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.

Microspheres of biodegradable polymers as a drug-delivery system in the vitreous.

Microspheres of biodegradable polymers were evaluated as a potential controlled-release drug-delivery system in the vitreous. The microspheres were prepared with polymers of poly(lactic acid) or copolymers of glycolic acid and lactic acid. The release of 5-fluorouracil (5-FU) from the microspheres was studied in vitro. Poly(lactic acid) microspheres released 70-85% of total 5-FU over 7 days. Microspheres of polymers with a smaller molecular weight released the drug more rapidly. Copolymer microspheres released 98% of 5-FU over 2 days. The rate of drug release was controllable by changing the molecular weight of the polymers or using a matrix of copolymer. The intravitreal kinetics of the microspheres were studied in ten rabbits in vivo. A suspension of microspheres was injected into the vitreous cavity of five normal eyes and five vitrectomized eyes. By 48 +/- 5.2 days after injection, the microspheres disappeared from the vitreous cavity in the five normal eyes. Clearance from the vitreous cavity was accelerated in the five rabbits that underwent vitrectomy (14 +/- 2.4 days; P less than 0.001). No difference was found in the b waves of electroretinograms before and after injection of the microspheres. The histologic study showed no abnormal findings as a result of the injection. These results suggested that microspheres of biodegradable polymers may be a potential delivery system for the controlled release of drugs in the vitreous.

Targeted delivery of anti-angiogenic agent TNP-470 using water-soluble polymer in the treatment of choroidal neovascularization.

PURPOSE: The conjugation of drugs with water-soluble polymers such as poly(vinyl alcohol) (PVA) tends to prolong the half-life of drugs and facilitate the accumulation of drugs in tissues involving neovascularization. The purpose of this study was to evaluate the effect of TNP-470-PVA conjugate on the proliferation of endothelial cells in vitro and on experimental choroidal neovascularization (CNV) in vivo. METHODS: TNP-470 was conjugated in PVA by a dimethylaminopyridine-catalyzed reaction. The effects of TNP-470-PVA and free TNP-470 on the proliferation of human umbilical vein endothelial cells (HUVECs) and bovine retinal pigment epithelial cells (BRPECs) were evaluated by the tetrazolium-based colorimetric assay (XTT assay). Experimental CNV was induced by subretinal injection of gelatin microspheres containing basic fibroblast growth factor, into rabbits. Thirty rabbits were intravenously treated either with TNP-470-PVA (n = 8), free TNP470 (n = 5), free PVA (n = 5), or saline (n = 12) daily for 3 days, 2 weeks after implantation of gelatin microspheres. Fluorescein angiography was performed to detect the area with CNV, and the evaluation was made by computerized measurement of digital images. These eyes were also examined histologically. To observe the accumulation of conjugate, 3 rabbits with CNV received rhodamine B isothiocyanate-binding PVA (RITC-PVA), and the lesion was studied 24 hours later by fluorescein microscopy. RESULTS: The TNP-470-PVA inhibited the growth of HUVECs, similar to that of free TNP-470. The BRPECs were less sensitive to TNP-470-PVA than were the HUVECs. TNP-470-PVA significantly inhibited the progression of CNV in rabbits (P = 0.001). Histologic studies at 4 weeks after treatment demonstrated that the degree of vascular formation and the number of vascular endothelial cells in the subretinal membrane of the eyes treated with TNP-470-PVA were less than those of the control eyes. RITC-PVA remained in the area with CNV 24 hours after administration. CONCLUSIONS: These results suggest that TNP-470-PVA inhibited the proliferation of HUVECs more sensitively than that of BRPECs, and the targeted delivery of TNP-470-PVA may have potential as a treatment modality for CNV.

A new vitreal drug delivery system using an implantable biodegradable polymeric device.

PURPOSE: The authors evaluated the feasibility of using an implantable biodegradable polymeric device to deliver drugs into the vitreous humor. METHODS: Two types of devices were prepared by compression-molding polymers of poly(DL-lactic acid) of two different molecular weights. The molecular weights of the poly(DL-lactic acid) used were 5,600 (device-1) and 9,100 (device-2). Sodium fluorescein (NaF) served as a hydrophilic drug marker. The release of the dye from the devices was studied in vitro. The intravitreal kinetics of NaF was evaluated in rabbits in vivo by fluorophotometry. The eyes were evaluated electrophysiologically and histologically to determine if there were toxic effects. RESULTS: Device-1 and device-2 released NaF for more than 25 and 45 days, respectively, in vitro. Detectable concentrations of NaF were present in the vitreous up to 17 days (device-1) and 28 days (device-2). Both types of devices were well tolerated, with no noted toxic effects. CONCLUSIONS: These results suggested that this device may be a potentially effective system to deliver drugs in the vitreous.

Treatment of malignant pleural effusions with doxorubicin hydrochloride-containing poly(L-lactic acid) microspheres.

For the treatment of malignant pleural effusions, we prepared doxorubicin hydrochloride (Adriamycin)-containing poly(L-lactic acid) microspheres (ADR-MS). In vitro, 50 percent and 100 percent release times of ADR from ADR-MS were 6.3 and 20 days, respectively. After intrapleural administration of ADR-MS for seven patients at an ADR dose of 40 mg, ADR was detected in the effusions for more than two weeks; however, ADR concentrations in serum were very small, consistent with minimal transpleural absorption of ADR. These results indicated the slow release of ADR into the pleural cavity. Furthermore, the amount of drained ADR was less than a few percent of the administered dose. In some cases, malignant cells in the effusion disappeared after the treatment. No complications related to the procedure occurred, and the patients developed no systemic symptoms. One patient died after four months, and the other six patients are alive after 21 to 31 months without reaccumulation of the effusion. The local administration of ADR-MS produces a localized high and systemic low concentration of ADR, which could potentially improve the patient's quality of life.

Traumatic sternal segment dislocation in a child.

We present an extremely rare case of traumatic dislocation of a sternal body segment in a child. We treated this sternal segment dislocation by means of open reduction using a newly developed pin made from poly-L-lactide containing 10 percent by weight hydroxyapatite. It is not necessary to remove this type of pin later because it is degradable and absorbed within the body within about one year after implantation. A two-year follow-up of this case revealed a good clinical result.

Bioabsorbable struts made from poly-L-lactide and their application for treatment of chest deformity.

Poly-L-lactide, a polymer of lactic acid, shows slow degradation in living tissue. Poly-L-lactide plate of high molecular weight maintains more than 90% of its initial mechanical properties for more than 3 months after implantation. Using struts made from poly-L-lactide plate, we performed chest wall reconstruction in 56 patients: for postoperative chronic sternal dehiscence in 23 and sternal elevation for pectus excavatum in 33 cases. The postoperative external appearances of the anterior chest were improved in comparison with the preoperative state in all cases. The internal features were evaluated by computed tomographic scan. Neither postoperative wound infection nor respiratory complication was observed, and no tendency for regression of the anterior chest occurred in any of the patients. In 3 of 56 cases (5.4%; one in the sternal dehiscence group and two in the pectus excavatum group), it was necessary to remove part of the strut because of overgrowth of granulation tissue around the implanted material after 4, 12, and 13 postoperative months, respectively. In the pectus excavatum group, the computed tomographic evaluations showed that poly-L-lactide strut maintained sufficient strength to support the thoracic wall 5 months after implantation. These findings suggest that the bioabsorbable poly-L-lactide strut is a promising material for surgical treatment of chest deformity.

Experimental study on a new tracheal prosthesis made from collagen-conjugated mesh.

A new tracheal prosthesis was made from fine Marlex mesh reinforced with a continuous polypropylene spiral. The mesh and spiral were covalently grafted and further coated with pig collagen with the aim of promoting connective tissue infiltration and providing initial airtightness. Complete surgical resection and replacement of a segment (2 cm in length, three to five tracheal rings) of the cervical trachea was performed in 13 adult mongrel dogs. Two dogs died of pneumonia about 2 months after operation, and eleven dogs were killed between 3 and 26 months. The prostheses in all dogs were promptly infiltrated by the surrounding connective tissue and completely incorporated by the host trachea. Formation of respiratory epithelium, which lined the prosthetic lumen, was seen to various degrees, and, in five dogs killed at 6 months or more after reconstruction, confluent epithelialization was confirmed histologically from the upper to the lower anastomotic site of the prosthesis. Marked stenosis of the prosthetic lumen caused by excessive scar tissue growth was seen in three dogs, and ulceration on the luminal surface was seen in two dogs. These results indicate that this tracheal prosthesis is highly biocompatible and promising for the repair of tracheal defects after further investigation.

Intrathoracic tracheal reconstruction with a collagen-conjugated prosthesis: evaluation of the efficacy of omental wrapping.

Reconstructions of the intrathoracic trachea in 24 dogs were done with the use of 50 mm long collagen-conjugated tracheal prostheses. Omental wrapping was also done in 14 of the dogs (omentopexy group) to evaluate the efficacy of this option in comparison with results in the other 10 dogs (control group). All 24 dogs had uneventful postoperative courses and were killed at 4 weeks or 3, 6, or 12 months after the operation. Better epithelialization and fewer complications, such as mesh exposure and luminal stenosis, were observed in the omentopexy group than in the control group. Angiography and analysis of regenerated blood vessels revealed that vessel ingrowth had started within 4 weeks and that vessel formation reached its maximal point within 6 to 12 months in the omentopexy group. In contrast, revascularization of the subepithelial region in the control group was poor even after 3 months, and vessel formation continued for as long as 12 months. The differences between the two groups were considered to be mainly a result of the speed of blood vessel ingrowth into the regenerated mucosa. We conclude that our prosthesis can be used safely for intrathoracic tracheal reconstruction and that omental wrapping is a useful supplementary method that reduces the occurrence of complications.

Surface modification of polymers for medical applications.

Most of the conventional materials do not meet the demands required for both their surface and bulk properties when used as biomaterials. An effective approach for developing a clinically applicable biomaterial is to modify the surface of the material which already has excellent biofunctionality and bulk properties. This review article focuses on the surface modification of polymers by grafting techniques, which have long been known in polymer chemistry but are not yet widely applied to biomaterials. A grafted surface can be produced primarily either by graft polymerization of monomers or covalent coupling reaction of existing polymer molecules onto the substrate polymer surface. The major surface properties that should be modified include two kinds of biocompatibility. One is the surface property that elicits the least foreign-body reactions and the other is the cell- and tissue-bonding capability. In addition, physiologically active surfaces with, for instance, selective adsorbability may be required. Attempts to produce these biocompatible or biospecific surfaces by grafting techniques are briefly overviewed in this article.

Effect of the size and surface charge of polymer microspheres on their phagocytosis by macrophage.

Polystyrene and phenylated polyacrolein microspheres of different diameters, as well as modified cellulose microspheres with different surface charges, were prepared in order to study the size and surface charge effect on their phagocytosis by mouse peritoneal macrophages. It was found that the maximal phagocytosis of polystyrene and phenylated polyacrolein microspheres took place when their size was in the range 1.0-2.0 microns. Microspheres with hydrophobic surfaces were more readily phagocytosed than those with hydrophilic surfaces. There was no significant difference in phagocytosis between cationic and the anionic surfaces when compared at a zeta potential of the same absolute value. The least phagocytosis was observed for cellulose microspheres with non-ionic hydrophilic surfaces. Addition of fetal calf serum to the culture medium resulted in decrease in phagocytosis for all microspheres.

Preparation of cross-linked hyaluronic acid films of low water content.

Hyaluronic acid (HA) was chemically cross-linked with poly(ethylene glycol) diglycidyl ether, a diepoxy compound (EX-810), to yield low water content and slowly degradable films when brought into contact with water. The cross-linking reaction was performed under acidic and neutral conditions, since the epoxy group is readily hydrolysed in alkaline media. To allow the reaction to proceed at high HA concentrations, a solution casting method was employed for the cross-linking of HA. The lowest water content of the cross-linked HA films obtained was 60 wt% when swollen with buffered saline at 37 degrees C. Alginic acid and poly(vinyl alcohol), which possess hydroxyl groups, similar to HA, were also found to undergo cross-linking with the diepoxy compound. Since IR spectra of the cross-linked films had no significantly new absorption, intermolecular formation of ether bonds between the hydroxyl groups belonging to different polysaccharide molecules was assumed to take place. It seemed too difficult to detect the ether bonds in the cross-linked HA films, because the virgin HA film itself contained ether bonds in the molecule. The cross-linked HA film with a water content of 60 wt% exhibited practically no weight loss after 10 days of immersion in phosphate-buffered saline (pH 7.4), while this film underwent in vivo degradation by 30% weight loss after 7 days of subcutaneous implantation in rats. The inflammation reaction elicited around the implanted film was not significant.

Vascularization effect of basic fibroblast growth factor released from gelatin hydrogels with different biodegradabilities.

Biodegradable gelatin hydrogels were prepared through the glutaraldehyde crosslinking of acidic gelatin with an isoelectric point (IEP) of 5.0 and the basic gelatin with an IEP of 9.0. The hydrogel water content was changed by the concentration of both gelatin and glutaraldehyde, used for hydrogel preparation. An aqueous solution of basic fibroblast growth factor (bFGF) was sorbed into the gelatin hydrogel freeze-dried to obtain a bFGF-incorporating gelatin hydrogel. Irrespective of the hydrogel water content, approximately 30% of the incorporated bFGF was released from the bFGF-incorporating acidic gelatin hydrogel, within the first day into phosphate-buffered saline solution at 37 degrees C, followed by no substantial release. Probably, the basic bFGF complexed with the acidic gelatin through poly-ion complexation would not be released under the in vitro non-degradation condition of gelatin. On the contrary, almost 100% of the incorporated bFGF was initially released from all types of basic gelatin hydrogels. This is due to the simple diffusion of bFGF because of no complexation between bFGF and the basic gelatin. When implanted subcutaneously into the mouse back, bFGF-incorporating acidic and basic gelatin hydrogels with higher water contents were degraded with time faster than those with lower water contents. Significant neovascularization was induced around the implanted site of the bFGF-incorporating acidic gelatin hydrogel. The induction period prolonged with the decrease in hydrogel water content. On the other hand, such a prolonged vascularization effect was not achieved by the bFGF-incorporating basic gelatin hydrogel and the hydrogel initially exhibited less enhanced effect, irrespective of the water content. These findings indicate that the controlled release of biologically active bFGF is caused by biodegradation of the acidic gelatin hydrogel, resulting in induction of vascularization effect dependent on the water content. It is possible that only the transient vascularization by the basic gelatin hydrogel is due to the initial large burst in bFGF release, probably because of the down regulation of bFGF receptor.

Covalent immobilization of proteins on to the surface of poly(vinyl alcohol) hydrogel.

Covalent immobilization of cell-adhesive proteins such as collagen and fibronectin on to the surface of poly(vinyl alcohol) hydrogel was investigated by using diisocyanates, polyisocyanates, and cyanogen bromide. It was found that 0.5 and 12 micrograms/cm2 of collagen were immobilized on to the surface by using hexamethylene diisocyanate and cyanogen bromide, respectively. The big difference in the graft amount between the two methods was ascribed to the different reaction media employed for the surface modifications; toluene for the reaction with hexamethylene diisocyanate and water for the reaction with cyanogen bromide.

Introduction of functional groups onto the surface of polyethylene for protein immobilization.

Amino and carboxyl groups could be introduced onto the surface of high-density polyethylene film by utilizing graft polymerization of acrylamide and the subsequent Hofmann degradation and alkaline hydrolysis of grafted polyacrylamide. Graft polymerization was carried out by immersing an argon-plasma treated film in an aqueous solution of the monomer, followed by heating after degassing the monomer/film mixture. The surface density of these functional groups could be increased up to 10(-7) mol/cm2. The surfaces having amino and carboxyl groups exhibited positive and negative zeta potentials, respectively, when contacted with KCl aqueous solution. Both of the functional groups introduced onto the polyethylene surface were found to be utilizable for covalent immobilization of protein using carbodiimide for the carboxylic group or mediators such as glutaraldehyde and ethylene glycol diglycidyl ether for the amino group.

Low-frictional catheter materials by photo-induced graft polymerization.

To develop low-frictional catheters, photo-induced graft polymerization of N,N-dimethylacrylamide (DMAA) was performed on to films and tubes of ethylene-vinyl acetate copolymer (EVA) and poly(vinyl chloride) (PVC). Their surfaces became very slippery, when the materials were preirradiated with UV from a low-pressure mercury lamp, followed by graft polymerization with DMAA in the presence of small amount of riboflavin without degassing. Although no significant difference was observed in surface lubrication between the EVA film and the tube, the latter required an additional procedure for the graft polymerization, that is, removal of air trapped inside the tube. The surfaces of EVA and PVC tubes grafted with DMAA were found to exhibit frictional forces around 0.5 N against a PVC and a silicone surface under wet conditions, whereas the frictional force of the ungrafted tubes against the same substrates was 10 and 20 N for PVC and EVA, respectively.

Production of interleukin 1 from macrophages incubated with poly (DL-lactic acid) granules containing ovalbumin.

The production profile of interleukin 1 (IL-1) from mouse peritoneal macrophages (M phi) was determined following their incubation with poly(DL-lactic acid) (PDLLA) granules containing ovalbumin (OVA). Upon incubation, M phi produced IL-1 at a significantly high rate compared with those incubated with OVA in the free form or OVA-free granules. A simple mixture of empty granules and free OVA exhibited the same level of IL-1 production as induced by free OVA alone. IL-1 production by the granules with a fixed OVA loading increased with an increase in their amount added to M phi. When incubated with a fixed amount of granules containing OVA of different loadings, M phi produced more IL-1 with an increase in the total OVA amount, but the IL-1 production decreased at OVA loadings higher than 10%. The presence of free OVA enhanced IL-1 production with the increased addition of empty granules, but the level induced by OVA loaded in granules was higher than that by mixtures of free OVA and empty granules, when compared at a similar OVA dose, irrespective of the absolute amount of PDLLA added. These findings indicate that the sustained release of OVA from the granules is critical to enhance the OVA-induced IL-1 production, in contrast to the OVA release accompanying a large initial burst, which reduced IL-1 production. It was concluded that the direct contact of PDLLA granules with M phi and the subsequent sustained release of OVA around M phi effectively activated M phi, resulting in enhanced IL-1 production.

Assessment of heat and storage conditions on gamma-ray and electron beam irradiated UHMWPE by electron spin resonance.

Electron spin resonance (ESR) spectroscopic study was undertaken to explore the nature of any remaining radicals in ultra-high molecular weight polyethylene (UHMWPE) after irradiation with gamma-rays and electron beams to a dose of 25 KGy in air or N2 environment. The decay of radicals was studied by observing the ESR intensity change as a function of time. The following post-irradiation conditions were employed: (1) storage in air or N2 at 25 degrees C, and (2) heat treatment in air or N2 at 80, 100, and 120 degrees C for time intervals up to 8 h. The study suggests that radicals remaining trapped in the matrix of UHMWPE could be dramatically scavenged by heat treatment, independently of the atmosphere during irradiation and the heating process. The melting temperature and mechanical properties of irradiated UHMWPE under the experimental conditions employed were shown not to alter significantly by heat treatment, except in the presence of air.

Effect of additives on gelation and tissue adhesion of gelatin-poly(L-glutamic acid) mixture.

Gelation and tissue adhesion of mixtures of gelatin and poly (L-glutamic acid) (PLGA) aqueous solution were investigated in the presence of additives following the addition of a water-soluble carbodiimide (WSC) that induced chemical cross linking between gelatin and PLGA. To prevent spontaneous gelation of the mixed solution through physical cross linking between gelatin molecules at room temperature, additives were added to the mixed solution. Among the additives studied, starch and urea were effective in preventing the spontaneous physical gelation. The mixed gelatin and PLGA solution set to a cross-linked hydrogel within scores of second by WSC addition, irrespective of the presence of urea, whereas the viscosity of the solution with added starch was too high to measure the gelation time. The cross-linked gelatin-PLGA hydrogels with and without urea showed higher bonding strength to soft tissues than fibrin glue. This was in marked contrast to gelatin-PLGA hydrogels with soluble starch. Irrespective of the presence of urea, the gelatin-PLGA hydrogels gradually biodegraded in the back subcutis of mice over 3 months and no severe inflammatory response to the hydrogels was observed. These findings indicate that urea is promising as an additive to prevent spontaneous physical gelation of the mixed gelatin and PLGA aqueous solution without changing the characteristics of WSC-induced cross linking and tissue adhesion of the formed hydrogel.

Hemostatic capability of rapidly curable glues from gelatin, poly(L-glutamic acid), and carbodiimide.

The hemostatic capability of rapidly curable glues composed of gelatin and poly(L-glutamic acid) (PLGA) was compared with that of the conventional fibrin glue. The hydrogels produced from mixed gelatin and PLGA aqueous solution within several seconds by addition of water-soluble carbodiimide (WSC) was applied to the dog spleen injured by needle pricking. The WSC-catalyzed gelatin-PLGA glues exhibited higher hemostatic capability than the fibrin glue. The total amount of bleeding from the injured spleen until hemostasis when the gelatin-PLGA hydrogel glues were applied was significantly smaller than that of the fibrin glue application. The gelatin-PLGA glue application enhanced the success rate of complete hemostasis to a significantly greater extent than the fibrin glue, while the frequency of glue applications until achieving complete hemostasis decreased. The gelatin PLGA hydrogels strongly adhered to the surface of dog spleen, whereas the fibrin hydrogel was easily detached from the spleen surface. It was concluded that this strong adhesion mechanically suppressed the bleeding, leading to enhanced hemostasis by the rapidly curable gelatin-PLGA glues.