Measurement of alveolar derecruitment in patients with acute lung injury: computerized tomography versus pressure–volume curve

Introduction  

Positive end-expiratory pressure (PEEP)-induced lung derecruitment can be assessed by a pressure–volume (P–V) curve method or by lung computed tomography (CT). However, only the first method can be used at the bedside. The aim of the study was to compare both methods for assessing alveolar derecruitment after the removal of PEEP in patients with acute lung injury or acute respiratory distress syndrome.     

Effect of biomaterial properties on bone healing in a rabbit tooth extraction socket model

In this work we sought to understand the effect of biomaterial properties upon healing bone tissue. We hypothesized that a hydrophilic polymer gel implanted into a bone tissue defect would impede the healing process owing to the biomaterial's prevention of protein adsorption and thus cell adhesion. To test this hypothesis, healing bone was investigated within a rabbit incisor extraction socket, a subcritical size bone defect that resists significant soft tissue invasion by virtue of its conformity. After removal of the incisor teeth, one tooth socket was left as an empty control, one was filled with crosslinked polymer networks formed from the hydrophobic polymer poly(propylene fumarate) (PPF), and one was filled with a hydrogel formed from the hydrophilic oligomer oligo(poly(ethylene glycol) fumarate) (OPF). At five different times (4 days as well as 1, 2, 4, and 8 weeks), jaw bone specimens containing the tooth sockets were removed. We analyzed bone healing by histomorphometrical analysis of hematoxylin and eosin stained sections as well as immunohistochemically stained sections. The proposed hypothesis, that a hydrophilic material would hinder bone healing, was supported by the histomorphometrical results. In addition, the immunohistochemical results reflect molecular signaling indicative of the early invasion of platelets, the vascularization of wound-healing tissue, the differentiation of migrating progenitor cells, and the formation and remodeling of bone tissue. Finally, the results emphasize the need to consider biomaterial properties and their differing effects upon endogenous growth factors, and thus bone healing, during the development of tissue engineering devices.     

In vitro degradation of polymeric networks of poly(propylene fumarate) and the crosslinking macromer poly(propylene fumarate)-diacrylate

Polymeric networks of poly(propylene fumarate) (PPF) crosslinked with poly(propylene fumarate)-diacrylate (PPF-DA) are currently being investigated as an injectable, biodegradable bone cement. This study examined the effect of crosslinking density, medium pH, and the incorporation of a beta-tricalcium phosphate (beta-TCP) filler on the in vitro degradation of PPF/PPF-DA. Cylindrical specimens were submerged in buffered saline at 37 degrees C and the change in weight, geometry, and compressive mechanical properties were monitored over a 52-week period. All formulations showed an initial increase in modulus and yield strength over the first 12 weeks, achieving maxima of 1307+/-101 and 51+/-3MPa, respectively, for the beta-TCP composite. PPF/PPF-DA networks with the lower crosslinking density demonstrated the greatest degradation with a 17% mass loss. Samples in the lower buffer pH 5.0 compared to physiological pH 7.4 did not show any differences in mass loss, but exhibited a faster decrease in the compressive strength over time. The beta-TCP composites maintained their mechanical properties at the level following their initial increase. These results show that the degradation of PPF/PPF-DA networks can be controlled by the crosslinking density, accelerated at a lower pH, and prolonged with the incorporation of the beta-TCP filler.     

Retinal pigment epithelial cell adhesion on novel micropatterned surfaces fabricated from synthetic biodegradable polymers

Novel synthetic biodegradable polymer substrates with specific chemical micropatterns were fabricated from poly(DL-lactic-coglycolic acid) (PLGA) and diblock copolymers of poly(ethylene glycol) and poly(DL-lactic acid) (PEG/PLA). Thin films of PLGA and PEG/PLA supported and inhibited, respectively, retinal pigment epithelial (RPE) cell proliferation, with a corresponding cell density of 352,900 and 850 cells/cm2 after 7 days (from an initial seeding density of 15,000 cells/cm2). A microcontact printing technique was used to define arrays of circular (diameter of 50 microm) PLGA domains surrounded and separated by regions (width of 50 microm) of PEG/PLA. Reversed patterns composed of PEG/PLA circular domains surrounded by PLGA regions were also fabricated. Both micropatterned surfaces were shown to affect initial RPE cell attachment, limit cell spreading, and promote the characteristic cuboidal cell morphology during the 8-h period of the experiments. In contrast, RPE cells on plain PLGA (control films) were elongated and appeared fibroblast-like. The reversed patterns had continuous PLGA regions that allowed cell-cell interactions and thus higher cell adhesion. These results demonstrate the feasibility of fabricating micropatterned synthetic biodegradable polymer surfaces to control RPE cell morphology.     

Use of stereolithography to manufacture critical-sized 3D biodegradable scaffolds for bone ingrowth

A novel approach to the manufacture of biodegradable polymeric scaffolds for tissue-engineering utilizing stereolithography (SLA) is presented. SLA is a three-dimensional (3D) printing method that uses an ultraviolet laser to photo-crosslink a liquid polymer substrate. The current generation of SLA devices provide a 3D printing resolution of 0.1 mm. The experiments utilized a biodegradable resin mixture of diethyl fumarate (DEF), poly(propylene fumarate) (PPF), and a photoinitiator, bisacylphosphine oxide (BAPO). The PPF is crosslinked with the use of the SLA's UV laser (325-nm wavelength). An SLA device was retrofitted with a custom fixture build tank enclosing an elevator-driven build table. A 3D prototype model testing the manufacturing control this device provides was created in a computer-aided-design package. The resulting geometric data were used to drive the SLA process, and a DEF/PPF prototype part was successfully manufactured. These scaffolds have application in the tissue engineering of bony substrates.     

In vitro osteogenic differentiation of marrow stromal cells encapsulated in biodegradable hydrogels

Novel hydrogel materials based on oligo(poly(ethylene glycol) fumarate) (OPF) crosslinked with a redox radical initiation system were recently developed in our laboratory as injectable cell carriers for orthopedic tissue engineering applications. The effect of OPF hydrogel material properties on in vitro osteogenic differentiation of encapsulated rat marrow stromal cells (MSCs) with and without the presence of osteogenic supplements (dexamethasone) was investigated. Two OPF formulations that resulted in hydrogels with different swelling properties were used to encapsulate rat MSCs (seeding density approximately 13 million cells/mL, samples 6 mm diameter x 0.5 mm thick before swelling) and osteogenic differentiation in these constructs over 28 days in vitro was determined via histology and biochemical assays for alkaline phosphatase, osteopontin and calcium. Evidence of MSC differentiation was apparent over the culture period for samples without dexamethasone, but there was large variability in calcium production between constructs using cells of the same source. Differentiation was also seen in samples cultured with osteogenic supplements, but calcium deposition varied depending on the source pool of MSCs. By day 28, osteopontin and calcium results suggested that, in the presence of dexamethasone, OPF hydrogels with greater swelling promoted embedded MSC differentiation over those that swelled less (43.7 +/- 16.5 microg calcium/sample and 16.4 +/- 2.8 microg calcium/sample, respectively). In histological sections, mineralized areas were apparent in all sample types many microns away from the cells. These experiments indicate that OPF hydrogels are promising materials for use as injectable MSC carriers and that hydrogel swelling properties can influence osteogenic differentiation of encapsulated progenitor cells.     

The spectrum of aldolase B (ALDOB) mutations and the prevalence of hereditary fructose intolerance in Central Europe

We investigated the molecular basis of hereditary fructose intolerance (HFI) in 80 patients from 72 families by means of a PCR-based mutation screening strategy, consisting of heteroduplex analysis, restriction enzyme digest, DNA single strand electrophoresis, and direct sequencing. For a subset of patients mutation screening with DHPLC was established which turned out to be as fast and as sensitive as the more conventional methods. Fifteen different mutations of the aldolase B (ALDOB) gene were identified in HFI patients. As in smaller previous studies, p.A150P (65%), p.A175D (11%) and p.N335K (8%) were the most common mutated alleles, followed by c.360_363delCAAA, p.R60X, p.Y204X, and c.865delC. Eight novel mutations were identified in eight families with HFI: a small indel mutation (c.1044_1049delTTCTGGinsACACT), two small deletions (c.345_372del28; c.841_842delAC), two splice site mutations (c.113-1G>A, c.799+2T>A), one nonsense mutation (c.612T>G (p.Y204X)), and two missense mutations (c.532T>C (p.C178R), c.851T>C (p.L284P)). By mutation screening for the three most common ALDOB mutations by DHPLC in 2,000 randomly selected newborns we detected 21 heterozygotes. Based on these data and after correction for less common and private ALDOB mutations, HFI prevalence in central Europe is estimated to be 1:26,100 (95% confidence interval 1: 12,600-79,000).     

Development of an injectable,in situ crosslinkable,degradable polymeric carrier for osteogenic cell populations. Part 2. Viability of encapsulated marrow stromal osteoblasts cultured on crosslinking poly(propylene fumarate)

The effect of temporary encapsulation of rat marrow stromal osteoblasts in crosslinked gelatin microparticles on cell viability and proliferation was investigated in this study for microparticles placed on a crosslinking poly(propylene fumarate) (PPF) composite over a 7 day time period. Encapsulated cells were seeded on crosslinking PPF composites at times up to 10 min following initiation of the crosslinking reaction, and also on fully crosslinked PPF composites and tissue culture polystyrene controls, with a cell seeding density of 5.3 x 10(4) cells/cm2. The crosslinked PPF composite exhibited an average gel point of 10.3 min and an average maximum crosslinking temperature of 47.5 degrees C. Cell viability and proliferation were assessed by DNA and 3H-thymidine assays and the results were compared with those for nonencapsulated cells. The results showed that the addition time of cells to a crosslinking PPF composite had a large effect on cell viability and proliferation for both encapsulated and nonencapsulated cells with more surviving cells added at later time points. Most importantly, the temporary encapsulation of cells significantly enhanced cell viability at earlier time points. The data indicate that the presence of gelatin microparticles does not affect the crosslinking of a PPF composite. They further suggest that the temporary encapsulation of cells in crosslinked gelatin microparticles may preserve the viability of cells contained in an actively crosslinking PPF composite used as an injectable polymeric scaffold serving also as a carrier for osteogenic cell populations.     

Synthesis and properties of photocross-linked poly(propylene fumarate) scaffolds

The photocross-linking of poly(propylene fumarate) (PPF) to form porous scaffolds for bone tissue engineering applications was investigated. PPF was cross-linked using the photoinitiator bis(2,4,6-trimethylbenzoyl) phenylphosphine oxide (BAPO) and exposure to 30 min of long wavelength ultraviolet (UV) light. The porous photocross-linked PPF scaffolds (6.5 mm diameter cylinders) were synthesized by including a NaCl porogen (70, 80, and 90 wt% at cross-linking) prior to photocross-linking. After UV exposure, the samples were placed in water to remove the soluble porogen, revealing the porous PPF scaffold. As porogen leaching has not been used often with cross-linked polymers, and even more rarely with photoinitiated cross-linking, a study of the efficacy of this strategy and the properties of the resulting material was required. Results show that the inclusion of a porogen does not significantly alter the photoinitiation process and the resulting scaffolds are homogeneously cross-linked throughout their diameter. It was also shown that porosity can be generally controlled by porogen content and that scaffolds synthesized with at least 80 wt% porogen possess an interconnected pore structure. Compressive mechanical testing showed scaffold strength to decrease with increasing porogen content. The strongest scaffolds with interconnected pores had an elastic modulus of 2.3+/-0.5 MPa and compressive strength at 1% yield of 0.11+/-0.02 MPa. This work has shown that a photocross-linking/porogen leaching technique is a viable method to form porous scaffolds from photoinitiated materials.     

Effect of fibronectin- and collagen I-coated titanium fiber mesh on proliferation and differentiation of osteogenic cells

The objective of this study was to evaluate the effects of fibronectin and collagen I coatings on titanium fiber mesh on the proliferation and osteogenic differentiation of rat bone marrow cells. Three main treatment groups were investigated in addition to uncoated titanium fiber meshes: meshes coated with fibronectin, meshes coated with collagen I, and meshes coated first with collagen I and then subsequently with fibronectin. Rat bone marrow cells were cultured for 1, 4, 8, and 16 days in plain and coated titanium fiber meshes. In addition, a portion of each of these coating treatment groups was cultured in the presence of antibodies against fibronectin and collagen I integrins. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, osteocalcin, and calcium content and alkaline phosphatase activity. There were no significant effects of the coatings on cellular proliferation as indicated by the DNA quantification analysis. When antibodies against fibronectin and collagen I integrins were used, a significant reduction (p < 0.05) in cell proliferation was observed for the uncoated titanium meshes, meshes coated with collagen, and meshes coated with collagen and fibronectin. The different coatings also did not affect the alkaline phosphatase activity of the cells seeded on the coated meshes. However, the presence of antibodies against fibronectin or collagen I integrins resulted in significantly delayed expression of alkaline phosphatase activity for uncoated titanium meshes, meshes coated with collagen, and meshes coated with collagen and fibronectin. Calcium measurements did not reveal a significant effect of fibronectin or collagen I coating on calcium deposition in the meshes. Also, no difference in calcium content was observed in the uncoated titanium meshes and meshes coated with fibronectin when antibodies against fibronectin or collagen I integrins were present. Meshes coated with both collagen I and fibronectin showed significantly higher calcium content when cultured in the presence of antibodies to collagen and fibronectin integrins. A similar phenomenon was also observed for collagen-coated meshes cultured in the presence of antibodies to fibronectin integrins. No significant differences in osteocalcin content were observed between the treatment groups. However, all groups exposed to antibodies against fibronectin integrins showed a significant decrease in osteocalcin content on day 16. These results show that a fibronectin or collagen I coating does not stimulate the differentiation of rat bone marrow cells seeded in a titanium fiber mesh.