Element release from titanium devices used in oral and maxillofacial surgery

Optical microscopy, scanning electron microscopy and X-ray microanalysis (EDS system) were used on c.p. titanium devices (21 grids and 10 plates) removed from 28 patients without signs of inflammation 6-24 months after surgery. Plates, grids and surrounding tissue were investigated to evaluate the titanium release and accumulation. Titanium was only present in the interfacial bone, probably due to fretting, and in all fibrous tissue surrounding the devices. Titanium content followed a decreasing gradient extending from the device surface and was not detected at a distance greater than 1mm. High titanium levels were found in blood cells in the connective tissue. In conclusion, titanium release from the devices stops only after bone is laid down on the titanium surfaces. Titanium release does not seem to interfere with the osteogenic process but perhaps may interact with it.     

Repair of the dura mater with processed collagen devices

BACKGROUND: We evaluated in a canine duraplasty model how specific differences in device physicomechanical properties, porosity, and crosslinking influenced the biological performance of three processed collagen dural substitutes. METHODS: Three collagen dural substitutes were studied: Dura-Guard, DuraGen, and Durepair. The initial strength, stiffness, and suture retention force were measured using standard mechanical test methods. The relative pore sizes of each device were assessed with a scanning electron microscope. Differential scanning calorimetry was used to measure their respective collagen denaturation temperatures. The biologic response and performance of the materials were evaluated via an acute (1 month) and long-term (3 and 6 months) canine bilateral duraplasty study. RESULTS: The mechanical properties of Dura-Guard and Durepair were similar to native dura. We could not quantify the mechanical properties of DuraGen because of its fragile nature. The denaturation temperature of DuraGen and Dura-Guard differed significantly from that reported for native collagens. The denaturation temperature of Durepair was comparable with the values reported for native collagens. All three materials were tolerated well by the animals. DuraGen did not maintain its structural integrity beyond 1 month. Dura-Guard and Durepair persisted for 6 months. Durepair was populated by fibroblasts and blood vessels, whereas Dura-Guard was not. CONCLUSIONS: The three dural substitutes tested were found to be safe and effective in healing surgically created defects in the dura mater. Although each of these dura substitutes are composed of collagen, differences in the collagen source and processing influenced device physicomechanical properties, porosity, and the nativity of the collagen polymer. These measured differences influenced device intraoperative handling and installation as well as the post-operative biological response, where differences in device resorption, cell penetration, vascularization, and collagen remodeling were observed.     

Binding and release of basic fibroblast growth factor from heparinized collagen matrices.

Endothelial cell seeding is a promising method to improve the performance of small-diameter vascular grafts. Growth of endothelial cells seeded on the luminal surface of synthetic vascular grafts, coated with a matrix suitable for cell seeding (e.g. collagen), can be accelerated by local, sustained release of basic fibroblast growth factor (bFGF). In this study two potential matrices for in vivo endothelial cell seeding were studied with respect to bFGF binding and release: collagen crosslinked using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), as well as heparinized EDC/NHS-crosslinked collagen. bFGF binding was determined after incubation of circular samples (10 mm diameter) with 0.25 ml bFGF solution for 90 min. Immobilization of increasing amounts of heparin, also using EDC and NHS, to crosslinked collagen containing 14 free primary amino groups per 1000 amino acid residues (E/N14C) resulted in binding of increasing amounts of bFGF. A plateau in bFGF binding was observed for heparinized E/N14C containing approximately 2.0-3.0 wt% of immobilized heparin which was obtained using a molar ratio of EDC to heparin-carboxylic acid groups of 0.4 during heparin immobilization (E/N14C-H(0.4)). At concentrations up to 840 ng bFGF/ml, 10% of the added bFGF bound to E/N14C, while binding of bFGF to E/N14C-H(0.4) amounted to 22%. Both E/N14C and E/N14C-H(0.4) pre-loaded with bFGF showed sustained bFGF release. A burst release of 30% in endothelial cell culture medium (CM) was observed for E/N14C during the first 6 h, compared to 2% release from E/N14C-H(0.4). After 28 days, the bFGF release from E/N14C and E/N14C-H(0.4) in CM amounted to 100 and 65%, respectively. Combined results of binding and release of bFGF indicate that compared to E/N14C, E/N14C-H(0.4) is the substrate of choice for bFGF pre-loading and subsequent endothelial cell seeding.     

Cellular automata model for drug release from binary matrix and reservoir polymeric devices

Kinetics of drug release from polymeric tablets, inserts and implants is an important and widely studied area. Here we present a new and widely applicable cellular automata model for diffusion and erosion processes occurring during drug release from polymeric drug release devices. The model divides a 2D representation of the release device into an array of cells. Each cell contains information about the material, drug, polymer or solvent that the domain contains. Cells are then allowed to rearrange according to statistical rules designed to match realistic drug release. Diffusion is modeled by a random walk of mobile cells and kinetics of chemical or physical processes by probabilities of conversion from one state to another. This is according to the basis of diffusion coefficients and kinetic rate constants, which are on fundamental level just probabilities for certain occurrences. The model is applied to three kinds of devices with different release mechanisms: erodable matrices, diffusion through channels or pores and membrane controlled release. The dissolution curves obtained are compared to analytical models from literature and the validity of the model is considered. The model is shown to be compatible with all three release devices, highlighting easy adaptability of the model to virtually any release system and geometry. Further extension and applications of the model are envisioned.     

Mechanical deformation of polymer matrix controlled release devices modulates drug release.

When magnetic fields were applied to polymer matrices of ethylene-vinyl acetate copolymer embedded with drug and a small magnet, drug release was increased up to 30-fold above baseline levels. It has been hypothesized that the effect of magnetic stimulation on the release of drugs from these matrices is the transduction of the applied magnetic field into a mechanical deformation of the matrix through motion of the magnet within the matrix. This current study provides support for this hypothesis by demonstrating that repeated pulsatile mechanical deformation of matrices can enhance the release of macromolecules from ethylene-vinyl acetate copolymer matrices. Furthermore, similar modulated release kinetics were obtained with mechanically compressed and magnetically stimulated matrices. We also established that modulation was dependent on the ratio of compression area to matrix volume and that modulation was maximized when this ratio was optimized.     

Metal ion release from fracture fixation devices: a potential marker of implant failure

Stainless steel is the alloy most frequently used for fracture fixation devices (FFD). We aimed to verify if the ion release evaluation could be a surrogate marker of performance and could allow an early detection of implant failure in patients with stainless steel FFD. We measured the nickel (Ni) and chromium (Cr) serum content in patients undergoing the retrieval of stainless steel plates (group I) or intramedullary nails (group II), because of consolidation or failure. Forty-five healthy donors were recruited as controls. Furthermore, the diagnostic performance of these values was evaluated: analysis power, sensitivity, specificity, positive and negative predictive values, likelihood ratios, and diagnostic accuracy were calculated. A significant increase of ion values was demonstrated in patients with failed plates, compared with the values recorded in patients with well-fixed plates (p = 0.002 for Cr and p = 0.002 for Ni), and in healthy subjects (p = 0.0002 for Cr and p = 0.003 for Ni). No significant difference was found between stable implants and controls (p = 0.8 for Cr and p = 0.06 for Ni). A high specificity (0.92 for Cr and 1.00 for Ni), positive predictive value (0.87 for Cr and 1.00 for Ni), and positive likelihood ratio (9.10 for Cr) were calculated for ion testing in plates. The substantial metal content elevations in patients with plates and the positive likelihood ratio above 5 for chromium testing suggest that ion dosage may be a useful surrogate marker for the presence of malfunctioning of these devices, perhaps before the onset of clinical and radiographic changes.     

Promotion of angiogenesis by sustained release of rhGM-CSF from heparinized collagen/chitosan scaffolds

A novel dermal substitute of combining recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) with a porous heparinized collagen/chitosan scaffolds was developed, considering the inadequate angiogenesis during repair of full-thickness skin defects. The physicochemical properties of heparinized collagen/chitosan scaffolds were examined and in vitro release pattern of rhGM-CSF from scaffolds was measured by ELISA. Four groups of composite scaffolds (heparinized or unheparinized scaffolds loaded with or without rhGM-CSF) were fabricated for subcutaneous implantation in young adult male Sprague-Dawley (SD) rats. Tissue specimens were harvested at different time points after implantation for histopathological, immunohistochemical observation, and Western blotting analysis. The heparinized scaffolds (H(1)E) showed slower biodegradation and sustained release of rhGM-CSF in vitro, although no significantly different release pattern was observed between the H(1)E and unheparinized scaffolds (H(0)E). In vivo investigation revealed that the heparinized scaffolds loaded with rhGM-CSF (H(1)E/rhGM-CSF) had the best cellular adhesion and migration, new vessel formation, and highest expression of VEGF and TGF-β1, indicating promoted angiogenesis. This study demonstrated that composite dermal substitute of combining rhGM-CSF with a porous heparinized collagen/chitosan scaffolds could be a potential therapeutic agent for full-thickness skin defects because of its sustained delivery of rhGM-CSF.     

A comparative study of the biologic and immunologic response to medical devices derived from dermal collagen

We examined collagen materials for soft tissue augmentation [Zyderm Collagen Implant (ZCI), glutaraldehyde cross-linked (GAX) collagen, and Koken Atelocollagen (Atelocollagen)]; hemostatic collagens [Gelfoam Gelatin Powder (Gelfoam), Avitene Microfibrillar Collagen Hemostat (Avitene), and Collastat Collagen Hemostat (Collastat)]; and reconstituted, intact fibrillar collagen from bovine skin in a subcutaneous guinea pig model. After 11, 25, and 39 days in situ, explants from animals injected with GAX collagen demonstrated greater wet-weight persistence than all other materials. Conversely, at all time points, the explants of Atelocollagen were the least persistent. Following 25 days in vivo, explants were examined using differential scanning calorimetry; ZCI and Atelocollagen displayed thermal transition temperatures of 58 degrees C. Avitene and Gelfoam explants displayed transition points of 30 degrees C and 32 degrees C, indicating denatured or cleaved collagen. By contrast, GAX collagen explants had a high (68 degrees C) transition temperature, reflecting its cross-linking. With respect to immunogenicity, day 39 sera from ZCI treated animals showed significantly lower titers in the ELISA to their respective implant collagen than all other groups examined, while antibody activity in the GAX collagen, Gelfoam, Atelocollagen, and intact collagen groups were not significantly different. Collastat elicited antibodies with a greater affinity than observed in these previous groups. Sera from Avitene treated animals demonstrated the highest antibody levels and were the only sera which reacted with bovine serum albumin. Thus, Avitene was the most immunogenic of the collagen materials examined, while GAX collagen demonstrated the greatest persistence and minimal immunogenicity, and ZCI was the least immunogenic.     

The impact of proteinase-induced matrix degradation on the release of VEGF from heparinized collagen matrices

The in vivo application of engineered matrices in human wound healing processes is often hampered by the slow rate of vascularization. Therefore much research is directed towards enhancing the angiogenic properties of such matrices. One approach for enhancing the vascularization is the incorporation of angiogenic growth factors. Recently, we and others have reported on immobilizing such factors into collagen matrices either by covalent attachment or by physical binding to covalently incorporated heparin. Especially the latter procedure has been shown to lead to substantial increase rates in vascularization in in vivo experiments. The increases have been proposed to depend on the sustained release of the incorporated angiogenic growth factors from the heparinized collagen matrices. In this paper, we report on investigations to study the release of vascular endothelial growth factor (VEGF) from collagen matrices under conditions which mimic potential in vivo situations. Relevant proteinase concentrations were deduced from in vitro experiments in which we evaluated the secretion of selected matrix metalloproteinases from fibroblasts in contact with collagen. The release of VEGF from non-modified, cross-linked and heparinized collagen matrices in the absence and in the presence of varying concentrations of proteinases was then determined by ELISA and liquid scintillation counting. The release behaviour appears to be controlled by both the presence of heparin and the levels of proteinases applied. Experiments with matrices containing radioactively labelled heparin suggest that VEGF release results from the consecutive and simultaneous release of three species of VEGF molecules that differ in their binding affinities to the differently modified collagen matrices. The species binding specifically to heparin most likely accounts for the previously observed increases in angiogenic potential between loading VEGF to non-heparinized and heparinized collagen matrices.     

Subtilisin Increases Macromolecular Efflux from the Oral Mucosa

The purpose of this study was to determine whether subtilisin, a potent serine proteinase derived from Bacillus species contaminating smokeless tobacco, increases macromolecular efflux from the oral mucosa and, if so, whether local elaboration of bradykinin mediates this response. Using intravital microscopy, I found that suffusion of subtilisin elicits significant, concentration-dependent leaky site formation and an increase in the clearance of fluorescein isothiocyanate-labeled dextran (molecular mass, 70 kDa) from the in situ hamster cheek pouch (P < 0.05). Heat-inactivated subtilisin had no significant effects on macromolecular efflux. Subtilisin-induced responses were significantly attenuated by Hoe 140 and NPC 17647, two structurally distinct selective bradykinin B₂ receptor antagonists, but not by des-Arg⁹-[Leu⁸]bradykinin, a selective bradykinin B₁ receptor antagonist, or CP-96,345, a selective neurokinin-1 receptor antagonist. Aprotinin, but not leupeptin, significantly attenuated subtilisin-induced increase in macromolecular efflux. Indomethacin had no significant effects on subtilisin-induced responses. Collectively, these data indicate that subtilisin increases the macromolecular efflux from the in situ hamster cheek pouch in a catalytic-site-dependent fashion through local elaboration of bradykinin. This response does not involve the stimulation of local afferent nerves or the production of prostaglandins.     

Simultaneous release of multiple molecules from poly(lactide-co-glycolide) nanoparticles assembled onto medical devices

Cell and tissue responses to implanted biomaterials often limit their effectiveness and lifetime. This is particularly true for materials implanted into the brain. We present here a new approach for the modification of materials to enable release of multiple agents, which might be useful in modulating tissue responses, without changing the properties of the underlying material, in this case, a silicon probe. Poly(lactide-co-glycolide) nanoparticles (NPs) were assembled onto silicon probe surfaces by electrostatic interactions. Charged NPs were fabricated by altering the properties of the surfactant. NPs formed with poly(ethylene-alt-maleic anhydride) (PEMA) were strongly negatively charged; these NPs assembled onto probes best when suspended at nearly physiological conditions (surface density ～ 83,600 ± 3000 particles/mm²). The percentage of surface area coverage by the NPs was estimated to be ～13% and was maintained over two weeks during constant exposure to PBS. Multiple fluorescent NP populations were attached to the same probe to allow visualization of simultaneous delivery of multiple agents by fluorescence microscopy. Release from NP coatings was reproducible and controllable. The distinct release profiles of each agent from the coatings were preserved upon attachment to the surfaces. The unique feature of this new system is that NPs encapsulating various molecules (i.e. drugs, proteins, or DNA) can be fabricated separately, in advance, and simply mixed prior to attachment. The versatility of this delivery system, therefore, makes it suitable for many applications.     

Macromolecular marionettes

Computer graphics can provide useful tools for molecular biology because pictorial displays are the most efficient way for computers to communicate the details of a complex molecular structure to human beings. The development of suitable hardware has led to the establishment of a number of computer graphics installations devoted to the investigation of molecular structure.We describe here the Evans and Sutherland $\text{LDS-1}\text{PDP-10}$ system of the Princeton University Computer Graphics Laboratory, and the program systems that we have developed for applications to the study of protein and nucleic acid structure and interaction. The hardware can generate displays of complicated three-dimensional line drawings. It allows interactive analysis and modification of a display by a human observer, as well as computational analysis of structural data by a powerful general-purpose digital computer, the PDP-10.We have developed programs for the study and analysis of proteins and nucleic acids of known structure, and for construction and experimentation with models of general polypeptides and polynucleotides. These model-building programs permit the generation of displays of polypeptides and polynucleotides of arbitrary sequence and conformation. It is possible to adjust all conformational angles interactively, or with the assistance of supportive software that can carry out a variety of structural manipulations, such as the folding of a selected region into a helical conformation.Although the purely computational problems encountered in this work are relatively straight-forward—a tribute to the power of the equipment—subtle problems arise in defining the role of a human being as a partner in the execution of an interactive program. Our attempts to achieve the most effective combination of human and computers depend on the recognition of the strengths and limitations of each, and of the efficiencies of the channels of communication between them.     

Improved collagen bilayer dressing for the controlled release of drugs

A novel bilayer dressing has been developed from bovine succinylated collagen. The dressing contains an antibiotic, Ciprofloxacin, for both immediate and time-regulated release for controlling the infection, as the infected open wounds need special care. The dressing consists of a sponge and a film, both prepared from succinylated bovine collagen. The sponge has a smooth surface on one side; its rough surface on the other side forms the bilayer system with the film. Both sponge and film act as an anionic reservoir to hold the cationic Ciprofloxacin. The drug, after dispersing in poly (N-vinyl-2-pyrrolidione) (PVP) solution is allowed to spread in the bilayer system by diffusion. The drug stays in the bilayer system because of ionic binding, but starts releasing when comes in contact with the wound. Release of the drug is immediate, but it is regulated by ionic binding between the drug and succinylated collagen. The wound exudates, and there is a polarity-controlled release of the drug from the bilayer system. The PVP and bilayer system permits only time-regulated release, and the system lasts up to 5 days with therapeutically sufficient drug availability.     

Sustained release of neurotrophin-3 and chondroitinase ABC from electrospun collagen nanofiber scaffold for spinal cord injury repair

Nerve regeneration after spinal cord injuries (SCI) remains suboptimal despite recent advances in the field. One major hurdle is the rapid clearance of drugs from the injury site, which greatly limits therapeutic outcomes. Nanofiber scaffolds represent a potential class of materials for enhancing nerve regeneration because of its biomimicking architecture. In this study, we investigated the feasibility of incorporating neurotrophin-3 (NT-3) and chondroitinase ABC (ChABC) onto electrospun collagen nanofibers for SCI treatment. By using microbial transglutaminase (mTG) mediated crosslinking, proteins were loaded onto electrospun collagen nanofibers at an efficiency of ～45-48%. By combining NT-3 with heparin during the protein incorporation process, a sustained release of NT-3 was obtained (～96% by day 28). As indicated by dorsal root ganglion outgrowth assay, NT-3 incorporated collagen scaffolds supported neuronal culture and neurite outgrowth for a longer time period than bolus delivery of NT-3. The presence of heparin also protected ChABC from degradation. Specifically, as evaluated by dimethylmethylene blue assay, bioactive ChABC was detected from collagen scaffolds for at least 32 days in vitro in the presence of heparin (～32% of bioactivity retained). In contrast, ChABC bioactivity was only ～1.9% by day 22 in the absence of heparin. Taken together, these results clearly demonstrated the feasibility of incorporating NT-3 and ChABC via mTG immobilization to produce protein-incorporated collagen nanofibers. Such biofunctional nanofiber constructs may find useful applications in SCI treatment by providing topographical signals and multiple biochemical cues that can promote nerve regeneration while antagonizing axonal growth inhibition for CNS regeneration.     

Sister chromatid exchanges and ion release in patients wearing fracture fixation devices

The quantification of sister chromatid exchange (SCE) during mitosis is a useful index for evaluating genotoxic effects in subjects occupationally or incidentally exposed to potentially toxic substances. The authors investigated the hypothesis that ions released by corrosion from prosthetic components of fracture fixation devices are associated with change in SCE incidence. In the present study, ten patients with implants were examined, and fifteen subjects with no implants were used as controls. SCE and high frequency cell (HFC) numbers were evaluated in circulating lymphocytes. In addition, nickel (Ni) and chromium (Cr) ion values in the serum were measured because, after iron, these metals are major components of stainless steel. A significant increase in SCE numbers was observed in patients compared to the control population (4.9 +/- 1.3 vs. 3.5 +/- 1.4). Ni concentration was 1.71 +/- 1.49 ng/mL in patients and 0.72 +/- 0.52 ng/mL in control subjects; Cr concentration was, respectively, 1.01 +/- 0.77 ng/mL and 0.19 +/- 0. 27 ng/mL. The increase of serum Cr and Ni was statistically significant. No correlation was found between the increased Cr concentrations and SCE number while Cr ion levels were found to be significantly correlated to HFC. An inverse correlation between Ni level and SCE numbers was observed. Our findings suggest that Cr release by stainless steel implants could have a genotoxic effect; thus it would be useful to carefully monitor implanted subjects with regard to serum ion dosage, SCE analysis, and HFC evaluation. In any case, it would be appropriate to remove the implant when fracture fixation is reached.     

Controlled release of vascular endothelial growth factor by use of collagen hydrogels

In vivo profile of vascular endothelial growth factor (VEGF) release from collagen hydrogels was investigated comparing that of hydrogel degradation while angiogenesis induced by the released VEGF was assessed. Collagen sponges were chemically cross-linked with different amounts of glutaraldehyde for various time periods. When 125I-labeled collagen hydrogels incorporating VEGF were subcutaneously implanted into the back subcutis of mice, the hydrogel radioactivity decreased with time, the decrement profile depending on the cross-linking conditions. The radioactivity was retained for longer time periods as the glutaraldehyde concentration and cross-linking time increased. Implantation study of collagen hydrogels incorporating 125I-labeled VEGF revealed that the remaining VEGF radioactivity decreased with time and the retention period was prolonged with the decreased hydrogel biodegradation. The slower the hydrogel degradation, the longer the period of VEGF retention. The collagen hydrogel incorporating VEGF induced significant angiogenesis around the implanted hydrogel, in marked contrast to VEGF in the solution form and VEGF-free empty hydrogel. The retention period of angiogenesis became longer with a decrease of the in vivo degradation rate of hydrogels. It is possible that the slower degraded hydrogel achieves a longer period of VEGF release, resulting in prolonged angiogenetic effect. We concluded that in our hydrogel system, biologically-active VEGF was released as a result of in vivo degradation of the hydrogel.     

Preparation of collagen/gelatin sponge scaffold for sustained release of bFGF

Artificial dermis (AD) has been used to regenerate dermis-like tissues in the treatment of full-thickness skin defects, but it takes 2 or 3 weeks to complete dermal regeneration. Our previous study demonstrated that injection of basic fibroblast growth factor (bFGF)-impregnated gelatin microspheres (MS) into the AD accelerates the regeneration of dermis-like tissue. However, injection of gelatin MS before clinical use is complicated and time consuming. This study investigated a new scaffold, in which collagen and gelatin are integrated, and which is capable of sustained bFGF release. We produced collagen/gelatin sponges with a gelatin concentration of 0wt%, 10wt%, 30wt%, and 50wt%. The mean pore size in each sponge decreased with the gelatin concentration. In an in vitro study, proliferation of fibroblasts in each sponge was not significantly different over 7 days of culture. As for in vivo sustained release of bFGF, a radioisotope study demonstrated that retention of bFGF in gelatin 10wt% and 30wt% sponges was significantly larger than that in gelatin 0wt% sponge. The collagen/gelatin sponges were grafted on full-thickness skin defects created on a rabbit ear, and we evaluated regeneration of dermis-like tissue by measuring the amount of hemoglobin and size of dermis-like tissue on histological sections. Seven days after implantation, the amount of hemoglobin in dermis-like tissue in gelatin 10wt% sponge was significantly larger than those in control and gelatin 50wt% sponge. Twenty-eight days after implantation, the area of dermis-like tissue in gelatin 10wt% sponge was significantly larger than those in the other specimens. We conclude that the collagen sponge integrated with 10wt% gelatin has the most potential for sustained release of bFGF and that the combination of collagen/gelatin 10wt% sponge and bFGF is a promising therapeutic modality for the treatment of full-thickness skin defects.     

Development of a temperature sensitive drug release system for polymeric implant devices

A low density polyethylene (LDPE) model surface was coated with poly(amino-p-xylylene) (amino-ppx) via chemical vapour deposition (CVD) polymerization. The functional surface was used to immobilize a polymeric drug release system consisting of poly(N-isopropylacrylamide) (NIPAAm)-co-poly(acrylic acid) (AAc). The coupled drug release system was used to incorporate the thrombin inhibitor r-hirudin. For the investigation of the concentration of incorporated r-hirudin and the release profile over a given period of time r-hirudin was labelled with 123I as well as fluorescein isothiocyanate (FITC). Using an incubation solution concentration of 0.2% at pH 5 and an ionic strength of 0.7 M a maximum concentration of 2.21 (+/- 0.11) nmol/cm2 of r-hirudin was detected. FITC-r-hirudin was almost quantitatively (2.08 +/- 10 nmol/cm2; 94%) released from the surface coating within a period of 14 days.     

Controlled release of vascular endothelial growth factor by use of collagen hydrogels

In vivo profile of vascular endothelial growth factor (VEGF) release from collagen hydrogels was investigated comparing that of hydrogel degradation while angiogenesis induced by the released VEGF was assessed. Collagen sponges were chemically cross-linked with different amounts of glutaraldehyde for various time periods. When ¹²⁵I-labeled collagen hydrogels incorporating VEGF were subcutaneously implanted into the back subcutis of mice, the hydrogel radioactivity decreased with time, the decrement profile depending on the cross-linking conditions. The radioactivity was retained for longer time periods as the glutaraldehyde concentration and cross-linking time increased. Implantation study of collagen hydrogels incorporating ¹²⁵I-labeled VEGF revealed that the remaining VEGF radioactivity decreased with time and the retention period was prolonged with the decreased hydrogel biodegradation. The slower the hydrogel degradation, the longer the period of VEGF retention. The collagen hydrogel incorporating VEGF induced significant angiogenesis around the implanted hydrogel, in marked contrast to VEGF in the solution form and VEGF-free empty hydrogel. The retention period of angiogenesis became longer with a decrease of the in vivo degradation rate of hydrogels. It is possible that the slower degraded hydrogel achieves a longer period of VEGF release, resulting in prolonged angiogenetic effect. We concluded that in our hydrogel system, biologically active VEGF was released as a result of in vivo degradation of the hydrogel.