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.

Effects of recombinant alpha-interferon-gelatin conjugate on in vivo murine tumor cell growth.

A recombinant human alpha-interferon A/D (IFN), also known to be effective in mice, was conjugated to gelatin with a water-soluble carbodiimide. The IFN-gelatin conjugate was much more efficient than free IFN in activating mouse peritoneal macrophages (Mø) in an in vitro experiment to inhibit the growth of IFN-resistant subline cells (RR1) of murine fibrosarcoma. A single i.p. injection of the conjugate administered to normal mice was also more effective than one of free IFN in activating peritoneal Mø and natural killer cells in peritoneal exudate cell and spleen cell populations. In the investigation on body distribution of the IFN-gelatin conjugate, an enhanced affinity to Mø as well as a prolonged retention were observed in comparison with free IFN. An injection of the IFN-gelatin conjugate i.p. was more effective than one of free IFN in suppressing the in vivo growth of not only IFN-sensitive SS2 cells but also RR1 cells in the peritoneal cavity of mice, although RR1 cells were only susceptible to the indirect effect of IFN via host cells, in contrast to SS2 cells. In addition to an increased recruitment of Mø to the peritoneal cavity in RR1-bearing mice receiving i.p. injection of the IFN-gelatin conjugate, these Mø were activated to inhibit the in vitro growth of RR1 cells. These results indicate that the IFN-gelatin conjugate is a promising antitumor agent that is much more effective than free IFN. The dose of IFN in the conjugate required for exerting the antitumor effects is much lower than that of free IFN, which leads to a reduction of adverse effects of IFN.

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.

Simple mixing of IFN with a polysaccharide having high liver affinity enables IFN to target to the liver.

Interferon (IFN) therapy is only one method that is clinically effective in controlling disease activity in patients with chronic hepatitis. A chelating residue (diethylenetriamine pentaacetic acid, DTPA) was introduced to pullulan, which is a polysaccharide with high liver affinity. This DTPA-pullulan could conjugate with IFN through Zn2+ coordination on mixing these three components. Intravenous injection of the IFN-DTPA-pullulan conjugate with Zn2+ coordination induced activity in the liver of an antiviral enzyme. 2',5'-oligoadenylate synthetase at IFN doses lower than those used for free IFN injection. In addition, synthetase induction by the conjugate continued for a longer time than did induction by free IFN. Liver targeting of IFN by this conjugation technique based on Zn2+ coordination opens a new method of IFN therapy.

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.

Injectable microspheres with controlled drug release for glaucoma filtering surgery.

The authors evaluated the effects of biodegradable poly (lactic acid) microspheres that provided the controlled release of the antimetabolic agent adriamycin (ADR) to prevent post surgical fibrosis after glaucoma filtering surgery. Fifty six eyes of 28 rabbits underwent posterior lip sclerotomy and received a 0.2 ml subconjunctival injection that contained microspheres 90 degrees from the filtering site immediately after surgery. Microspheres containing ADR (100 or 200 micrograms) were randomly administered to one eye. The fellow eyes served as controls and received microspheres without the drug. Intraocular pressure in the eyes treated with the microspheres that contained the drug was significantly lower than that in the control eyes from days 7-12 in the 100 micrograms group and from days 6-16 in the 200 micrograms group (P < .05). Eyes that received ADR had a significantly longer patent filtering bleb compared with the control eyes (P < .05). No corneal complications were observed in the eyes treated with 100 micrograms of ADR and the control eyes. Peripheral corneal opacities (25%) and epithelial erosion (17%) were observed in the eyes that received the 200 micrograms dose, but the cornea returned to normal after 4 wk. These results suggest that controlled-drug-release microspheres with an antimetabolic agent may be promising for preventing fibrosis after surgery.

Biodegradable microspheres containing adriamycin in the treatment of proliferative vitreoretinopathy.

The use of biodegradable polymer microspheres containing adriamycin for the treatment of proliferative vitreoretinopathy (PVR) in an experimental rabbit model was investigated. A single injection of microspheres containing 10 micrograms of adriamycin effectively decreased traction retinal detachment to 10% (n = 10), whereas 50% of eyes injected with blank microspheres (n = 10) developed retinal detachment (P < 0.05). A single injection of microspheres, containing 3 micrograms of adriamycin, did not suppress retinal detachment. Electroretinographic and histologic studies confirmed that the 10 micrograms injection of adriamycin in microspheres was not toxic to the retina, although the injection of the same amount of free adriamycin caused retinal necrosis and detachment. Thus, microspheres containing adriamycin hold promise as a new treatment modality for PVR.

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.

Effect of focal X-ray irradiation on experimental choroidal neovascularization.

PURPOSE: Radiation therapy has been used to treat choroidal neovascularization (CNV) in patients with age-related macular degeneration. The in vivo effect of applying focal x-ray irradiation to the eye of rabbits with experimental CNV was investigated. METHODS: CNV was induced in the rabbit eyes by subretinal implantation of gelatin hydrogel microspheres impregnated with basic fibroblast growth factor. Three weeks after implantation, 17 of 34 eyes with CNV lesions accompanied by fluorescein leakage were irradiated with a single dose of 20 Gy; the other 17 eyes were not irradiated and served as the controls. The eyes were examined before irradiation and 1, 2, and 4 weeks after irradiation, by indirect ophthalmoscopy and fluorescein angiography. The degree of a decreasing amount of fluorescein leakage from the CNV lesions after irradiation was graded using a computerized image analysis system and was compared in the irradiated and nonirradiated eyes. These eyes were also examined histologically and immunohistochemically. RESULTS: Fluorescein leakage from the CNV lesions had significantly decreased in the eyes irradiated with 20 Gy compared with the control eyes, throughout the study period (P < 0.05). Histologic and immunohistochemical studies at 4 weeks after irradiation demonstrated that the degree of vascular formation and the number of vascular endothelial cells in the subretinal membrane of the irradiated eyes were less than those of the control eyes. CONCLUSIONS: Focal x-ray irradiation at the ocular region effectively reduced experimental CNV activity. These results support the possibility that radiation therapy may be beneficial in treating 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.

A new model of subretinal neovascularization in the rabbit.

PURPOSE: To establish a new model of subretinal neovascularization (SRN) in the rabbit by implanting basic fibroblast growth factor (bFGF)-impregnated gelatin microspheres beneath the retina. METHODS: Basic fibroblast growth factor-impregnated gelatin microspheres were prepared by forming a polyion complex between gelatin and bFGF. The microspheres, containing 2.5 micrograms of bFGF, were injected into the subretinal space of rabbit eyes (n = 29). Control eyes (n = 10) received bFGF-free gelatin microspheres. Eyes were followed up for 3 days to 8 weeks by ophthalmoscopy, photography, fluorescein angiography, light microscopy, and transmission electron microscopy. RESULTS: Twenty of 24 experimental eyes (83%) showed fluorescein leakage from SRN 2 weeks after implantation of the bFGF-impregnated microspheres. This leakage continued for 2 to 6 more weeks. In striking contrast, control eyes showed no fluorescein leakage. Histologic examination revealed SRN in all the experimental eyes but in none of the control eyes. CONCLUSIONS: Subretinal implantation of bFGF-impregnated gelatin microspheres induces reproducible SRN in the rabbit.

Cellular response in subretinal neovascularization induced by bFGF-impregnated microspheres.

PURPOSE: To determine the sequence of cellular changes associated with a new rabbit model of subretinal neovascularization (SRN) induced by subretinal injection of basic fibroblast growth factor (bFGF)-impregnated microspheres. METHODS: bFGF-impregnated gelatin microspheres, prepared by forming a polyion complex between gelatin and bFGF, were subretinally implanted into rabbit eyes. The eyes were studied by immunochemistry at 3 days to 8 weeks after implantation. Antibodies to CD4, CD8, cytokeratin, CD31, glial fibrillary acidic protein (GFAP), and RAM11 were used. RESULTS: Cytokeratin-positive retinal pigment epithelial (RPE) cells appeared on day 3 and continued to increase in number in the subretinal space throughout the growth of the SRN membrane, becoming the predominant cell type. Macrophages (RAM11-positive) appeared early, but most disappeared within 7 days. GFAP-positive Müller cells were evident early in the retina but migrated into the subretinal space after 7 days; the gliotic adhesion they formed between the retina and the SRN membrane was prominent at 8 weeks. CD31-positive endothelial cells were first evident at 14 days and formed neovascular channels that were still present for up to 8 weeks. CD4- and CD8-positive lymphocytes appeared in the early stages but were few in number. CONCLUSIONS: SRN membranes are primarily composed of RPE cells and vascular endothelial cells. The membrane adheres to the retina by a gliotic band. The cellular components involved in the membrane of this model resemble those found in SRN membranes removed from patients with age-related macular degeneration.

Drug delivery systems for antitumor activation of macrophages.

Macrophages, one of the immunologically competent cells centrally contributing to host defense mechanisms, can be stimulated by a variety of immunomodulatory agents to exert antitumor activity. However, many problems, such as serious side effects attributed to the high-dosage regimens of the drugs, should be addressed for successful tumor immunotherapy using immunomodulatory agents. Thus, it is essential to develop carrier systems that use much lower doses of the drugs. This article reviews the carrier systems of drugs for effective immunopotentiation, especially macrophage activation of antitumor function. Since research in this field has just started, few investigations, including ours, have been reported.

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.