Sealing effects of cross-linked gelatin.

Surgical sealants form gel when applied to tissues. Currently, fibrin sealant has been successfully used in many surgical fields, but it has several disadvantages, including possible virus transmission, low adhesive strength, and high cost. In this study, gelatin and glutaraldehyde (GA) solutions were chosen to demonstrate the effectiveness of cross-linked gelatin gel as sealant and barrier, both of which have long been used in medical applications. It was found that the gelatin gel prepared from 26 wt% gelatin and 1 wt% GA solutions exhibited bonding strength almost three times higher than that of fibrin glue. The bonding strength increased with the increasing gelatin and GA concentrations. When a needle hole on PTFE vascular grafts was sealed with the gelatin gel, the water-resistant pressure significantly increased upon rubbing and was twice higher than that of fibrin glue. The cytotoxicity of gelatin gel was found to be much lower than that of albumin glue prepared at the same composition as commercially available BioGlue®. The gelatin gel was found to be also effective as barrier to prevent adhesion in a rat cecum abrasion model.

Alveolar bone regeneration using absorbable poly(L-lactide-co-epsilon-caprolactone)/beta-tricalcium phosphate membrane and gelatin sponge incorporating basic fibroblast growth factor.

The aim of this study was to evaluate the effects of combining a porous poly(L-lactide-co-epsilon-caprolactone)/beta-tricalcium phosphate membrane and gelatin sponge incorporating basic fibroblastic growth factor (bFGF) on bone regeneration in mandibular ridges. Four full-thickness saddle-type defects (10 mm long x 5 mm deep) were symmetrically created in both edentulous mandibular alveolar ridges of 6 beagles. The dome-shaped membrane was secured to each defect site, and a gelatin sponge containing 200 microg bFGF was implanted on the left side of each defect (experimental group). Only the membranes (control group) were secured to the defect sites on the right. Three and 6 months later, 3 animals were killed. Bone regeneration was analyzed by soft X-ray photographs, micro-computed tomography (CT) images, and peripheral quantitative CT (pQCT), and then examined histologically. Soft X-ray examination revealed an increase in new bone volume in the experimental group 6 months postoperatively. pQCT showed that immature bone density was higher in the experimental group. Micro-CT images revealed well formed new bone along the original contour of the dome-shaped membrane in the experimental group. Histologically, inflammatory infiltration of tissue surrounding the membranes was slight. These results suggest that combining the poly(L-lactide-co-epsilon-caprolactone)/beta-tricalcium phosphate membrane and bFGF-gelatin sponge is promising for alveolar ridge reconstruction.

Effect of a static magnetic field on ion transport in a cellulose membrane.

A cellulose membrane was exposed to the static magnetic field (SMF) in the presence of KCl solution and ion transport through the membrane was measured before and after the SMF exposure. SMF at 0.24 T significantly enhanced the rate of ion transport, especially after the first exposure (p<0.05), while the increased ion transport rate did not return to the initial basal level after exchange of the aqueous medium. These results suggest that an irreversible, temporal conformation change took place on the cellulose membrane or on the water bound to the cellulose surface. The accelerating effect of SMF on the ion transport seems to have occurred as a result of stabilized hydration layer on the cellulose surface.

Usefulness of microspheres composed of gelatin with various cross-linking density.

The release rate of insulin, as a model peptide, from gelatin microspheres (GM) prepared with gelatin having various cross-linking densities in vitro was examined. The release of insulin from GM showed the burst effect, followed by a slow release phase regardless of the cross-linking density of gelatin. The total amount of insulin released in 2 weeks decreased with increasing cross-linking density of gelatin. The release rate of insulin within 6 h was well correlated with the cross-linking density of gelatin. The remaining amounts of both insulin and GM after injection of insulin incorporated in GM to mice femoral muscle tissue were also examined in vivo. Both insulin and GM rapidly disappeared from the injection site within 1 day, and thereafter slowly disappeared over 14 days. The time courses of the remaining amounts were fairly similar to each other. Furthermore, the remaining amount of insulin 1 day after administration was well correlated with the cross-linking density of gelatin. These data suggest that insulin was released from GM with the degradation of GM in mice muscular tissue and that the release rate of insulin can be controlled by modifying the cross-linking density of gelatin. In conclusion, the control of the release rate of insulin from GM can be achieved under both in vitro and in vivo conditions by gelatin through the alteration of cross-linking conditions.

Laser-perforated membranous biomaterials induced pore size-dependent bone induction when used as a new BMP carrier.

Previously we found that laser perforation of a collagen membrane (35 microm thickness, Koken Co., Tokyo) produced an effective bone morphogenetic protein (BMP) carrier, if the created pore sizes were larger than 0.5 mm. In this study we applied the same technique to create pores of 0.2 and 1.0 mm in a thicker (1.2 mm thickness) porous biodegradable membrane made of polylactic acid and an epsilon-caprolactone copolymer (PLA-CL) to obtain an effective membranous BMP carrier with higher mechanical strength. Pieces of PLA-CL (0.5 x 1.0 x 0.12 cm) combined with rhBMP-2 (5 microg) were implanted subcutaneously into rats and processed for analyses at 1-3 weeks. The laser-perforated PLA-CL membranes equipped with 1.0 mm pores induced mineralization beginning from the margins of the pores judging from the X-ray patterns, but bone formation seemed to proceed irregularly inside the pores. In the perforated PLA-CL membrane with 1.0-mm pores bone formation did not significantly increase compared with the nonperforated one. This was due to the fact that the PLA-CL membrane was already a porous structure (85% porosity). In contrast with laser-perforated PLA-CL 0.2 mm pores, bone was induced on the collagen fibers and fiber bundles inside the pores. The different patterns of bone formation between the PLA-CL membranes with 1.0 and 0.2 mm pores seemed to be related to the active formation of perpendicular collagen fibers through the 0.2 mm pores.

Static magnetic field effects on bone formation of rats with an ischemic bone model.

Effects of a static magnetic field were studied on bone formation using an ischemic rat femur model. Metal rods were prepared from magnetized and unmagnetized samariun cobalt to have tapered structure, both with the same geometrical dimension, and were implanted transcortically into the middle diaphysis of 88 rat femurs. Both sides of the rat femoral artery were ligated to create an ischemic bone model, followed by implantation of the tapered rod to the femur. The bone mineral density (BMD) and weight of the femurs were measured at 1st and 3rd week after implantation. The result at the 3rd week post-implantation revealed that the BMD and weight of the ischemic bone model rats were significantly reduced, compared with that of non-operated femur. It was also found that the magnetized group had significantly higher bone weights than the unmagnetized (p<0.05). The BMD of the rats implanted with the magnetized rods were similar to those of the non-operated (p>0.05). This enhancement of the femoral bone formation of the ischemic rat model by the static magnetic field seems to be due to the improved blood circulation of the femur.

A trial to prepare biodegradable collagen-hydroxyapatite composites for bone repair.

This paper is a trial to prepare collagen-hydroxyapatite composites in vitro by an alternate immersion method. Collagen sponges of different biodegradabilities were prepared through chemical cross-linking of Type I collagen with glutaraldehyde (GA) at concentrations of 0.2, 1.0, and 2.0 wt%. The sponges were immersed at 37 degrees C in Tris-HCl-buffered solution containing 200 mM CaCl2 (pH 7.4) for 2 h and then in an aqueous solution of 120 mM Na2HPO4 (pH 9.3) for a 2 h further (one immersion cycle). The alternate immersion cycle was repeated for different times to obtain collagen-hydroxyapatite composites. The characterization of the resulting composites was performed by Fourier transform infrared spectroscopy (FT-IR). X-ray diffraction (XRD), and scanning electron microscopy (SEM). The weight of composites increased with an increase in immersion cycles and the rate of increase became greater with higher GA cross-linking levels for collagen sponge preparation. The pH of the phosphate solution decreased with the immersion cycle, which suggests H+ generation accompanied hydroxyapatite formation. Irrespective of the GA concentration and immersion cycle, every composite showed IR absorption bands attributable to phosphate and hydroxyl groups at 950-1100 or 550-650 and 3000-3500 cm(-1) and broad peaks specific to hydroxyapatite on the XRD charts. SEM study revealed small white clusters of hydroxyapatite interspersed uniformly on/in the collagen framework without any preferential orientation. The composite prepared from 0.2 wt% GA cross-linked collagen sponge which showed favourable characteristics was applied to a rat skull defect to evaluate its osteoconductivity as well as biodegradability. The formation of new bone tissue was histologically observed at the defect 12 weeks after application in marked contrast to the collagen sponge alone. The composite degraded without any inflammation reaction. It is concluded that the collagen-hydroxyapatite composite prepared by the present method is a biodegradable biomaterial of osteoconductivity applicable to bone repair.

Development of an artificial dermis preparation capable of silver sulfadiazine release.

This article describes the antibacterial effects of an artificial dermis impregnated with silver sulfadiazine (Ag-SD) in vitro as well as in vivo. In the in vitro test, silver release from the artificial dermis impregnated with Ag-SD, by immersion in collagenase solution was controlled by the degradation of the collagen sponge. The artificial dermis impregnated with 3% or higher doses of Ag-SD completely suppressed the growth of Pseudomonas aeruginosa (Ps.) or Staphylococcus aureus (St.). The cytotoxicity test revealed that impregnation of 5% or higher doses of Ag-SD suppressed the growth of fibroblasts. However, when the artificial dermis impregnated with Ag-SD was implanted into full-thickness skin defects on the backs of guinea pigs, no tissue damage was histologically observed around the implanted site of the dermis. In the in vivo test, the artificial dermis impregnated with 10% Ag-SD, which was grafted on experimentally contaminated wounds in the backs of guinea pigs, macroscopically suppressed degradation of the collagen sponge, and significantly reduced the growth of both Ps. and St., compared with artificial dermis without Ag-SD. We conclude that collagen sponge impregnated with Ag-SD is a promising artificial dermis applicable to treat contaminated wounds.

The pH dependence of monofilament sutures on hydrolytic degradation.

Hydrolytic degradation of two nonabsorbable sutures, four absorbable sutures, and a new type of absorbable suture was studied in buffered media of various pHs at 37 degrees C. The pH levels fixed in this study were 1.0, 7.4, 8.5, and 10.5. Physical measurements were made on the retention of tensile strength and melting temperature of the sutures after hydrolysis for 12 weeks. Sutures containing glycolic acid as a comonomer exhibited enhanced degradation in alkaline media, similar to polyglycolide multifilament sutures. Poly-p-dioxanone (PDS II) suture lost strength to a significant extent at pH 1.0, suggesting that care should be taken when this suture is used for closing tissues in contact with acidic media, such as the stomach. In marked contrast, the degradation of lactide-epsilon-caprolactone copolymer [P(LA/CL)] suture was not sensitive to the pH of media. The surface morphology of hydrolyzed sutures varied, depending on the pH of media. Particularly, moon-crater-shaped impressions were observed on glycolide-epsilon-caprolactone copolymer (MONOCRYL) and glycolide-trimethylene carbonate-dioxanone copolymer (BIOSYN) sutures. Among the nonabsorbable sutures, nylon (ETHILON) exhibited the fastest loss of strength in acidic buffer solution, and polypropylene (PROLENE) suture retained most of its initial strength at all pHs studied.

Tissue-engineered vascular autograft: inferior vena cava replacement in a dog model.

Tissue-engineered vascular autografts (TEVAs) were made by seeding 4-6 x 10(6) of mixed cells obtained from femoral veins of mongrel dogs onto tube-shaped biodegradable polymer scaffolds composed of a polyglycolid acid (PGA) nonwoven fabric sheet and a copolymer of L-lactide and caprolactone (n = 4). After 7 days, the inferior vena cavas (IVCs) of the same dogs were replaced with TEVAs. After 3, 4, 5, and 6 months, angiographies were performed, and the dogs were sacrificed. The implanted TEVAs were examined both grossly and immunohistologically. The implanted TEVAs showed no evidence of stenosis or dilatation. No thrombus was found inside the TEVAs, even without any anticoagulation therapy. Remnants of the polymer scaffolds were not observed in all specimens, and the overall gross appearance similar to that of native IVCs. Immunohistological staining revealed the presence of factor VIII positive nucleated cells at the luminal surface of the TEVAs. In addition, lesions were observed where alpha-smooth muscle actin and desmin positive cells existed. Implanted TEVAs contained a sufficient amount of extracellular matrix, and showed neither occlusion nor aneurysmal formation. In addition, endothelial cells were found to line the luminal surface of each TEVA. These results strongly suggest that "ideal" venous grafts with antithrombogenicity can be produced.

Interaction of poly(2-acrylamido 2-methylpropane sulfonate)-grafted polystyrene beads with cationic complement proteins.

Influence of various biomaterials on the complement system in serum has been intensively studied by many research groups, since activation of the complement pathway in vivo has been known to give rise to some pathological conditions, such as inflammation and anaphylaxis. Much effort has been devoted to develop new materials that do not activate or deteriorate the complement system. The present work is aimed at revealing the mode of reactions of anionic poly(2-acrylamido 2-methylpropane sulfonate) grafted on polystyrene bead (PAMPS-g-bead) with serum complement. Complement activity assay, determination of complement proteins levels, and immunoblot analysis were carried out for sera pretreated with PAMPS-g-beads. The results clearly showed that, when PAMPS-g-beads were incubated with serum, those beads adsorbed several complement proteins, i.e. C1q, factor D, factor P, C6, and C8, but the generation of activation fragments of complement components was not observed. Especially, factor D was most effectively removed from serum, resulting in potential inhibition of the alternative pathway. A larger amount of PAMPS-g-beads was needed to decrease the serum CH50 level. That may be caused by removal of C6. Although some polyanions, such as dextran sulfate, were reported to activate the complement system, the obtained results indicate that the PAMPS-g-bead is not an activator of the complement pathway, but acts as an adsorbent of complement components. One possible clinical application of the PAMPS-g-beads is adsorption of serum factor D by extracorporeal treatment of patients with renal failure with a high level of factor D, because the increased quantity of factor D in serum may cause consistent activation of the alternative pathway.

Liver targeting of interferon-beta with a liver-affinity polysaccharide based on metal coordination in mice.

Frequent and high-dose i.v. injections of interferon-beta (IFN-beta) have been used clinically to treat patients with viral hepatitis despite various side effects. Because side effects are caused by the systemic effects of IFN-beta, the purpose of this study was to target the drug specifically to the liver, thus reducing the adverse events. A chelating residue, diethylenetriaminepentaacetic acid (DTPA), was introduced to pullulan, a water-soluble polysaccharide with a high affinity for the liver. Murine IFN-beta could be coordinately conjugated with the DTPA-pullulan by simple mixing in an aqueous solution containing zinc ion (Zn2+). Intravenous injection of the IFN-beta-DTPA-pullulan conjugate with Zn2+ coordination enhanced liver induction of an antiviral enzyme, 2',5'-oligoadenylate synthetase (2-5AS), to a greater extent than that by free IFN-beta, although the 2-5AS levels in the liver depended on the mixing ratio of the IFN-beta/DTPA residue of DTPA-pullulan/Zn2+. In addition, the duration of the liver 2-5AS induction by the IFN-beta-DTPA-pullulan conjugate with Zn2+ coordination was longer than that by free IFN-beta. The liver targeting of IFN-beta by DTPA-pullulan with Zn2+ coordination may be a promising IFN therapy.

An oral drug delivery system targeting immune-regulating cells ameliorates mucosal injury in trinitrobenzene sulfonic acid-induced colitis.

Control of immune-regulating cells in the colonic mucosa is important in the treatment of patients with inflammatory bowel disease (IBD). The aim of study was to examine the therapeutic effect of dexamethasone (DX) microspheres on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats, a model for human Crohn's disease. DX microspheres and DX alone were administered orally to rats with TNBS-induced colitis. The macroscopic score, histological score, myeloperoxidase (MPO) activity, nitric oxide (NO) production, and gene expressions of proinflammatory cytokines, cyclooxygenase (COX)-1, and COX-2 in the colonic tissue were determined. Proliferating cell nuclear antigen (PCNA) staining and expression of nuclear transcription factor (NF)-kappaB in colonic tissues were also investigated. Macroscopic score, histological score, MPO activity, and NO production in rats treated with DX microspheres were significantly lower than in those treated with DX alone. The gene expression of proinflammatory cytokines and COX-2 in rats treated with DX microspheres was down-regulated, compared with that in rats treated with DX alone. The number of PCNA-positive cells in the DX microsphere group was larger than in the group treated with DX alone. DX microspheres suppressed NF-kappaB activation in TNBS-induced colitis more strongly than DX alone. Oral administration of DX microspheres appears to ameliorate mucosal injury in TNBS-induced colitis. This drug delivery system could be an ideal therapy for human IBD.

Use of a bioabsorbable implant for the repair of severed digital flexor tendons in four horses.

A new bioabsorbable implant composed of poly-L-lactic acid was used to repair the severed digital flexor tendons of four horses. The limbs were immobilised with distal casts which were changed after six to eight weeks and removed after 12 to 16 weeks. The horses were followed clinically and ultrasonographically for from seven to 19 months after the surgery. The ultrasonographic examination after the cast had been removed showed that the implants had been well incorporated into scar tissue. Two of the horses were mildly lame at the trot seven months after the surgery, but had returned to work after 12 months. The other two horses are still lame. No complications were observed with the implant.

Controlled release of growth factors based on biodegradation of gelatin hydrogel.

To develop a carrier for the controlled release of biologically-active growth factors, biodegradable hydrogels were prepared through glutaraldehyde cross-linking of gelatin with isoelectric points (IEP) of 5.0 and 9.0, i.e. 'acidic' and 'basic' gelatins, respectively. Radioiodinated growth factors were used to investigate their sorption and desorption from the hydrogel of both types of gelatin. Basic fibroblast growth factor (bFGF) and transforming growth factor-beta1 (TGF-beta1) were well sorbed with time to the acidic gelatin hydrogel, while less sorption was observed for the basic gelatin hydrogel. This could be explained in terms of the electrostatic interaction between the growth factors and the acidic gelatin. However, bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF), though their IEPs are higher than 7.0, were sorbed to the acidic gelatin hydrogel to a smaller extent than the two other growth factors. Under in vitro non-degradation conditions, approximately 20% of the incorporated bFGF and TGF-beta1 was desorbed from the hydrogels within the initial 40 min, followed by no further substantial desorption, whereas large initial desorption was observed for BMP-2 and VEGF. When implanted in the back subcutis of mice, gelatin hydrogels were degraded over time. Each growth factor was retained in vivo being incorporated in the acidic gelatin hydrogel: the smaller the in vitro desorption amount from the hydrogel, the longer the in vivo retention time. The in vivo profile of bFGF and TGF-beta1 retention was in good accordance with that of the hydrogel. These findings indicate that the growth factor immobilized to the acidic gelatin hydrogel through ionic interaction was released in vivo as a result of hydrogel degradation.

Evaluation of gelatin microspheres for nasal and intramuscular administrations of salmon calcitonin.

The suitability of gelatin microspheres for nasal and intramuscular delivery of salmon calcitonin (sCT) was examined. Negatively and positively charged gelatin microspheres were prepared using acidic gelatin [isoelectric point (IEP) value of 5.0] and basic gelatin (IEP=9.0), respectively. The average diameters of positively charged gelatin microspheres in their dried state were 3.4, 11.2, 22.5 and 71.5 microm, while that of negatively charged gelatin microspheres was 10.9 microm. Both types of gelatin microspheres were capable of adhering to the nasal mucosa. The mucoadhesion of positively charged gelatin microspheres was significantly higher than that of their negatively charged counterparts. The absorption of sCT after intranasal and intramuscular administration was evaluated by calculating the area above the hypocalcemic-time curve (AAC) in rats. The AAC values after nasal administration of sCT in positively and negatively charged gelatin microspheres were significantly greater than that in pH 7.0 PBS. Therefore, the nasal absorption of sCT was enhanced by both types of gelatin microspheres. The hypocalcemic effect after administration of sCT in positively charged gelatin microspheres of 11.2 microm was significantly greater than that of negatively charged gelatin microspheres of the same size. On the other hand, AAC values were not affected by their particle sizes. The AAC values after the intramuscular administration of sCT in positively and negatively charged gelatin microspheres were significantly increased compared to that in PBS. Furthermore, the time-courses of the plasma calcium levels differed between positively and negatively charged gelatin microspheres. The hypocalcemic effect of the negatively charged gelatin microspheres tended to appear more slowly and last longer compared to that of positively charged gelatin microspheres. The hypocalcemic effects after intramuscular administration of sCT in gelatin microspheres were not affected by their particle sizes as well as those after intranasal administration. In conclusion, the gelatin microspheres have been shown to be a useful vehicle for nasal or intramuscular delivery of sCT.

Acceleration of fracture healing in nonhuman primates by fibroblast growth factor-2.

One of the greatest needs in the clinical bone field is a bioactive agent to stimulate bone formation. We previously reported that fibroblast growth factor-2 (FGF-2) exhibited strong anabolic actions on bone formation in models of rodents and dogs. Aiming at a clinical application, this study was undertaken to clarify the effect of a single local application of recombinant human FGF-2 on fracture healing in nonhuman primates. After a fracture was created at the midshaft of the right ulna of animals and stabilized with an intramedullary nail, gelatin hydrogel alone (n = 10) or gelatin hydrogel containing 200 microg FGF-2 (n = 10) was injected into the fracture site. Although 4 of 10 animals treated with the vehicle alone remained in a nonunion state even after 10 weeks, bone union was complete at 6 weeks in all 10 animals treated with FGF-2. Significant differences in bone mineral content and density at the fracture site between the vehicle and FGF-2 groups were seen at 6 weeks and thereafter. FGF-2 also increased the mechanical property of the fracture site. We conclude that FGF-2 accelerates fracture healing and prevents nonunion in primates, and therefore propose that it is a potent bone anabolic agent for clinical use.

The efficacy of prevascularization by basic FGF for hepatocyte transplantation using polymer devices in rats.

This study used polymer devices implanted in rats to investigate the effect of prevascularization by basic fibroblast growth factor (bFGF) on hepatocyte transplantation (HTx). Lewis rats served as both donors and recipients. Polyvinyl alcohol (PVA) sponges with either hydrogel containing bFGF (bFGF group) or distilled water (control group) were implanted between the mesenteric leaves of recipient rats. Hepatotrophic stimulation was induced by a portacaval shunt and a 70% partial hepatectomy. After 1 week of prevascularization, hepatocytes harvested from the donor Lewis rats using a collagenase digestive method were injected into the sponges. Specimens were harvested at 2 weeks, 1 month, and 2 months after HTx. Histologic examination revealed that the control groups contained small numbers of hepatocytes restricted to the peripheral areas of the sponges. However, a large number of hepatocytes, including clusters, was found distributed uniformly in the bFGF group. In the bFGF group at 2 weeks, 1 month, and 2 months, the percentage of the sponge occupied by hepatocytes was 7.21+/-2.64%, 6.98+/-2.59%, and 5.58+/-3.77%, respectively. The corresponding ratios for the control group were 0.40+/-0.39%, 0.40+/-0.40%, and 0.87+/-1.51%. In addition, the mean number of new blood vessels in the bFGF group was significantly greater than that in the control group at 0 days, 2 weeks, and 1 month after HTx. These results suggest that bFGF strongly induced vascularization, which enabled a large number of hepatocytes to survive in the polymer devices.

Derivation of pharmacokinetics equations for quantitative evaluation of bioartificial liver functions.

A bioartificial liver (BAL) is an extracorporeal medical device incorporating living hepatocytes in a cartridge. A variety of BALs have been developed and new devices are being introduced. Some of them have been clinically applied and from the results obtained they are claimed to be useful devices for assisting the liver functions of patients. However, there is still uncertainty as to their efficacy and their limitations are not clear. It is important to establish methods to quantitatively evaluate the metabolic and synthetic functions of BAL. In this paper, we derive simple equations for the quantitative evaluation of BAL functions on the basis of pharmacokinetics. Pharmacokinetics was originally developed to understand the processes of absorption, distribution, and elimination of administered drugs. Metabolic functions of the natural liver have been analyzed using pharmacokinetics and values of the useful parameters, clearance (CL) and intrinsic clearance (CLint), have been reported. The metabolic functions of the BAL expressed using values of CL and CLint are easily compared with those of the normal human liver. We believe that our method provides a useful basis for estimating the clinical effectiveness of BAL.