedic trauma; muscle injury; articular cartilage defects and osteoarthritis; meniscal injuries; bone disease; nerve, tendon, and ligament injuries; spinal cord injuries; intervertebral disc problems; pediatrics; and rotator cuff repair. The use of MSCs in orthopedics may transition the practice in the field from predominately surgical replacement and reconstruction to bioregeneration and prevention. However, additional research is necessary to explore the safety and effectiveness of MSC treatment in orthopedics, as well as applications in other medical specialties.

Follow-up of a New Arthroscopic Technique for Implantation of Matrix-Encapsulated Autologous Chondrocytes in the Knee

PURPOSE The purpose of this study was to evaluate the clinical and sequential imaging follow-up results at a mean of 36 months after an arthroscopic technique for implantation of matrix-encapsulated autologous chondrocytes for the treatment of articular cartilage lesions on the femoral condyles. METHODS Ten patients underwent arthroscopic implantation of autologous chondrocytes seeded onto a bioabsorbable scaffold. The patients were evaluated clinically using a visual analog scale (VAS) for pain and International Knee Documentation Committee (IKDC), Lysholm, and Tegner scores. Magnetic resonance imaging (MRI) T2-mapping and magnetic resonance observation of cartilage repair tissue (MOCART) evaluations were also performed. Second-look arthroscopic evaluation using the International Cartilage Repair Society (ICRS) grading classification was performed at 12 months. RESULTS Compared with their preoperative values, at 36 months mean values ± standard deviation for the VAS scale for pain were 6.0 ± 1.5 to 0.3 ± 0.4. Improvement in clinical scores between preoperative values and 36-month follow-up values in subjective IKDC scores was 46.9 ± 18.5 to 77.2 ± 12.8; in Lysholm scores, it was 51.8 ± 25.1 to 87.9 ± 6.5, and in the Tegner activity scale it was 2.9 ± 1.7 to 5.9 ± 1.9. Mean T2 mapping and MOCART scores improved over time to 38.1 ± 4.4 ms and 72.5 ± 10, respectively. Mean ICRS score by second-look arthroscopy at 1 year was 10.4 ± 0.1. CONCLUSIONS All clinical scores improved over time compared with the preoperative values. Clinical results are comparable with MRI T2 mapping and ICRS evaluations, suggesting that this arthroscopic technique for cell-based cartilage repair is efficacious and reproducible at a mean of 36 months of follow-up. LEVEL OF EVIDENCE Level IV, therapeutic case series.

Use of irradiated human amnion as a matrix for limbal stem cell culture

Several ocular diseases affect the corneal surface; the development of effective technologies for the treatment of corneal lesions has brought about an improvement in the quality of life of affected patients. The aim of this study is to culture and characterize limbal stem cells cultured on gamma (60Co) radiosterilized human amnion (RHA). Limbal stem cells were isolated from ten preserved samples of corneal transplant. The cells were cultured since primary culture until expanded cells on RHA and stained with monoclonal antibodies to establish their immunophenotype, after which cytokeratin 12 and Vimentin were positive by immunohistochemistry. The immunophenotype remained constant since primary culture until expanded cells in RHA. The RHA and cells construct were structurally integrated. Immunohistochemistry was cytokeratin 12, Vimentin positive, and cytokeratin 19 negative. In vitro limbal cells maintain a constant epithelial transition immunophenotype in culture up to primary culture until expanded cells on RHA.

Ingeniería de tejidos y osteoartritis

Articular cartilage lesions predispose to the development of early osteoarthritis. Most current surgical techniques give rise to the formation of fibrocartilage with biochemical and biomechanical properties inferior to those or articular cartilage. Tissue engineering could offer a modern alternative to the treatment of these lesions and in this way, prevent the development of early osteoarthritis in young active patients. Different tissue engineering approaches rely on the current use of autologous chondrocytes, or the potential use of mesenchymal stem cells. Other variables rely on the type of scaffold to use such as synthetic biodegradable polymers, fibrin or collagen-derived scaffolds of different sources, bovine, porcine, rat tail, etc, in the form of gels, sponges, mesh, etc, and all of these with or without growth factors. The use of autologous chondrocytes is a reality at the present time, whether injected under a periosteum patch or seeded on collagen. However, most investigators and biotech companies are in search of onestep surgical procedures, for which reason stem cells have to be kept in mind, as well as systems that will allow arthroscopic implantation.Las lesiones de cartilago articular predisponen al desarrollo precoz de osteoartritis. La mayoria de las tecnicas quirurgicas actuales para el tratamiento de lesiones condrales dan lugar a la formacion de fibrocartilago con propiedades bioquimicas y biomecanicas inferiores a las del cartilago articular. La ingenieria de tejidos puede ofrecer una alternativa moderna para el tratamiento de estas lesiones y de esta forma prevenir el desarrollo de osteoartritis en pacientes jovenes activos. Existen diferentes alternativas en cuanto al tipo de celulas para implantar como tratamiento, ya sean el actual uso de condrocitos autologos o celulas troncales mesenquimales. La otra variable es el tipo de andamio sobre el cual cultivar o sembrar la celulas para su implante: materiales sinteticos biocompatibles y bioabsorbibles, los derivados de fibrina o de colageno de diferentes fuentes (bovina, porcina, de cola de rata, etc.), en forma de geles, esponjas, mallas, etc., y todas ellas con o sin la adicion de factores de crecimiento. En la actualidad, el uso de condrocitos autologos es una realidad, ya sea inyectados en suspension bajo un parche de periostio o sembrados en colageno. Casi todos los investigadores y las empresas de biotecnologia estan buscando tecnicas para las que no se requieran dos intervenciones quirurgicas, por lo cual muy probablemente habra que pensar en celulas troncales y con sistemas de implantacion artroscopicos

Expression of MIG‐6, WNT‐9A, and WNT‐7B During Osteoarthritis

Abstract:  Although the molecular mechanisms for initiation of cartilage destruction in osteoarthritis (OA) are unknown, it has been demonstrated that disruption of mitogen‐inducible gene 6 (Mig‐6) in mice leads to the onset of a degenerative joint disease like OA. On this basis, we correlated gene expression of Mig‐6 with Wnt‐9a and Wnt‐7b genes; we showed downregulation of Mig‐6, Wnt‐7b, and Wnt‐9a during OA, while Wnt‐7b was expressed also in osteoblast‐like cells. Here we suggest that Aggrecan degradation occurs before the downregulation of Mig‐6. It remains to be proven whether there is any relation between Wnt signaling and Aggrecan degradation.