Randomized controlled study of aerosolized hypertonic xylitol versus hypertonic saline in hospitalized patients with pulmonary exacerbation of cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) lung disease is characterized by chronic bacterial infection and recurrent pulmonary exacerbations. Xylitol is a 5-carbon sugar that can lower the airway surface salt concentration and augment innate immunity. We examined the safety and efficacy of aerosolized xylitol use for 2 weeks in subjects hospitalized with a pulmonary exacerbation of CF. METHODS: In a 2-week study, 60 subjects with cystic fibrosis and FEV1 > 30% predicted were enrolled to receive aerosolized 7% hypertonic saline (4 ml) or 15% xylitol (5 ml) twice a day for 14 days. Outcomes assessed included change from baseline in FEV1% predicted, change in sputum microbial density, revised CF quality of life questionnaire including the respiratory symptom score, time to next hospitalization for a pulmonary exacerbation, and frequency of adverse events. RESULTS: 59 subjects completed the study (one subject in the saline group withdrew before any study product administration). No significant differences were noted between the 2 arms in mean changes in lung function, sputum microbial density for Pseudomonas aeruginosa and Staphylococcus aureus, body weight, quality of life, and frequency of adverse events. CONCLUSIONS: Aerosolized hypertonic xylitol was well-tolerated among subjects hospitalized for CF pulmonary exacerbation. Future studies examining efficacy for long term use in patients with CF lung disease would be worthwhile. The clinical trial registration number for this study is NCT00928135.

Sonographic evidence of abnormal tracheal cartilage ring structure in cystic fibrosis.

OBJECTIVES/HYPOTHESIS: Tracheal cartilage ring structural abnormalities have been reported in cystic fibrosis (CF) mice and pigs. Whether similar findings are present in humans with CF is unknown. We hypothesized that tracheal cartilage ring shape and size would be different in people with CF. STUDY DESIGN: Tracheal cartilage ring size and shape were measured in adults with (n = 21) and without CF (n = 18). METHODS: Ultrasonography was used in human subjects to noninvasively assess tracheal cartilage ring structure in both the sagittal and the transverse planes. Tracheal cartilage ring thickness was also determined from histological sections obtained from newborn non-CF and CF pigs. These values were compared with human data. RESULTS: Human CF tracheas had a greater width and were less circular in shape compared to non-CF subjects. CF tracheal cartilage rings had a greater midline cross-sectional area and were thicker compared to non-CF rings. Maximal tracheal cartilage ring thickness was also greater in both newborn CF pigs and human adults with CF, compared to non-CF controls. CONCLUSIONS: Our findings demonstrate that structural differences exist in tracheal cartilage rings in adults with CF. Comparison with newborn CF pig data suggests that some of these changes may be congenital in nature. LEVEL OF EVIDENCE: 3b

SERI surgical scaffold, prospective clinical trial of a silk-derived biological scaffold in two-stage breast reconstruction: 1-year data.

BACKGROUND: SERI Surgical Scaffold is a long-term bioresorbable silk-derived biological scaffold developed to provide soft-tissue support and repair. METHODS: SURE-001 (ClinicalTrials.gov identification no. NCT01256502) is a prospective, single-arm study in the United States of patients undergoing two-stage, implant-based breast reconstruction using SERI. RESULTS: A total of 139 patients were enrolled and will be followed for 2 years; in this article, the authors report interim data on 71 patients followed for 1 year. Investigator satisfaction scores (mean ± SD) at 6 and 12 months were 9.2 ± 0.98 and 9.4 ± 0.91, respectively (10 = very satisfied). SERI was rated easy/very easy to use in 98 percent or more of cases across five categories in stage I surgery. Patient satisfaction with the treated breast(s) (mean ± SD) was higher at 6 (4.3 ± 0.87; 5 = very satisfied) and 12 months (4.5 ± 0.82) compared with screening (3.6 ± 1.09; p < 0.0001). Key complication rates (per breast) were tissue necrosis (6.7 percent), seroma (5.7 percent), hematoma (4.8 percent), implant loss (3.8 percent), capsular contracture (1.9 percent), and breast infection (1.0 percent). None were attributed to SERI by the investigators. In 13 patients (14 breasts) who underwent unplanned radiation therapy, one complication was reported. CONCLUSIONS: In this interim report, high levels of investigator and patient satisfaction, and ease of use of SERI were reported. Prospectively collected complication rates were similar to those reported in primarily retrospective studies of two-stage, implant-based breast reconstructions using other implantable soft-tissue support materials such as acellular dermal matrices. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

An evaluation of SERI surgical scaffold for soft-tissue support and repair in an ovine model of two-stage breast reconstruction.

This study was designed to evaluate the SERI Surgical Scaffold, a silk-derived bioresorbable scaffold, in an ovine model of two-stage breast reconstruction. Sheep were implanted bilaterally with either SERI or sham sutures during the stage 1 procedure. The SERI group underwent an exchange procedure for a breast implant at 3 months; animals in the sham group were killed at 3 months. The sham samples were significantly weaker than the SERI plus tissue samples by 3 months. At all endpoints, SERI plus tissue samples were greater than or equal to 150 percent of native ovine fascial strength. Histologic evaluation of SERI samples showed evidence of bioresorption through 12 months. SERI provided adequate soft-tissue support with progressive bioresorption. By 12 months, newly formed tissue had assumed the majority of load-bearing responsibility.

Neonates with cystic fibrosis have a reduced nasal liquid pH; a small pilot study.

BACKGROUND: Disrupted HCO3(-) transport and reduced airway surface liquid (ASL) pH in cystic fibrosis (CF) may initiate airway disease. We hypothesized that ASL pH is reduced in neonates with CF. METHODS: In neonates with and without CF, we measured pH of nasal ASL. We also measured nasal pH in older children and adults. RESULTS: In neonates with CF, nasal ASL (pH5.2 ± 0.3) was more acidic than in non-CF neonates (pH6.4 ± 0.2). In contrast, nasal pH of CF children and adults was similar to values measured in people without CF. CONCLUSIONS: At an age when infection, inflammation and airway wall remodeling are minimal, neonates with CF had an acidic nasal ASL compared to babies without CF. The CF:non-CF pH difference disappeared in older individuals, perhaps because secondary manifestations of disease increase ASL pH. These results aid understanding of CF pathogenesis and suggest opportunities for therapeutic intervention and monitoring of disease.

Design and characterization of a scaffold for anterior cruciate ligament engineering.

Advances in biomedical engineering have led to an understanding of the human body's capacity for anterior cruciate ligament (ACL) healing if provided the correct impetus--a long-term bioresorbable scaffold that anticipates the defect site's requirements. Tissue engineering an ACL requires a scaffold that can meet multiple and often conflicting mechanical and biological design requirements. The design and characterization of a hydrophilic silk scaffold is presented as an example of the preclinical testing required to fully characterize a scaffold for ACL reconstruction. We hypothesize that by providing a structural scaffold which anticipates ACL repair mechanisms, an "engineered" autologous ligament with excellent functional integrity can be developed by the body itself. Mechanical, biological, and patient-clinician testing demonstrate that the hydrophilic silk scaffold is a mechanically robust, biocompatible, long-term bioresorbable ACL scaffold with demonstrated safety that can be implanted in accordance with standard surgical procedures.

Sequential biochemical and mechanical stimulation in the development of tissue-engineered ligaments.

Application of stimuli in sequence to developing cultures in vitro offers the potential to intricately direct cell development and differentiation by following the template of native tissue behavior. We hypothesize that administration of mechanical stimulation at the peak of growth factor-induced cell activity will differentiate bone marrow stromal cells (BMSCs) along a fibroblast lineage and enhance in vitro ligament development through enhanced matrix ingrowth, matrix metalloproteinase-2 (MMP-2) production, collagen type I production, and extracellular matrix (ECM) alignment. BMSC-seeded silk matrices were cultured in a static growth-factor-free environment for 5 days prior to loading into bioreactor vessels to first establish an appropriate dynamic rotational regime, as determined through assessment of cell activity, histology, and surface topography. Once the regime was determined, seeded matrices initially cultured in basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or growth-factor-free control medium for 5 days were loaded into the bioreactor for 9 days of mechanical stimulation. Our findings indicated that the sequential application of mechanical stimulation following growth factor supplemented static culture-induced cell differentiation toward a fibroblast lineage, enhancing matrix ingrowth, cell and ECM alignment, and total collagen type I produced compared to respective static cultures. The current results suggest a dynamic culturing regime in the development of engineered tissues.

The use of long-term bioresorbable scaffolds for anterior cruciate ligament repair.

The absence of adequate options to restore full knee joint function through anterior cruciate ligament reconstruction prompts the need to develop new ligament replacement strategies. Recent focus within the ligament engineering field has been on the establishment of appropriate anterior cruciate ligament graft design requirements and evaluation methods. A range of biomaterials and graft constructions has been explored in an attempt to identify the optimal ligament replacement. Thorough and standardized evaluation methods are required throughout all phases of development, from initial in vitro bench screening through a large animal in vivo model. The initial positive clinical, gross pathologic, histologic, and mechanical results from a 12-month in vivo goat study demonstrate the potential of bioengineered ligament devices.

Yarn design for functional tissue engineering.

Tissue engineering requires the ability to design scaffolds with mechanical properties similar to those of the native tissue. Here, B. mori silk yarns are used as a model system to demonstrate the potential benefits and drawbacks of several textile methods used to fabricate tissue engineering scaffolds. Fibers are plied, twisted, cabled, braided, and/or textured to form several geometries with a wide range of mechanical outcomes. Predictable changes in ultimate tensile strength and stiffness are demonstrated following processing and as a function of test environment. The mechanical effects of increasing turns per inch and combining groups of fibers into higher-order yarn structures are demonstrated. Braids, one of the most commonly used textile structures, are shown to be limited by a change in stiffness following the locking-angle and therefore, potentially not the ideal structure for tissue engineering. Cabled yarns appear to allow the most flexibility in mechanical outcomes with a highly organized geometry. Twisted yarns, while more economical than cabled yarns, result in a higher stiffness and lower percent elongation at break than cabled yarns.

Monitoring mesenchymal stromal cell developmental stage to apply on-time mechanical stimulation for ligament tissue engineering.

To evaluate the appropriate time frame for applying mechanical stimuli to induce mesenchymal stromal cell (MSC) differentiation for ligament tissue engineering, developmental cell phenotypes were monitored during a period of in vitro culture. MSCs were seeded onto surface-modified silk fibroin fiber matrices and cultured in Petri dishes for 15 days. Cell metabolic activity, morphology, and gene expression of extracellular matrix (ECM) proteins (collagen type I and III and fibronectin), ECM receptors (integrins alpha-2, alpha-5, and beta-1), and heat-shock protein 70 (HSP-70) were monitored during the culture of MSC. MSCs showed fluctuations in cell metabolic activity, ECM, integrin, and HSP-70 transcription potentially correlating to innate developmental processes. Cellular response to mechanical stimulation was dependent on the stage of cell development. At day 9, when levels of cell metabolic activity, ECM, integrin, and HSP-70 transcription peaked, mechanical stimulation increased MSC metabolic activity, alignment, and collagen production. Mechanical stimulation applied at day 1 and 3 showed detrimental effects on MSCs seeded on silk matrices. The results presented in this study identify a unique correlation between innate MSC development processes on a surface-modified silk matrix and dynamic environmental signaling.

Sequential growth factor application in bone marrow stromal cell ligament engineering.

In vitro bone marrow stromal cell (BMSC) growth may be enhanced through culture medium supplementation, mimicking the biochemical environment in which cells optimally proliferate and differentiate. We hypothesize that the sequential administration of growth factors to first proliferate and then differentiate BMSCs cultured on silk fiber matrices will support the enhanced development of ligament tissue in vitro. Confluent second passage (P2) BMSCs obtained from purified bone marrow aspirates were seeded on RGD-modified silk matrices. Seeded matrices were divided into three groups for 5 days of static culture, with medium supplement of basic fibroblast growth factor (B) (1 ng/mL), epidermal growth factor (E; 1 ng/mL), or growth factor-free control (C). After day 5, medium supplementation was changed to transforming growth factor-beta1 (T; 5 ng/mL) or C for an additional 9 days of culture. Real-time RT-PCR, SEM, MTT, histology, and ELISA for collagen type I of all sample groups were performed. Results indicated that BT supported the greatest cell ingrowth after 14 days of culture in addition to the greatest cumulative collagen type I expression measured by ELISA. Sequential growth factor application promoted significant increases in collagen type I transcript expression from day 5 of culture to day 14, for five of six groups tested. All T-supplemented samples surpassed their respective control samples in both cell ingrowth and collagen deposition. All samples supported spindle-shaped, fibroblast cell morphology, aligning with the direction of silk fibers. These findings indicate significant in vitro ligament development after only 14 days of culture when using a sequential growth factor approach.

In vitro degradation of silk fibroin.

A significant need exists for long-term degradable biomaterials which can slowly and predictably transfer a load-bearing burden to developing biological tissue. In this study Bombyx mori silk fibroin yarns were incubated in 1mg/ml Protease XIV at 37 degrees C to create an in vitro model system of proteolytic degradation. Samples were harvested at designated time points up to 12 weeks and (1) prepared for scanning electron microscopy (SEM), (2) lyophilized and weighed, (3) mechanical properties determined using a servohydraulic Instron 8511, (4) dissolved and run on a SDS-PAGE gel, and (5) characterized with Fourier transform infrared spectroscopy. Control samples were incubated in phosphate-buffered saline. Fibroin was shown to proteolytically degrade with predictable rates of change in fibroin diameter, failure strength, cycles to failure, and mass. SEM indicated increasing fragmentation of individual fibroin filaments from protease-digested samples with time of exposure to the enzyme; particulate debris was present within 7 days of incubation. Gel electrophoresis indicated a decreasing amount of the silk 25 kDa light chain and a shift in the molecular weight of the heavy chain with increasing incubation time in protease. Results support that silk is a mechanically robust biomaterial with predictable long-term degradation characteristics.

Tissue engineering of ligaments.

Tissue engineering is emerging as a significant clinical option to address tissue and organ failure by implanting biological substitutes for the compromised tissues. As compared to the transplantation of cells alone, engineered tissues offer the potential advantage of immediate functionality. Engineered tissues can also serve as physiologically relevant models for controlled studies of cells and tissues designed to distinguish the effects of specific signals from the complex milieu of factors present in vivo. A high number of ligament failures and the lack of adequate options to fully restore joint functions have prompted the need to develop new tissue engineering strategies. We discuss the requirements for ligament reconstruction, the available treatment options and their limitations, and then focus on the tissue engineering of ligaments. One representative tissue engineering system involving the integrated use of adult human stem cells, custom-designed scaffolds, and advanced bioreactors with dynamic loading is described.

Human bone marrow stromal cell and ligament fibroblast responses on RGD-modified silk fibers.

Adhesion, spreading, proliferation, and collagen matrix production of human bone marrow stromal cells (BMSCs) on an RGD-modified silk matrix was studied. Anterior cruciate ligament fibroblasts (ACLFs) were used as a control cell source. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the modified silk matrices support improved BMSC and ACLF attachment and show higher cell density over 14 days in culture when compared with the non-RGD-modified matrices. Collagen type I transcript levels (at day 7) and content (at day 14) was significantly higher on the RGD-modified substrate than on the nonmodified group. The ability of RGD-coupled silk matrices to support BMSC attachment, which leads to higher cell density and collagen matrix production in vitro, combined with mechanical, fatigue, and biocompatibility properties of the silk protein matrix, suggest potential for use of this biomaterial for tissue engineering.

Silk-based biomaterials.

Silk from the silkworm, Bombyx mori, has been used as biomedical suture material for centuries. The unique mechanical properties of these fibers provided important clinical repair options for many applications. During the past 20 years, some biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue-like proteins) was the likely cause. More recent studies with well-defined silkworm silk fibers and films suggest that the core silk fibroin fibers exhibit comparable biocompatibility in vitro and in vivo with other commonly used biomaterials such as polylactic acid and collagen. Furthermore, the unique mechanical properties of the silk fibers, the diversity of side chain chemistries for 'decoration' with growth and adhesion factors, and the ability to genetically tailor the protein provide additional rationale for the exploration of this family of fibrous proteins for biomaterial applications. For example, in designing scaffolds for tissue engineering these properties are particularly relevant and recent results with bone and ligament formation in vitro support the potential role for this biomaterial in future applications. To date, studies with silks to address biomaterial and matrix scaffold needs have focused on silkworm silk. With the diversity of silk-like fibrous proteins from spiders and insects, a range of native or bioengineered variants can be expected for application to a diverse set of clinical needs.

Silk matrix for tissue engineered anterior cruciate ligaments.

A silk-fiber matrix was studied as a suitable material for tissue engineering anterior cruciate ligaments (ACL). The matrix was successfully designed to match the complex and demanding mechanical requirements of a native human ACL, including adequate fatigue performance. This protein matrix supported the attachment, expansion and differentiation of adult human progenitor bone marrow stromal cells based on scanning electron microscopy, DNA quantitation and the expression of collagen types I and III and tenascin-C markers. The results support the conclusion that properly prepared silkworm fiber matrices, aside from providing unique benefits in terms of mechanical properties as well as biocompatibility and slow degradability, can provide suitable biomaterial matrices for the support of adult stem cell differentiation toward ligament lineages. These results point toward this matrix as a new option for ACL repair to overcome current limitations with synthetic and other degradable materials.

Cell differentiation by mechanical stress.

Growth factors, hormones, and other regulatory molecules are traditionally required in tissue engineering studies to direct the differentiation of progenitor cells along specific lineages. We demonstrate that mechanical stimulation in vitro, without ligament-selective exogenous growth and differentiation factors, induces the differentiation of mesenchymal progenitor cells from the bone marrow into a ligament cell lineage in preference to alternative paths (i.e., bone or cartilage cell lineages). A bioreactor was designed to permit the controlled application of ligament-like multidimensional mechanical strains (translational and rotational strain) to the undifferentiated cells embedded in a collagen gel. The application of mechanical stress over a period of 21 days up-regulated ligament fibroblast markers, including collagen types I and III and tenascin-C, fostered statistically significant cell alignment and density and resulted in the formation of oriented collagen fibers, all features characteristic of ligament cells. At the same time, no up-regulation of bone or cartilage-specific cell markers was observed.

Advanced bioreactor with controlled application of multi-dimensional strain for tissue engineering.

Advanced bioreactors are essential for meeting the complex requirements of in vitro engineering functional skeletal tissues. To address this need, we have developed a computer controlled bench-top bioreactor system with capability to apply complex concurrent mechanical strains to three-dimensional matrices independently housed in 24 reactor vessels, in conjunction with enhanced environmental and fluidic control. We demonstrate the potential of this new system to address needs in tissue engineering, specifically toward the development of a tissue engineered anterior cruciate ligament from human bone-marrow stromal cells (hBMSC), where complex mechanical and biochemical environment control is essential to tissue function. Well-controlled mechanical strains (resolution of < 0.1 micron for translational and < 0.1 degree for rotational strain) and dissolved oxygen tension (between 0%-95% +/- 1%) could be applied to the developing tissue, while maintaining temperature at 37 +/- 0.2 degrees C about developing tissue over prolonged periods of operation. A total of 48 reactor vessels containing cell culture medium and silk fiber matrices were run for up to 21 days under 90 degrees rotational and 2 mm translational deformations at 0.0167 Hz with only one succumbing to contamination due to a leak at an medium outlet port. Twenty-four silk fiber matrices seeded with human bone marrow stromal cells (hBMSCs) housed within reactor vessels were maintained at constant temperature (37 +/- 0.2 degrees C), pH (7.4 +/- 0.02), and pO2 (20 +/- 0.5%) over 14 days in culture. The system supported cell spreading and growth on the silk fiber matrices based on SEM characterization, as well as the differentiation of the cells into ligament-like cells and tissue (Altman et al., 2001).