Effect of a freeze-dried CMC/PLGA microsphere matrix of rhBMP-2 on bone healing.

The hypothesis of this research was that implants of poly(lactide-co-glycolide) (PLGA) microspheres loaded with bone morphogenetic protein-2 (rhBMP-2) and distributed in a freeze-dried carboxymethylcellulose (CMC) matrix would produce more new bone than would matrix implants of non-protein-loaded microspheres or matrix implants of only CMC. To test this hypothesis it was necessary to fashion microsphere-loaded CMC implants that were simple to insert, fit precisely into a defect, and would not elicit swelling. Microspheres were produced via a water-in-oil-in-water double-emulsion system and were loaded with rhBMP-2 by soaking them in a buffered solution of the protein at a concentration of 5.4 mg protein per gram of PLGA. Following recovery of the loaded microspheres by lyophilization, matrices for implantation were prepared by lyophilizing a suspension of the microspheres in 2% CMC in flat-bottom tissue culture plates. Similar matrices were made with 2% CMC and with 2% CMC containing blank microspheres. A full-thickness calvarial defect model in New Zealand white rabbits was used to assess bone growth. Implants fit the defect well, allowing for direct application. Six weeks postsurgery, defects were collected and processed for undecalcified histology. In vitro, 60% of the loaded rhBMP-2 released from devices or microspheres in 5 to 7 days, with the unembedded microspheres releasing faster than those embedded in CMC. In vivo, the rhBMP-2 microspheres greatly enhanced bone healing, whereas nonloaded PLGA microspheres in the CMC implants had little effect. The results showed that a lyophilized device of rhBMP-2/PLGA microspheres in CMC was an effective implantable protein-delivery system for use in bone repair.

Influence of dietary protein and lipid on weight loss in obese ovariohysterectomized cats.

OBJECTIVE: To determine effects of dietary lipid and protein on development of hepatic lipidosis (HL) and on physical and biochemical indices following rapid weight loss in cats. ANIMALS: 24 ovariohysterectomized cats. PROCEDURE: Cats were fed a high energy diet until they gained 30% of their ideal body weight and then randomly assigned to receive 1 of 4 weight-reduction diets (6 cats/diet) at 25% of maintenance energy requirements per day. Diets contained a low or high quality protein source and a lipid source deficient or sufficient in long chain essential fatty acids (LCEFA). Serum and plasma samples and liver biopsy specimens were obtained for biochemical analyses and determination of hepatic lipid content before and after weight gain and during and after weight loss. RESULTS: Irrespective of weight-reduction diet fed, all cats lost weight at a comparable rate (4.51 to 5.00 g/d/kg of obese body weight). Three cats developed hepatic lipidosis. Significant changes in plasma insulin, cholesterol, triglyceride, and serum glucose concentrations were detected after weight gain and weight loss in all diet groups, but values for these variables did not differ among groups. CONCLUSIONS AND CLINICAL RELEVANCE: Cats can lose 25 to 30% of their obese body weight over 7 to 9 weeks without developing overt clinical signs of HL, provided that weight-reduction diets are highly palatable, contain a high quality protein, have a source of LCEFA, and are fortified with vitamins and microminerals. However, rapid weight loss may increase risk factors associated with development of diabetes mellitus.

Two lyophilized polymer matrix recombinant human bone morphogenetic protein-2 carriers in rabbit calvarial defects.

We have developed a lyophilized bone morphogenetic protein (BMP) delivery device that can be formulated to control release over 2 to 8 weeks. Bioerodible poly (d,l lactide-co-glycolide) particles loaded with 90 micrograms recombinant human BMP-2 were suspended in either carboxymethylcellulose (CMC) or methylcellulose (MC) implants. Plain CMC and MC implants served as controls, as did a nonimplanted group. A total of 40 rabbits was evaluated histologically 2, 4, or 8 weeks after receiving circular full-thickness 15-mm calvarial defects. MC appeared to prevent prolapse of periosteum and dura into the defects and did not elicit bone growth. Addition of BMP improved the result. CMC implants appeared to encourage bone growth even in the absence of BMP. When BMP was added, new bone formed earlier. CMC may influence new bone formation because it is hydrophilic. MC is less hydrophilic and may cause undue inflammation. Either can be combined with BMP to produce unitary devices that are easy to make and use.

Fish oil dietary supplementation for prevention of indomethacin induced gastric and small bowel toxicity in healthy volunteers.

OBJECTIVE: To determine if N-3 fatty acid (fish oil) dietary supplements could favorably alter indomethacin induced gastric and small bowel toxicity related to use of nonsteroidal antiinflammatory drugs (NSAID). METHODS: Healthy volunteers consumed 8 g of N-3 fatty acids for 16 weeks, while controls consumed corn oil. Subjects ingested indomethacin 50 mg tid between Weeks 12 and 16. Upper gastrointestinal (GI) endoscopy with biopsies and Cr-EDTA swallows were performed at Week 12 and again at Week 16. Biopsy specimens were graded for inflammation and endoscopic scores were recorded. RESULTS: No significant differences were seen between groups in any study variable, although the direction of change favored the fish oil subjects for inflammation scores in both the stomach and duodenum (fish oil subjects -0.50 +/- 1.2 stomach, -0.28 +/- 0.97 duodenum; and corn oil subjects +0.10 +/- 0.84 stomach, +0.20 +/- 0.79 duodenum; p = 0.086). Direction of change in stomach inflammation showed a trend favoring fish oil (p = 0.056 by chi square). CONCLUSION: Although no significant differences were seen between groups, the changes observed in inflammation suggest a possible benefit of fish oil in the amelioration of NSAID induced GI inflammation.

Relationship of phosphatidylcholine to hydrophobic surfactant on rat intestinal chylomicron secretion.

Hydrophobic surfactants such as Poloxalene inhibit triglyceride secretion into lymph by enterocytes. The inhibitory effect of these agents on triglyceride secretion is reversed when lipid presented for absorption is exclusively in the form of phosphatidylcholine (PC) and not triglyceride. The present investigation performed in conscious mesenteric lymph fistula rats was designed to determine whether various mixtures of triglyceride and PC given intraduodenally with Poloxalene would also reverse the inhibitory effect of Poloxalene on triglyceride secretion into lymph. A 50-50 mixture of triolein (TO) and PC resulted in normal triglyceride secretion into lymph. However, when the mixture of lipids was 75-25, TO to PC, results for triglyceride recovery in lymph were considerably reduced. The transport rate for triglyceride into lymph was not as depressed, however, as observed for Poloxalene treated rats given lipid for absorption basically in the triglyceride form. Substitution of phosphatidylethanolamine for PC had no beneficial effect on triglyceride secretion in Poloxalene treated rats. It is concluded that PC can reverse the inhibitory effect of Poloxalene on triglyceride secretion into lymph even when considerable amounts of triglyceride along with PC are presented for absorption.

The evaluation of lyophilized polymer matrices for administering recombinant human bone morphogenetic protein-2.

Novel unitary devices, prepared by lyophilization of viscous solutions of sodium carboxymethylcellulose (CMC) and methylcellulose (MC), were evaluated as sustained-release delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). In vitro characterization of the unitary devices, which contained rhBMP-2-loaded poly (d,l lactide-co-glycolide) (PLGA) bioerodible particles (BEPs), was conducted over a 2-month period. Determinations included buffer uptake, mass and molecular weight loss and rhBMP-2 release from the unitary devices. CMC devices imbibed approximately 16 times their weight of buffer, while with MC, equilibrium uptake was approximately 6 times the dry weight of the devices. Overall mass loss percentages were approximately 55 and 35%, respectively, for CMC and MC devices. rhBMP-2 release from the devices was essentially a triphasic process: an initial phase during which "free" protein (rhBMP-2 present on the surface and within the pores of the PLGA BEPs) was released, a lag period during which no release was discerned, and then release of "bound" rhBMP-2 (protein adsorbed to the BEPs). The release of bound protein correlated with the mass loss of the polymer which began after 3 weeks. Release from the unitary devices was lower than that from the BEPs alone, due to a retardation effect of the gelled CMC/MC polymers. In rabbits in which full-thickness cranial bone defects were created, the implants were well tolerated and induced significant new bone growth during an 8-week evaluation period. The CMC devices appear to have induced bone earlier (at 2 weeks), but this did not affect eventual 8-week results. CMC devices without rhBMP-2 appeared to provide some bone conduction, in contrast to the blank MC devices.

Estrus detection by using vaginal cytologic examination in miniature swine.

Vaginal smears were obtained from four Yucatan miniature swine daily for 69 days and stained with hematologic stain. Epithelial cells were categorized as superficial, large intermediate, small intermediate, or parabasal. Leukocytes were also quantitated. External signs of estrus were recorded, including swelling, discharge, restlessness, or vocalization. Mean age of three of the swine was 147 days at the beginning of the study. The fourth pig was 317 days old. The three younger swine had their first observed estrus at the age of 178 days (range, 167 to 196 days). Mean cycle length was 17 to 21 days. The moving mean leukocyte count (i.e., each value was averaged with the values for the previous day and the following day) always exceeded the epithelial cell count (regardless of type), except during the 3 to 4 days when the pigs exhibited external signs of estrus. Further, epithelial cells were at their peak during estrus, decreasing markedly during diestrus, and increasing again during proestrus. The combined superficial plus large intermediate cell counts were significantly higher during estrus than during diestrus or proestrus. We conclude that daily vaginal smears can be used to determine the stage of estrus in Yucatan pigs.

Experimental rectus abdominis myocutaneous and rectus abdominis myoperitoneal flaps as urinary bladder wall substitutes in miniature swine.

An experiment was performed in Yucatan miniature swine to determine the feasibility and characteristics of musculocutaneous or musculoperitoneal flaps as urinary bladder wall substitutes. In five swine, a single-pedicle skin island flap (rectus abdominis myocutaneous, RAM/C) was sutured into the bladder. In five other swine the flap was a peritoneum island (rectus abdominis myoperitoneal, RAM/P). Three swine were sham-operated controls. The patches were in place for 20 weeks, remaining viable and elastic. Inflammation, maceration, and infection were absent. Skin patch histology was unchanged. The peritoneal patches became resurfaced with uroepithelium. The sham bladder volume (ml/kg body weight) did not differ significantly from RAM/P bladder volume (p = 0.54). RAM/C bladders were slightly smaller than shams (p = 0.11) and significantly smaller than RAM/P bladders (p = 0.03). Substitution of the bladder wall with RAM patch flaps is feasible. This is an important preliminary step toward our goal of nonenteral urinary bladder wall substitution.

Peritoneal dialysis therapy for patients with liver and renal failure with ascites.

Two patients with severe liver disease complicated with ascites were recently treated at our institution. Both rapidly developed renal failure. In one patient, liver disease was the result of alcohol abuse, and in the other, was due to malnutrition associated with obesity and acute weight loss. The only reasonable therapeutic approach for these patients was believed to be a course of peritoneal dialysis, along with other supportive measures. In both cases, the management was successful. Furthermore, it was possible to discontinue dialysis at the time of discharge. We conclude that peritoneal dialysis can be a life-saving procedure in patients with severe liver disease and ascites complicated by renal failure.

Carbohydrate content of apolipoprotein B-48 from rat chylomicrons of varying density.

Monosaccharide composition was determined in apolipoprotein B-48 (apoB) of chylomicrons of rat mesenteric lymph. Chylomicrons were separated into three fractions based on density. Triglyceride and apolipoprotein content were determined in each. ApoB was isolated and quantified using precipitation with isopropanol. Chylomicrons were collected in lymph under normal conditions, and with Poloxalene 2930 when chylomicron secretion was inhibited. Most of the triglyceride was carried in the least dense fraction, while the highest apoB content was in the most dense fraction under normal conditions. Mannose and galactosamine contents of apoB were similar in all fractions while contents of both glucosamine and galactose were highest in the least dense fraction. When chylomicron secretion was inhibited by Poloxalene, the amount of triglyceride recovered in the least dense fraction was significantly reduced. Despite the inhibition of lipid transport in the least dense fraction of chylomicrons by Poloxalene, there was little change in apoB recoveries and in the relative content of various monosaccharides in the apoB from each of the three fractions as compared to results obtained during lipid absorption under normal conditions. In conclusion, carbohydrate composition of apoB of chylomicrons is heterogeneous and varies with chylomicron density.

Hydrophobic surfactant inhibits hypercholesterolemia in pair-fed rabbits on a cholesterol-free, low-fat diet.

Poloxalene, a hydrophobic surfactant, is known to prevent hypercholesterolemia in animals fed a high-fat, high-cholesterol diet. It has not been demonstrated, however, whether this agent is of benefit when hypercholesterolemia is induced in animals by means other than the feeding of a high-fat diet. In this study, hypercholesterolemia was produced in rabbits by feeding a low-fat, cholesterol-free diet with dietary protein supplied by casein for a period of 8 weeks. Controls were given this diet without poloxalene and experimentals were given the diet with poloxalene. Total serum cholesterol levels increased in both groups, but the rise was greater for the control group. Lipoprotein analysis performed at the conclusion of the study showed significantly greater low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels in the control group as compared to the experimental group. Total protein and apolipoprotein B (apo B) were also greater in control LDL. It was concluded that poloxalene favorably affects this model of hypercholesterolemia as total serum cholesterol, LDL cholesterol, and LDL apo B were all less and the HDL cholesterol to LDL cholesterol ratio was higher in surfactant-treated rabbits.

Alterations of secretory pattern of intestinal lipoproteins by the benzoyl ester derivative of poloxalene surfactant (BEP).

Hydrophobic surfactant BEP was administered intraduodenally as part of lipid emulsion to rats with cannulated mesenteric lymphatic duct. The effect on the size and composition of intestinal triglyceride-rich lipoproteins (TRLp) was assessed by comparing the results with those obtained during infusion of the lipid emulsion alone. Administration of BEP decreased intestinal capacity to transport triglyceride and cholesterol in large TRLp, SF greater than 2000, and resulted in a significant reduction of total triglyceride in lymph. Non-apoB apolipoproteins decreased significantly in large and increased in small TRLp without appreciable change in total content. Contrary to these findings total apoB protein content increased significantly, primarily due to an increase in small TRLp. Changes in lipid and protein content of apolipoproteins produced by BEP resulted in increased ratios of apolipoproteins to lipids in TRLp. It was therefore concluded that inhibition of lipid transport by BEP was not a result of apolipoprotein deficiency. Discontinuation of BEP administration resulted in a prompt recovery of the intestinal lipid transport system.

Hydrophobic surfactant effects on aortic cholesterol accumulation and atherosclerosis in hypercholesterolemic rabbits.

Studies were performed in hypercholesterolemic rabbits to determine whether the hydrophobic surfactant, Poloxalene 2930 (Pol), is of benefit under these conditions. Lipoprotein analyses plus chemical and morphologic studies of the aorta were performed to evaluate the results. In one study, rabbits were made hypercholesterolemic by dietary means and then divided into two groups and given a cholesterol-free diet with one group additionally given Pol with treatment continued for 10 weeks. Pol treatment resulted in less atherosclerosis but the mechanism for this effect was not apparent from lipoprotein analysis. In the other study 3 groups of rabbits were given a cholesterol-rich diet for 16 weeks. Two groups received Pol supplement with one of these groups receiving a dose that was too small to prevent hypercholesterolemia. In this group plus the group on diet alone comparable degrees of hypercholesterolemia were maintained throughout the study. Lipoprotein abnormalities were similar in these two groups except that those on Pol had a more normal cholesterol to apolipoprotein B ratio. The amount of atherosclerosis in both groups was mild but aortic cholesterol content was much less for the Pol group. It is concluded that Pol limits cholesterol accumulation in the aortic wall of hypercholesterolemic rabbits and can retard the development of atherosclerosis.

Poloxalene 2930, a hydrophobic surfactant that prevents atherosclerosis, alters composition of rabbit lipoproteins.

Poloxalene 2930, a hydrophobic surfactant, was incorporated into an atherogenic diet at dose levels, 0.5% and 1% of the diet, and fed to rabbits for 10 weeks. Another group received plain atherogenic diet alone and a control group was fed chow. After this period, serum lipoproteins were separated by centrifugation and analyzed. The composition of lipoproteins from rabbits on atherogenic diet was abnormal. The cholesterol: triglyceride ratio of every lipoprotein fraction was significantly increased as was the cholesterol: protein ratio of very low density lipoproteins. Supplementing the diet with Poloxalene 2930 prevented these alterations. In all groups on the atherogenic diet the percentage of apolipoprotein E in VLDL and HDL increased. In the case of Poloxalene-treated rabbits this developed even though serum cholesterol levels were normal or slightly increased. It is concluded that Poloxalene 2930 has a systemic effect on lipoproteins which may contribute to its antiatherogenic action.

Absorption and excretion of the hydrophobic surfactant, 14C-poloxalene 2930, in the rat.

Absorption and excretion of 14C-Poloxalene 2930 (PX), a nonionic hydrophobic surfactant of large molecular weight, were studied using bile fistula rats. Approximately half of the dose infused intraduodenally was absorbed and some of the absorbed surfactant was excreted in bile. The remainder was excreted in urine. Only trace quantities of the 14C-PX were recovered in liver and carcass at termination of the study. Two studies were also performed with 14C-PX incorporated into the diet. In the first feeding study of 7 days duration, most of the agent was excreted via the gastrointestinal tract within 72 hr of discontinuing treatment. In the second study, rats were fed dietary 14C-PX for 7, 14, or 23 days to determine whether the surfactant continued to accumulate in the body as the test period was extended. Further accumulation did occur between the 7th and 14th days but not when feeding was continued for a total of 23 days. Of the amount of 14C-PX ingested after 23 days of feeding, essentially all was excreted by the end of 7 days after discontinuing treatment. These studies indicate that despite its large molecular weight of about 3,000 some 14C-PX is absorbed. Furthermore, absorbed material is promptly excreted in bile and urine with little retained in body tissues.

Effect of surfactant poloxalene 2930 on food intake, lipid absorption, and serum cholesterol in rats.

Effect of hydrophobic surfactant, poloxalene 2930, on lipid absorption was studied in rats. Under acute conditions with surfactant infused intraduodenally with a lipid meal absorbed lipid accumulated abnormally in the enterocytes. This effect was quickly reversed after terminating treatment. Long-term administration of poloxalene given in semipurified diets resulted in changes in food intake, weight gain, fecal fat output, and serum cholesterol concentrations. The composition of the diet used as the vehicle for administration had a considerable effect on these results. When semipurified diets were used, food intake and weight gain were greatest when the dietary fat content was at the highest level. When the surfactant was given in ground chow, food intake was not affected and weight gain was only slightly, but significantly, less than the controls as a result of mild fat malabsorption. It is concluded that poloxalene 2930 affects lipid absorption, food intake, and serum cholesterol concentration but that results of this treatment are considerably affected by dietary factors.