Joaquin Cortiella

Influence of Acellular Natural Lung Matrix on Murine Embryonic Stem Cell Differentiation and Tissue Formation

We report here the first attempt to produce and use whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine embryonic stem cells (mESCs). We compared the influence of AC lung, Gelfoam, Matrigel, and a collagen I hydrogel matrix on the mESC attachment, differentiation, and subsequent formation of complex tissue. We found that AC lung allowed for better retention of cells with more differentiation of mESCs into epithelial and endothelial lineages. In constructs produced on whole AC lung, we saw indications of organization of differentiating ESC into three-dimensional structures reminiscent of complex tissues. We also saw expression of thyroid transcription factor-1, an immature lung epithelial cell marker; pro-surfactant protein C, a type II pneumocyte marker; PECAM-1/CD31, an endothelial cell marker; cytokeratin 18; alpha-actin, a smooth muscle marker; CD140a or platelet-derived growth factor receptor-alpha; and Clara cell protein 10. There was also evidence of site-specific differentiation in the trachea with the formation of sheets of cytokeratin-positive cells and Clara cell protein 10-expressing Clara cells. Our findings support the utility of AC lung as a matrix for engineering lung tissue and highlight the critical role played by matrix or scaffold-associated cues in guiding ESC differentiation toward lung-specific lineages.

Testosterone injection stimulates net protein synthesis but not tissue amino acid transport

Testosterone administration (T) increases lean body mass and muscle protein synthesis. We investigated the effects of short-term T on leg muscle protein kinetics and transport of selected amino acids by use of a model based on arteriovenous sampling and muscle biopsy. Fractional synthesis (FSR) and breakdown (FBR) rates of skeletal muscle protein were also directly calculated. Seven healthy men were studied before and 5 days after intramuscular injection of 200 mg of testosterone enanthate. Protein synthesis increased twofold after injection (P < 0.05), whereas protein breakdown was unchanged. FSR and FBR calculations were in accordance, because FSR increased twofold (P < 0.05) without a concomitant change in FBR. Net balance between synthesis and breakdown became more positive with both methodologies (P < 0.05) and was not different from zero. T injection increased arteriovenous essential and nonessential nitrogen balance across the leg (P < 0.05) in the fasted state, without increasing amino acid transport. Thus T administration leads to an increased net protein synthesis and reutilization of intracellular amino acids in skeletal muscle.

A composite tissue-engineered trachea using sheep nasal chondrocyte and epithelial cells

This study evaluates the feasibility of producing a composite engineered tracheal equivalent composed of cylindrical cartilaginous structures with lumens lined with nasal epithelial cells. Chondrocytes and epithelial cells isolated from sheep nasal septum were cultured in Hams F12 media. After 2 wk, chondrocyte suspensions were seeded onto a matrix of polyglycolic acid. Cell‐polymer constructs were wrapped around silicon tubes and cultured in vitro for 1 wk, followed by implanting into subcutaneous pockets on the backs of nude mice. After 6 wk, epithelial cells were suspended in a hydrogel and injected into the embedded cartilaginous cylinders following removal of the silicon tube. Implants were harvested 4 wk later and analyzed. The morphology of implants resembles that of native sheep trachea. H&E staining shows the presence of mature cartilage and formation of a pseudostratified columnar epithelium, with a distinct interface between tissue‐engineered cartilage and epithelium. Safranin‐O staining shows that tissue‐engineered cartilage is organized into lobules with round, angular lacunae, each containing a single chondrocyte. Proteoglycan and hydroxyproline contents are similar to native cartilage. This study demonstrates the feasibility of recreating the cartilage and epithelial portion of the trachea using tissue harvested in a single procedure. This has the potential to facilitate an autologous repair of segmental tracheal defects.—Kojima, K., Bonassar, L. J., Roy, A. K., Mizuno, H., Cortiella, J., Vacanti, C. A. A composite tissue‐engineered trachea using sheep nasal chondrocyte and epithelial cells. FASEB J. 17, 823–828 (2003)

In vitro analog of human bone marrow from 3D scaffolds with biomimetic inverted colloidal crystal geometry

In vitro replicas of bone marrow can potentially provide a continuous source of blood cells for transplantation and serve as a laboratory model to examine human immune system dysfunctions and drug toxicology. Here we report the development of an in vitro artificial bone marrow based on a 3D scaffold with inverted colloidal crystal (ICC) geometry mimicking the structural topology of actual bone marrow matrix. To facilitate adhesion of cells, scaffolds were coated with a layer of transparent nanocomposite. After seeding with hematopoietic stem cells (HSCs), ICC scaffolds were capable of supporting expansion of CD34+ HSCs with B-lymphocyte differentiation. Three-dimensional organization was shown to be critical for production of B cells and antigen-specific antibodies. Functionality of bone marrow constructs was confirmed by implantation of matrices containing human CD34+ cells onto the backs of severe combined immunodeficiency (SCID) mice with subsequent generation of human immune cells.

Identification and initial characterization of spore-like cells in adult mammals

We describe the identification and initial characterization of a novel cell type that seems to be present in all tissues. To date we have isolated what we term “spore‐like cells” based on the characteristics described below. They are extremely small, in the range of less than 5 μm, and appear to lie dormant and to be dispersed throughout the parenchyma of virtually every tissue in the body. Being dormant, they survive in extremely low oxygen environments, as evidenced by their viability in tissues (even in metabolically very active tissues such as the brain or spinal cord) for several days after sacrifice of an animal without delivery of oxygen or nutrients. The spore‐like cells described in this report have an exceptional ability to survive in hostile conditions, known to be detrimental to mammalian cells, including extremes of temperature. Spore‐like cells remain viable in unprepared tissue, frozen at −86°C (using no special preservation techniques) and then thawed, or heated to 85°C for more than 30 min. Preliminary characterization of these cells utilizing basic and special stains, as well as scanning and transmission electron microscopy reveal very small undifferentiated cells, which contain predominantly nucleus within a small amount of cytoplasm and a few mitochondria. Focal periodic acid‐Schiff and mucicarmine stains suggest a coating of glycolipid and mucopolysaccharide. In vitro, these structures have the capacity to enlarge, develop, and differentiate into cell types expressing characteristics appropriate to the tissue environment from which they were initially isolated. We believe that these unique cells lie dormant until activated by injury or disease, and that they have the potential to regenerate tissues lost to disease or damage. J. Cell. Biochem. 80:455–460, 2001.

Adjunctive methods of pain control in burns

Opiates remain the most common form of analgesic therapy in the burn patient today. Because of increased opiate requirements, optimal relief of burn pain continues to be a problem for these patients. The purpose of this article is to summarize those alternative pain control methods that appear in the literature. For instance, in minor burns acetominophen continues to be a useful first line analgesic. Non-steroidal anti-inflammatory drugs (NSAID) and benzodiazepine are generally combined with opiates while entonox seems to be used commonly in the adolescent patients to relieve procedural pain. Antidepressants appear to enhance opiate-induced analgesia while anticonvulsants are useful in the treatment of sympathetically maintained pain following burns. Ketamine has been extensively used during burn dressing changes but its psychological side-effects have limited its use. Clonidine, however, has shown promise in reducing pain without causing pruritus or respiratory depression. Other forms such as transcutaneous electrical nerve stimulation (TENS), psychological techniques, topical and systemic local anaesthetics are also useful adjuncts.

Engineering of a complex organ: progress toward development of a tissue-engineered lung.

Although there has been slow progress in the engineering of the lung, recent advances in the use of stem or progenitor cells leading to the reliable production of component parts of the lung show promise for the future development of engineered lung tissue. Progress toward the goal of developing an engineered lung will only be accomplished through the parallel development of effective and functional tissue-engineered components that include both upper and lower respiratory tract as well as scaffold material suitable for use in the lung. The knowledge acquired from developing each individual component of lung will, over time, be integrated to allow for the development of larger complex organ structures. To accomplish the goal of developing engineered lung for regenerative medicine, many advances will be required in scaffold design and production, including improved biocompatibility, improved elasticity, and better control of scaffold ultrastructure and porosity. Development of new materials designed to meet the anatomic and physiologic needs of the lung must occur before we can begin to realize the goal of engineering functional lung tissue. Better understanding of factors promoting cell adhesion, migration, differentiation, and vascularization of grafts and lung regeneration as a whole is also needed. Advances in the development of mathematical models to examine the conditions that promote lung morphogenesis and tissue growth for computational investigations of tissue development will also be necessary if we are to realistically evaluate the production of lung tissue strictly from the engineering perspective. It is obvious that engineering of lung tissue will require a multidisciplinary approach if we are to eventually succeed in our attempts to produce tissues worthy of clinical application in the future.

High frequency percussive ventilation in pediatric patients with inhalation injury.

The objective of this study was to present data that showed high frequency percussive ventilation (HFPV) was superior to traditional mechanical ventilation for the treatment of children with inhalation injuries. Inhalation injuries continue to be the number one cause of death of patients with thermal injuries in the United States. Therapy for this condition has consisted of conservative pulmonary toilet and mechanical ventilation. Despite improvements in the management of burn injury, patients with inhalation injury develop pneumonia and pneumothorax, leading to adult respiratory distress syndrome. Unfortunately, inhalation injury that is complicated by pneumonia has been shown to increase mortality by 60% in these patients. Cioffi has shown that prophylactic use of HFPV in adult patients with inhalation injury has been a successful method of reducing the incidence of pneumonia and mortality. The effects of HFPV on the incidence of pneumonia, peak inspiratory pressures, and arterial partial pressure of oxygen/fraction of inspired concentration of oxygen (P/F) ratios were retrospectively studied in 13 children with inhalation injuries and compared with historic controls treated with conventional mechanical ventilation. All patients were treated with our standard inhalation injury protocol and extubated when they met standard extubation criteria. Patients ranged in age from 6 to 9 years, and most had burns covering greater than 50% of their total body surface areas. No deaths occurred in either group, but the patients who were treated with HFPV had no cases of pneumonia (P < .05), better P/F ratios (P < .05), lower peak inspiratory pressures, and less work of breathing (P < .05) as compared with our control group. On the basis of our clinical experience and data, the use of HFPV seems to be an effective treatment for the reduction of pulmonary morbidity in pediatric patients with inhalation injuries.

A COMPARISON OF ORAL TRANSMUCOSAL FENTANYL CITRATE AND ORAL HYDROMORPHONE FOR INPATIENT PEDIATRIC BURN WOUND CARE ANALGESIA

The ideal oral wound care analgesic for children should be palatable, provide potent analgesia of rapid onset and short duration, and require minimal, yet appropriate, monitoring. With use of a double-blinded crossover design, we compared the efficacy and safety of oral transmucosal fentanyl citrate (OTFC) (approximately 10 micrograms/kg) with the efficacy and safety of oral hydromorphone (60 micrograms/kg) in 14 pediatric inpatients (ages 4 to 17 years) undergoing daily burn wound care in a ward setting. Pulse oximetry, vital signs, side effects, patient pain scores, and observer scores for cooperation, anxiety, and sedation were recorded. Pulse oximetry, vital signs, cooperation, sedation, incidence of nausea or vomiting, and the amount of time it took to resume normal activities were similar in both treatment groups. OTFC resulted in improved pain scores before wound care and improved anxiolysis during wound care, but at other points it was similar in effect to hydromorphone. We conclude that OTFC is a safe and effective analgesic, that it may provide minor improvements in analgesia and anxiolysis compared with hydromorphone, and that it offers a palatable alternative route of opioid administration without intravenous access for wound care procedures in children.

Assessment of Protein Nutritional Status

An evaluation of protein status can be approached by use of anthropometric, clinical, and biochemical data, either singly or in combination, and further aided with dietary data. Each of these approaches has advantages and limitations. Biochemical evaluation has the potential of being the most objective and quantitative. Indicators that have been or might be used include plasma hormone responses to reduced protein intake, plasma levels of specific proteins or specific amino acids, urinary excretion of specific amino acids and other nitrogen-containing compounds, anthropometric and physical measurements of body muscle mass, and functional tests of muscle strength. Several measurements can be combined to produce nutritional indices of broader potential value. The importance of concomitant infection and inflammation, with its many effects on protein metabolism, cannot be ignored in making these assessments. Unfortunately, no single test or group of tests can be recommended at this time as a routine and reliable indicator of protein status. Nonetheless, our increasing knowledge of the metabolism and functions of proteins, together with the recent use of noninvasive stable isotope techniques and of sophisticated physicochemical measurements, provides encouragement that more appropriate indicators are in the offing.