Roger S. Crowther

In vivo degradation of porous poly(propylene fumarate)/poly(DL-lactic-co-glycolic acid) composite scaffolds

This study investigated the in vivo degradation of poly(propylene fumarate) (PPF)/poly(DL-lactic-co-glycolic acid) (PLGA) composite scaffolds designed for controlled release of osteogenic factors. PPF/PLGA composites were implanted into 15.0mm segmental defects in the rabbit radius, harvested after 12 and 18 weeks, and analyzed using histological techniques to assess the extent of polymer degradation as well as the tissue response within the pores of the scaffolds. Polymer degradation was limited to micro-fragmentation of the scaffold at the ends and edges of the implant at both 12 and 18 weeks. The tissue within the pores of the scaffold consisted of fibrous tissue, blood vessels and some inflammatory cells. In areas where polymer breakdown was evident, an increased inflammatory response was observed. In contrast, areas of bone ingrowth into the polymer scaffold were characterized by minimal inflammatory response and polymer degradation. Our results show that minimal degradation of porous PPF occurs within 18 weeks of implantation in a rabbit model. Further, the in vivo degradation data of porous PPF/PLGA scaffolds are comparable with earlier obtained in vitro data.

Controlled release of an osteogenic peptide from injectable biodegradable polymeric composites.

Poly(D,L-lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) blend microparticles loaded with the osteogenic peptide TP508 were added to a mixture of poly(propylene fumarate) (PPF), poly(propylene fumarate)-diacrylate (PPF-DA), and sodium chloride (NaCl) for the fabrication of PPF composite scaffolds that could allow for tissue ingrowth as well as for the controlled release of TP508 when implanted in an orthopedic defect site. In this study, PPF composites were fabricated and the in vitro release kinetics of TP508 were determined. TP508 loading within the PLGA/PEG microparticles, PEG content within the PLGA/PEG microparticles, the microparticle content of the PPF composite polymer component, and the leachable porogen initial mass percent of the PPF composites were varied according to a fractional factorial design and the effect of each variable on the release kinetics was determined for up to 28 days. Each composite formulation released TP508 with a unique release profile. The initial release (release through day 1) of the PLGA/PEG microparticles was reduced upon inclusion in the PPF composite formulations. Day 1 normalized cumulative mass release from PPF composites ranged from 0.14+/-0.01 to 0.41+/-0.01, whereas the release from PLGA/PEG microparticles ranged from 0.31+/-0.02 to 0.58+/-0.01. After 28 days, PPF composites released 53+/-4% to 86+/-2% of the entrapped peptide resulting in cumulative mass releases ranging from 0.14+/-0.01 microg TP508/mm(3) scaffold to 2.46+/-0.05 microg TP508/mm(3) scaffold. The results presented here demonstrate that PPF composites can be used for the controlled release of TP508 and that alterations in the composites composition can lead to modulation of the TP508 release kinetics. These composites can be used to explore the effects varied release kinetics and dosages on the formation of bone in vivo.

Serial experimental and clinical studies on the pathogenesis of multiple organ dysfunction syndrome (MODS) in severe burns

These serial clinical and experimental studies were designed to clarify the pathogenesis of postburn MODS. Both animal and clinical studies were performed. In animal experiments, 46 male cross-bred dogs were cannulated with Swan-Ganz catheters and 39 of them were inflicted with 50% TBSA third degree burns (7 were used as controls). The burned dogs were randomly divided into 4 groups: immediate infusion, delayed infusion, delayed fast infusion and delayed fast infusion combined with ginsenosides. All dogs were kept under constant barbiturate sedation during the whole study period. Hemodynamics, visceral MDA, mitochondrial respiratory control rate (RCR) and ADP/O ratio, ATP, succinic dehydrogenase (SDH), organ water content as well as light and electron microscopy of visceral tissues were determined. In the clinical study, 61 patients with extensive deep burns were chosen, of which 16 sustained MODS. Plasma TXB2/6-keto-PGF1alpha ratio, TNF, SOD, MDA, circulatory platelet aggregate ratio (CPAR), PGE2, interleukin-1, total organ water content and pathological observations of visceral tissues from patients who died of MODS were carried out. Results demonstrated that ischemic-reperfusion damage due to severe shock, sepsis and inhalation injury are three main causes of postburn death. All inflammatory mediators increased markedly in both animals and patients who sustained organ damage or MODS. SDH, RCR, ADP/O and ATP decreased significantly. These findings suggested that ischemic damage and systemic inflammatory response syndrome (SIRS) initiated by mediators or cytokines might be important in the pathogenesis of postburn MODS.

Improvement of early postburn cardiac function by use of Panax notoginseng and immediate total eschar excision in one operation

Cardiac dysfunction development in the early stage postburn has been an important problem in burn treatment. However, no effective therapies are available for use in clinical practice. In this study, we sought to determine whether early total eschar excision (EEE) in one operation and the traditional Chinese herb Panax notoginseng (PNS) would be helpful in improving early postburn cardiac function. 160 Wistar rats were randomly divided into burn (burn group, n = 50), burn treated with EEE (EEE group, n = 50), burn treated with PNS (PNS group, n = 50) groups and normal controls (n = 10). All rats except the normal control were given a 30% TBSA full skin thickness burn and resuscitated with Ringers lactate. EEE was performed immediately after the burn group received the first intraperitoneal injection of Ringers lactate. The wound was covered with homoskin from normal rats. In the PNS group, two doses of PNS (200 mg/kg for each dose) were given intraperitoneally immediately and 4 h postburn. Cardiac contractile function and cardiac troponin T (TnT) were determined at 1, 3, 6, 12 and 24 h postburn. Results showed that cardiac contractile parameters including AOSP, AODP, LVSP and +dp/dt(max) all declined and were still significantly lower than the control values at 24 h postburn. Cardiac TnT was elevated markedly and reached a level 25 times higher than control at 12 h postburn. In EEE and PNS groups, the reduction of cardiac contractile function was limited as compared with that in the burn group. Levels of TnT in both EEE and PNS groups were significantly lower than in the burn group 6 h postburn later. The findings of this study demonstrated that both EEE and PNS were effective in improving early postburn cardiac function.

Calcium carbonate in cholesterol gallstones : polymorphism, distribution, and hypotheses about pathogenesis

This study of sets of cholesterol gallstones collected consecutively from 222 patients in La Paz, Bolivia, and Mexico City, Mexico, has developed a reliable infrared (IR) spectroscopic method for the detection of calcium carbonate in cholesterol gallstones and provided the basis for simultaneous identification of each of its three polymorphs: calcite, vaterite, and aragonite. The peaks in the 854 to 876 cm−1 region demonstrated 98% sensitivity and specificity for carbonate detection. As little as 3% carbonate by weight could be detected using these peaks. The overall incidence of carbonate was 19% in these populations containing a high proportion of Amerinds. Infrared microspectroscopy of 10 to 50 μm particles, dissected from stones, allowed a ring‐by‐ring examination of 11 carbonate‐containing stones. It was determined that different carbonate polymorphs, when present in the same gallstone, almost always occurred in separate rings. In approximately half of the gallstones, different polymorphs were present in successive layers in the same stone, indicating that conditions governing stone growth changed cyclically. Carbonates were usually precipitated in peripheral layers rather than in the center, supporting the theory that formation of calcium carbonates may be related to episodes of intermittent obstruction of the cystic duct, as opposed to being a major factor in stone nidation. (Hepatology 1995;22:488–496.)

Effects of Early Eschar Excision En Masse at One Operation for Prevention and Treatment of Organ Dysfunction in Severely Burned Patients

Abstract. We sought to determine whether early eschar excision en masse (EEE) at one operation would be effective in the prevention and treatment of postburn organ dysfunction (OD) and multiple organ dysfunction syndrome (MODS). A total of 60 patients, with total body surface burned area over 35% and a third degree burn area over 20% were studied and divided into two groups, the EEE group (35 cases) and the group treated with repeated escharectomies by stages (repeated escharectomy group, 25 cases). Other than the different operations undertaken, the patients in both groups received identical conventional treatment. Before, during, and after operation the hemodynamic and blood gas indices, plasma levels of endotoxin and tumor necrosis factor (TNF), and the injurious effects of burn patients sera on endothelial cells in vitro were determined in patients of the EEE group. The incidence of OD and MODS was decreased significantly (11.4%) in patients of the EEE group, and the cure rate increased greatly (85.7%). The cardiac output dropped to 77.8% of its preoperative level at the end of escharectomy but began to rise at 2 hours and returned to its baseline levels at 24 hours after operation. Plasma levels of endotoxin and TNF and levels of lactic dehydrogenase and 6-keto-prostaglandin F1|ga in the endothelial cell culture media were all reduced profoundly. The cultured endothelial cells maintained their original morphology. The findings substantiate the hypothesis that eschar excision en masse at one operation is feasible and effective in preventing and treating early postburn OD and MODS, mainly by alleviating systemic inflammatory response syndrome and endothelial cell injury.

Thrombin peptide (TP508) treatment of rat growth plate cartilage cells promotes proliferation and retention of the chondrocytic phenotype while blocking terminal endochondral differentiation

A synthetic peptide representing the receptor‐binding domain of human thrombin (TP508, also known as Chrysalin®) accelerates fracture repair in rats via endochondral ossification and promotes repair of rabbit cartilage defects. To understand how this peptide might stimulate cartilage and bone formation, we employed an established in vitro model of growth plate cartilage regulation. Rat costochondral cartilage resting zone and growth zone chondrocytes were treated with 0, 0.07, 0.7, or 7 μg/ml TP508 or a scrambled peptide, TP508‐SP. Proliferation ([3H]‐thymidine incorporation) was examined in pre‐confluent cultures; effects on cell number, alkaline phosphatase activity, [35S]‐sulfate incorporation, and responsiveness to vitamin D metabolites were tested using confluent cultures. TP508 did not affect proliferation of resting zone cells but it caused a dose‐dependent increase in cell number and DNA synthesis of growth zone cells. Alkaline phosphatase specific activity of resting zone cells was reduced by TP508, whereas [35S]‐sulfate incorporation was increased. Neither parameter was affected in growth zone cell cultures. TP508 treatment for 24 h did not induce resting zone cells to respond to 1α,25(OH)2D3, either with respect to alkaline phosphatase activity or proteoglycan production. In contrast, TP508 treatment reduced the stimulatory effect of 24R,25(OH)2D3 on alkaline phosphatase but it did not alter the stimulatory effect of 24R,25(OH)2D3 on [35S]‐sulfate incorporation. In cultures treated for 48, 72, or 140 h with TP508, 1α,25(OH)2D3 restored alkaline phosphatase activity to control levels but did not stimulate activity over levels observed in untreated control cultures. The stimulatory effect of TP508 on [35S]‐sulfate incorporation was evident up to 48 h post‐confluence but at later time points, proteoglycan production was comparable to that seen in control cultures, control cultures challenged with 1α,25(OH)2D3, and cultures treated with TP508 followed by 1α,25(OH)2D3. TP508‐SP had no effect on any of the parameters tested. These results indicate that TP508 exerts maturation specific effects on chondrocytes in the endochondral lineage, promoting cartilage extracellular matrix synthesis over endochondral differentiation in resting zone cells and proliferation over differentiation of growth zone cells.

Glycochenodeoxycholic acid inhibits calcium phosphate precipitation in vitro by preventing the transformation of amorphous calcium phosphate to calcium hydroxyapatite.

Calcium hydroxyapatite can be a significant component of black pigment gallstones. Diverse molecules that bind calcium phosphate inhibit hydroxyapatite precipitation. Because glycine-conjugated bile acids, but not their taurine counterparts, bind calcium phosphate, we studied whether glycochenodeoxycholic acid inhibits calcium hydroxyapatite formation. Glycochenodeoxycholic acid (2 mM) totally inhibited transformation of amorphous calcium phosphate microprecipitates to macroscopic crystalline calcium hydroxyapatite. This inhibition was not mediated by decreased Ca2+ activity. Taurocholic acid (2-12 mM) did not affect hydroxyapatite formation, but antagonized glycochenodeoxycholic acid. Both amorphous and crystalline precipitates contained a surface fraction relatively rich in phosphate. The surface phosphate content was diminish by increasing glycochenodeoxycholic acid concentrations, and this relationship was interpreted as competition between bile acid and HPO4(-4) for binding sites on the calcium phosphate surface. A phosphate-rich crystal surface was associated with rapid transition from amorphous to crystalline states. These results indicate that glycochenodeoxycholic acid prevents transformation of amorphous calcium phosphate to crystalline hydroxyapatite by competitively inhibiting the accumulation of phosphate on the crystal embryo surface.

Activation of human neutrophils by calcium carbonate polymorphs.

Gallstone formation is frequently accompanied byinflammation of the gallbladder mucosa. Some gallstonecomponents such as cholesterol, calcium bilirubinate,and calcium hydroxyapatite have been previously shown to activate neutrophils. We investigatedthe effect on neutrophils of the calcium carbonatepolymorphs aragonite, calcite, and vaterite (all foundin gallstones). By chemiluminescence, superoxide, and degranulation assay, all three crystalswere shown to cause rapid activation of neutrophils. Thepotency of the crystals was aragonite > vaterite >calcite. In vivo, crystals may be plasma-protein-coated before they encounter neutrophils; thereforesome experiments were repeated using crystals that hadbeen preincubated with plasma. For aragonite andvaterite, protein adsorption decreased thechemiluminescence response by approximately 50%. In contrast,protein-coated calcite crystals elicited a greaterchemiluminescence response than did uncoated crystals.In summary, the calcium carbonate polymorphs are potent activators of neutrophils and thus have thepotential to contribute to gallstone-associatedcholecystitis.

Interaction of human gallbladder mucin with calcium hydroxyapatite: Binding studies and the effect on hydroxyapatite formation

Calcium hydroxyapatite (HAP) crystals formed in vitro in the presence of polymeric human gallbladder mucin (1.0 mg/mL) were smaller (0.75 ± 0.39 μm) than control crystals (7.86 ± 2.76 μm), but the mucin did not affect the kinetics of crystal formation or alter the amount of mineral phase present at equilibrium. In contrast, glycopeptide subunits produced by proteolysis of the native mucin had no effect on HAP crystal size. Both native mucin and glycopeptides bound to mature HAP crystals, but the glycopeptides were much more readily displaced by phosphate ions. Therefore, in experiments where HAP was being formed, the phosphate ions inhibited the interaction of glycopeptides with the nascent HAP. These results indicate that gallbladder mucin may modulate HAP formation in vivo, and that this ability may be altered during pathological states, such as neutrophil infiltration or bacterial colonization, that may cause the release of proteinases capable of digesting mucin.