Controlled Releases of FGF-2 and Paclitaxel from Chitosan Hydrogels and their Subsequent Effects

Current Drug Delivery, 2006, 3, 351-3583511567-2018/06 $50.00+.00© 2006 Bentham Science Publishers Ltd.Controlled Releases of FGF-2 and Paclitaxel from Chitosan Hydrogels and their Subsequent Effects on Wound Repair, Angiogenesis, and Tumor GrowthMasayuki Ishihara *, Masanori Fujita 1, Kiyohaya Obara 1, Hidemi Hattori, Shingo Nakamura 1,Masaki Nambu 2, Tomoharu Kiyosawa 2, Yasuhiro Kanatani, Bonpei Takase, Makoto Kikuchi and Tadaaki Maehara 1Research Institute, 1Dept. of Surgery II, and 2Dept. of Plastic Surgery, National Defense Medical Collage, 3-2 Namiki,Tokorozawa, Saitama 359-8513, JapanAbstract: A photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted in an insoluble hydrogel like a soft rub-ber within 30 sec of ultraviolet light (UV)-irradiation. The photocrosslinked chitosan hydrogel showed strong sealing strength and potential use as a new tissue adhesive in surgical application. Paclitaxel, which is an anti-tumor reagent and a vascularization-inhibitor, retained in the photocrosslinked chitosan hydrogel, and were gradually released from the pho-tocrosslinked chitosan hydrogel in vivo upon the degradation of the hydrogel. The paclitaxel-incorporated pho-tocrosslinked chitosan hydrogels effectively inhibited tumor growth and angiogenesis in mice. On the other hand, the fi-broblast growth factor (FGF)-2 molecules also retained in both the photocrosslinked chitosan and an injectable chito-san/IO 4-heparin hydrogels, and were gradually released from the hydrogels upon their in vivo biodegradations. The activ-ity of FGF-2 in the hydrogels was stable for long time (more than 14 days). The controlled release of biologically active FGF-2 molecules from the hydrogels caused an induction of the angiogenesis and, possibly, collateral circulation occurred in the healing-impaired diabetic (db/db) mice and the ischemic limbs of rats. The purpose of this review is to describe the effectiveness of the chitosan hydrogels (photocrosslinkable chitosan hydrogel and chitosan/IO 4-heparin hydrogel) as a lo-cal drug delivery carrier for FGF-2 and paclitaxel to control wound repair, tumor growth, and angiogenesis. It is thus pro-posed that the chitosan hydrogels may be a promising new local carrier for drugs such as FGF-2 and paclitaxel.Keywords: Photocrosslinked chitosan hydrogel, Chitosan/IO 4-heparin hydrogel, Angiogenesis, Drug delivery carrier, Con-trolled release, Paclitaxel, Fibroblast growth factor-2 (FGF-2).INTRODUCTIONSystemic and regional chemotherapies to inhibit tumor growth and angiogenesis are used in the management of can-cer patients. Drug delivery to the tumor core following a systemic intravenous injection involves three processes, i.e.distribution through vascular space, transport across mi-crovessel walls, and diffusion through interstitial space within the tumor tissue [1, 2]. Anti-tumor activity of che-motherapeutic agents may be enhanced by an alteration of the drug administration, particularly focusing on delivery to the tumor site [1, 2]. When the drug is directly injected into a tumor such as by intratumoral injection or by direct instilla-tion into peritumoral space in intravesical therapy of superfi-cial bladder cancer [2] and in intraperitoneal dialysis of ovarian cancer [2, 3], the delivery is primarily occurred by diffusion through interstitial space. Moreover, regional de-livery of anti-tumor drugs is also expected to provide high local concentration and to decrease the incidences of side effects commonly observed with systemic therapy [4], and such regional delivery has been achieved with the use of biodegradable polymeric carrier [5] such as chitosan hydro-gel.*Address correspondence to this author at the Research Institute, National Defense Medical Collage, 3-2 Namiki, Tokorozawa, Saitama 359-8513,Japan; Tel: +81-429-95-1211; Fax: +81-429-91-1611;E-mail: ishihara@ndmc.ac.jpOn the other hand, preclinical studies have demonstrated that angiogenic growth factors can stimulate the develop-ment of collateral arteries in animal models of peripheral and myocardial ischemia, a concept called therapeutic angio-genesis [6, 7]. Although many studies using growth factors have been carried out in the field of angiogenesis, their use has not always been successful in vivo [8]. One of the rea-sons for this difficulty is the high diffusibility and very short half-life during which growth factors retain their biological activity in vivo . Thus, it is necessary to enhance their in vivo activities of growth factors using biodegradable polymeric carrier for angiogenic therapy.Polymeric carrier should be biocompatible and biode-gradable, and the degradation product should be non-toxic.Polymeric carrier can be developed using a variety of mate-rials including synthetic or natural polymers [9]. Among these materials, chiotsan, the deacetylated derivative of chi-tin, is promising candidate; it is biodegradable in the pres-ence of lysozyme, an enzyme present in the body and also produced by macrophages during wound healing. However,chitosan has low mechanical integrity and degrades rapidly.Glutaraldehyde is widely used to improve the structural properties of chitosan and other polymers such as collagen and gelatin by crosslinking the amino groups [10]. However,glutaraldehyde is generally considered to be toxic [11] and alternative crosslinkers are sought. Dimethyl 3, 3, dithio bis352 Current Drug Delivery, 2006, Vol. 3, No. 4Ishihara et al.propionimidate (DTBP) is a potential alternative crosslinking agent, that was successfully used to crosslink collagen [12,13] and chitosan [9].We have previously reported the preparation and charac-terization of a novel photocrosslinkable chitosan (Az-CH-LA) hydrogel [14, 15]. The material is a viscous solution and is easily crosslinked upon ultraviolet light (UV-) irradiation,resulting in an insoluble hydrogel within 30 sec. The pho-tocrosslinked chitosan hydrogel is a strong tissue-adhesive and, when compared with a fibrin glue, is more effective in sealing air leakage from pinholes on isolated small intestines and aorta, as well as from incisions on isolated trachea [14,15]. On the other hand, additions of non-anticoagulant hepa-rin (periodate-oxidized (IO 4-) heparin) to the viscous water-soluble chitosan (CH-LA) aqueous solution produces an in-soluble and injectable chitosan/IO 4-heparin hydrogel [16,17].Controlled release of paclitaxel from the pho-tocrosslinked chitosan hydrogel in vitro was described, and its anti-angiogenesis and anti-tumor effects in vivo were shown to evaluate the paclitaxel-incorporated pho-tocrosslinked chitosan hydrogel as a regional delivery for tumor treatment [18]. In contrast, when FGF-2 was added to each hydrogel, almost all of the FGF-2 molecules retained in the hydrogels (photocrosslinked chitosan hydrogel and chito-san/IO 4-heparin hydrogel), and were gradually released from each hydrogel upon the in vivo biodegradation of the hydro-gels [16, 19]. The present studies have shown that the con-trolled release of biologically active FGF-2 molecules from either FGF-2-incorporated photocrosslinked chitosan hydro-gel or chitosan/IO 4-heparin hydrogel causes an induction ofangiogenesis in the healing-impaired diabetic (db/db) mice [20, 21] and the ischemic limbs of rats [17]. Thus, the pho-tocrosslinked chitosan hydrogel and the injectable chito-san/IO 4-heparin hydrogel may be excellent carriers for con-trolled release of the drug reagents such as FGF-2 and pacli-taxel.PHOTOCROSSLINKED CHITOSAN HYDROGEL AS A BIOLOGICAL ADHESIVEIn situ-formed hydrogel prepared from its aqueous solu-tion can be used to fabricate injectable hybrid matrix. The materials, which are induced to form a hydrogel by a physiologically permitted stimulus such as temperature or pH change and light irradiation, have been utilized for artifi-cial three-dimensional matrix and drug delivery vehicles. We have previously reported on a photocrosslinkable chitosan molecule (Az-CH-LA) that contains both lactose moieties (lactobionic acid) and photoreactive azide groups (p-azidebenzoic acid) (Fig. 1) [14]. Lactose (lactobionic acid)moieties have been introduced through a condensation reac-tion with amino groups of the chitosan. The chitosan to which 2% lactobionate was introduced (CH-LA) exhibited a good aqueous solubility at neutral pH. Furthermore, applica-tion of ultraviolet light (UV-) irradiation at a lamp distance of 2 cm to photocrosslinkable chitosan (Az-CH-LA) aqueous solution to which 2.5% p-azidebenzoic acid was introduced produced an insoluble hydrogel like soft rubber within 30sec. The hydrogel firmly adhered two pieces of ham with each other, depending upon the Az-CH-LA concentration (Fig. 1).Fig. (1).Photocrosslinkable chitosan hydrogel.Controlled Releases of FGF-2 and Paclitaxel Current Drug Delivery, 2006, Vol. 3, No. 4 353The binding strength and sealing strength of the pho-tocrosslinked chitosan hydrogel prepared from 2 - 4 w% Az-CH-LA aqueous solution was superior to that of fibrin glue (Beriplast P: Hoechst-Marion-Roussel, Tokyo, Japan) [15]. The bursting pressures of the photocrosslinked chitosan hy-drogel were more than that of the fibrin glue on lung, small intestine, trachea, and thoracic aorta as shown in Table 1 [14, 15]. These results suggest that the sealing strength of the photocrosslinked chitosan hydrogel may be sufficient to stop arterial bleeding and air leakage from the lung or trachea in surgical applications.Table 1.Air-Sealing Strength of Photocrosslinked Chitosan Hydrogel and Fibrin GlueSealing strength (mmHg)Chitosan hydrogel Fibrin glue Lung51±11 (n=4)12±2 (n=4) Small intestine65±5 (n=6)48±7 (n=6) Trachea77±29 (n=6)44±16 (n=6) Thoracic aorta225±25 (n=6)65±15 (n=6)The data represent the mean ± SDAlthough most bleeding in surgical procedures can be controlled by appropriate sutures, hemostasis is uncontrolla-ble under certain conditions, such as coagulopathy, medica-tion of anticoagulants, inflammation, infection, and severe adhesion [15]. In addition, intractable air leakage in lung surgery has often been found, especially in emphysematous lung disease [15]. In many cases of such uncontrollable bleedings and intractable air leakages, a number of adhesives have been utilized in hemostasis and air sealing, i.e. chemi-cally crosslinkable gelatins [22], cyanoacrylate polymers [23, 24], and fibrin glues [25, 26].Requirements for such adhesives are non-irritating locally and non-toxic systemati-cally, an appropriate flexibility, and bio-degradability. How-ever, cytotoxicity and severe tissue irritability have been found when using resorcinol, formaldehyde, or carbodi-imides for the crosslink-reaction of gelatins[22] or due to the formation of formaldehyde by degradation of cyanoacrylate [23, 24]. Fibrin glue, which contains fibrinogen, thrombin, factor XIII, and a protease inhibitor, utilizes the blood co-agulation system for sealing tissues and currently is the most widely used surgical adhesive. Its effective hemostatic and air sealing abilities have been reported by many investigators [25, 26]. However, fibrin glue has a disadvantage in its in-dustrial production, since human blood is used as its source. Furthermore, when using the biological materials, it is diffi-cult to fully prevent infectious contaminations. Thus, poly-saccharides, such as chitosan, having a hydrogel-forming property are considered to be more advantageous in their application as adhesive materials [15].PACLITAXEL-INCORPORATED PHOTOCROSS-LINKED CHITOSAN HYDROGEL FOR TUMOR TREATMENTTo prepare a paclitaxel-incorporated photocrosslinked chitosan hydrogel, 1 ml of Taxol® (Bristol Pharmaceuticals K. K.) containing paclitaxel (6 mg/ml) in a vehicle composed of Cremophor® EL and ethanol at 50:50 (v/v) ratio was mixed into 1 ml of 40 mg/ml Az-CH-LA aqueous solution (finally 20 mg/ml Az-CH-LA solution) with vortex. UV-laser irradiation for 30 sec was able to convert to the same insoluble hydrogel by inserting optical crystal fiber con-nected with He-Cd laser into the injected viscous Az-CH-LA aqueous solution (0.2 ml). While about 35 - 40% of both the paclitaxel and the vehicle were released from the pho-tocrosslinked hydrogel within 1 day, and the releases were observed over a period of 4 days with half-releasing time of 45 h [18].Paclitaxel is a potent inhibitor of angiogenesis, cell mi-gration, and collagenase production in addition to its anti-proliferative effect for tumor cells [27-29]. Paclitaxel-incorporated photocrosslinked chitosan hydrogel was found to be able to inhibit Lewis lung cancer cells (3LL), human umbilical vein endothelial cells (HUVEC), and human der-mal microvascular endothelial cells (HMVEC) growth with low concentrations (>15 ng/ml). The washings of the pacli-taxel-incorporated photocrosslinked chitosan hydrogels with the culture medium for longer than 15 days resulted in a loss of ability to inhibit those cell growth except HMVEC growth. HMVEC growth could be inhibited in the presence of the washed paclitaxel-incorporated photocrosslinked chi-tosan hydrogel for longer than 21 days. On the other hand, the paclitaxel-incorporated photocrosslinked chitosan hydro-gel had significantly lower inhibitory effect on the fibroblast (human dermal fibroblast) growth [18].A measurable tumor volume (about 100 mm3) was formed at 12 days after subcutaneous implantation of 3LL cells. Paclitaxel-incorporated photocrosslinked chitosan hy-drogel and the controls was subcutaneously administered beneath the tumor using 18G needle and disposable (1 ml) syringe and UV laser irradiated for 30 sec. As shown in (Fig.2), administrations of paclitaxel alone and photocrosslinked chitosan hydrogels alone reduced subcutaneous induced tu-mor growth of 3LL cells to various extents during 7 days. The paclitaxel-incorporated photocrosslinked chitosan hy-drogel more strongly inhibited tumor growth to less than 5% of the control group than paclitaxel alone and pho-tocrosslinked chitosan hydrogel alone [18]. The inhibitory effect on tumor growth by the paclitaxel-incorporated pho-tocrosslinked chitosan hydrogel last during 14 days and sub-sequently the tumor in almost all mice grew again. However, second application of the paclitaxel-incorporated pho-tocrosslinked chitosan hydrogel on 10 days after first appli-cation could have an additional anti-tumour effect.To evaluate the effect of paclitaxel-incorporated pho-tocrosslinked chitosan hydrogel on anti-angiogenesis, im-muno-histochemical staining of murine CD34 of paclitaxel-incorporated photocrosslinked chitosan hydrogel-treated, photocrosslinked chitosan hydrogel-treated, paclitaxel-treated, and control tumors of 3LL cells were carried out [18]. In paclitaxel-treated and control mice on day 8, many CD34 positive stained vessels were diffusely located and clearly formed tube-like structures in the tumor. On the other hand, CD34 positive stained vessels were significantly less in the paclitaxel-incorporated photocrosslinked chitosan hy-drogel-treated tumors. As shown in Fig. 3, paclitaxel-354 Current Drug Delivery, 2006, Vol. 3, No. 4Ishihara et al.Fig. (2). Inhibitory effect of paclitaxel-incorporated pho-tocrosslinked chitosan hydrogel on 3LL-tumor growth. Tumor cells were implanted into the dorsal subcutis of mice. After tumors reached a measurable size (about 100 mm3), 200µl of paclitaxel-incorporated photocrosslinked chitosan hydrogel were administered beneath the tumor. Data were compared with the average tumor volume of the PBS treated group on day 12, defined as 100%. Fig. (3). Effect of paclitaxel-incorporated photocrosslinked chitosan hydrogel on 3LL-tumor vascularization. Vascularization of the 3LL-tumor, evaluated immuno-histochemically with anti-murine CD34, markedly decreased in paclitaxel-incorporated pho-tocrosslinked chitosan hydrogel-treated and photocrosslinked chito-san hydrogel-treated 3LL-tumors when compared with peclitaxel-treated and PBS-treated 3LL-tumors.incorporated photocrosslinked chitosan hydrogel signifi-cantly reduced the number of CD34 positive vessels com-pared with other treatments, suggesting that paclitaxel-incorporated photocrosslinked chitosan hydrogel signifi-cantly inhibited angiogenesis in tumors. Application of pho-tocrosslinked chitosan hydrogel showed intermediate effect of anti-angiogenesis. It is thus proposed that the paclitaxel-incorporated photocrosslinked chitosan hydrogel may be a promising new biomaterial to strongly inhibit vascularization and tumor growth.It should also be noted that application of the pho-tocrosslinked chitosan hydrogel alone significantly inhibited tumor growth, although the inhibitory activity was lower than the paclitaxel-incorporated photocrosslinked chitosan hydrogel. Chitosan has been observed to accelerate wound healing[30, 31] by inducing infiltration of inflammatory cells into a wound area[32], activation of macrophages[33], production of cytokines[34], as well as possessing an anti-infection activity[35]. In addition, it has also shown a growth-inhibition effect on tumor cells[36], inhibition of tumor-induced angiogenesis and tumor metastasis [37], and chitosan directly inhibits tumor cell proliferation by inducing apoptosis [38].FGF-2-INCORPORATED PHOTOCROSSLINKED CHITOSAN HYDROGEL FOR A WOUND MANA-GEMENTWound dressings before the 1960s were considered to be only the so-called passive products having a minimal role in healing process [39]. The pioneering research of Winter[40] initiated the concept of an active involvement of a wound dressing in establishing and maintaining an optimal envi-ronment for wound repair. This awareness resulted in the development of wound dressings from traditional passive materials to functional active dressings which, through the interaction with the wounds they cover, create and maintain a moist and healing environment. An ideal wound dressing should protect the wound from bacterial infection, provide a moist and healing environment, and be biocompatible [39].Among the fibroblast growth factors, FGF-2 is well char-acterized[41]. It is a potent modulator of cell proliferation, motility, differentiation, and survival, as well as plays an important role in normal regeneration processes in vivo, i.e. embryonic development[42, 43], angiogenesis[44], os-teogenesis[45, 46], and wound repair[30, 31]. FGF-2 is known to be stored in various sites of the body, interacting with glycosaminoglycans such as heparin and heparan sul-fate of the extracellular matrix [47-49]. FGF-2 specifically binds to heparin and heparan sulfate with a high affinity, and both its mitogenic activity and biological stability are modulated by heparin[49, 50] and chitosan [51]. Heparin enhances the mitogenic activity of FGF-1 and FGF-2[52, 53]. Heparin and chitosan also protect FGF-2 from inactiva-tion by acid and heat, as well as from degradation by prote-ases[49-51]. Other studies have shown evidence that heparin and heparan sulfate serve as a co-factor to promote binding of FGF-2 to high-affinity receptors, enhancing its activity [52, 53].Biodegradable polymer macromolecules are classified as either synthetic or natural. Polyactic acid and polyglycolic acid belong to the former group, whereas collagen, gelatin, alginate, and chitosan belong to the latter. Based on the in vivo storage mechanism, controlled release of the heparin-binding growth factors has been described from heparin-Controlled Releases of FGF-2 and Paclitaxel Current Drug Delivery, 2006, Vol. 3, No. 4 355carrying polystyrene-bound collagen substrata[54], acidic gelatin hydrogels[55], alginate gels containing heparin[56], and photocrosslinked chitosan hydrogels [19-21]. Lopez et al. reported the sustained release of FGF-2 using alginate gel containing heparin [57]. Because alginate is a poorly biode-gradable polysaccharide, it may difficult to control the car-rier degradation and the subsequent FGF-2 release. Further-more, collagen and gelatin have a disadvantage in its indus-trial production, since animal tissues are used as its source. When using the biological materials, it is difficult to fully prevent infectious contaminations. Polysaccharides, e.g. chitosan, have been considered to be advantageous in their application as a wound dressing material [19, 20, 58]. In contrast, chitosan with FGF-2 has many useful and advanta-geous biological properties in the application as a biosafty as well as a wound dressing, namely biocompatibility, biode-gradability, hemostatic activity, anti-infectional activity and property to accelerate wound-healing [19, 20, 58].To prepare a FGF-2-incorporated photocrosslinkable chitosan (Az-CH-LA), 1 ml of 50µg human recombinant FGF-2 in PBS was mixed into 1 ml of 40 mg/ml Az-CH-LA aqueous solution (finally 20 mg/ml Az-CH-LA solution) with vortex [19, 20]. Approximately 20% of the incorporated FGF-2 molecules was found to be released to the washing medium in vitro from the photocrosslinked chitosan hydro-gels within the first day, followed by no substantial further release after that [19, 20]. Initial small release of FGF-2 (about 20%) from the photocrosslinked chitosan hydrogels may be explained in terms of this molecular diffusion.It has been shown that the application of the pho-tocrosslinked chitosan hydrogel into open wounds induces a significant wound contraction, thereby accelerating the wound closure and healing process in a normal mouse model for wound repair [30, 31]. In addition, the photocrosslinked chitosan hydrogel showed the ability of controlled release of various growth factors serving as a novel carrier and induc-ing neovascularization in vivo [19-21]. FGF-2 interacted with Az-CH-LA molecules and the FGF-2 molecules incor-porated into the photocrosslinked chitosan hydrogel were gradually released upon in vivo biodegradation of the hydro-gel itself. We also evaluated the effect of FGF-2-incorporated photocrosslinked chitosan hydrogel on the wound healing process using healing impaired diabetic db/db mice (Fig. 4) [20, 21]. The FGF-2-incorporated pho-tocrosslinked chitosan hydrogels show a substantial effect to induce vascularization and granulation tissue formation and to improve wound healing in the db/db mice [20, 21].It is interesting to note that that the addition of FGF-2 to the photocrosslinked chitosan hydrogel had only a minor effect on the degree of healing in normal (db/+ : their normal littermates) mice (data not shown). Although the mechanism responsible for the minor effect of FGF-2 in normal (db/+) mice is not completely understood, it is likely that the pres-ence of macrophages has a significant effect on the forma-tion of wound granulation tissue[20, 21], and that macro-phage accumulation is enough in db/+ mice. Furthermore, a defect in vascular endothelial growth factor (VEGF) expres-sion is suggested to be associated with a wound-healing dis-Fig. (4). Wound closure of FGF-2-incorporated photocrosslinked chitosan hydrogel-treated db/db mice. Open wound areas of FGF-2-incorporated photocrosslinked chitosan hydrogel-treated, photocrosslinked chitosan hydrogel-treated, and control (none) wounds were de-termined every 2 day after initial wounding. The data represent the mean±SE of eight mice. *Student t test, p<0.001, n=8. The photographsof the wound at day 2, day 4, or day 16 are representative of eight mice in each group.356 Current Drug Delivery, 2006, Vol. 3, No. 4Ishihara et al.order[20, 21]. Thus, it is possible that db/+ mice have a suf-ficient amount of growth factors in the wound. INJECTABLE CHITOSAN/IO4-HEPARIN HYDRO-GELThe photocrosslinkable chitosan (Az-CH-LA) is viscous soluble solution and the photocrosslinked chitosan hydrogel with UV-irradiation is not injectable. In this study, we have prepared the injectable FGF-2-incorporated chitosan/IO4-heparin hydrogel. Water-soluble chitosan molecules (CH-LA) have been prepared as described above. The CH-LA aqueous solution is a viscous solution and is easily gelled upon mixing with a non-anticoagulant (IO4-) heparin solu-tion, resulting in an injectable hydrogel, probably due to ionic interaction of chitosan (plus charged) with non-anticoagulant (IO4-) heparin (minus charged). Non-anticoagulant (IO4-) heparin was prepared using native hepa-rin from porcine intestine, as has been reported previously [16, 17]. One ml of phosphate-buffered saline (PBS) con-taining 50 _g/ml FGF-2 and 4 mg/ml IO4-heparin was mixed into 1 ml of 40 mg/ml CH-LA aqueous solution (finally 20 mg/ml CH-LA solution) using vortex. Only a minor amount of IO4-heparin released from the injectable FGF-2-incorporated chitosan/IO4-heparin hydrogel, and approxi-mately 20% of FGF-2 released within the first day, followed by no further substantial release after that [16, 17]. INJECTABLE FGF-2-INCORPORATED CHITOSAN/ IO4-HEPARIN HYDROGEL FOR THERAPEUTIC ANGIOGENESIS IN HIND LIMB ISCHEMIA Tissue hemoglobin in subcutaneous tissue in db/db mice around the injected sites of the FGF-2-incorporated chito-san/IO4-heparin hydrogels was measured in order to evaluate the controlled release of FGF-2 from the hydrogels and its neovascularization. The tissue hemoglobin amount increased until 4-8 days after injection of FGF-2-incorporated chito-san/IO4-heparin hydrogel and then slightly decreased from day 15. Since injections of FGF-2 solution, IO4-heparin so-lution, FGF-2/IO4-heparin solution, FGF-2/chitosan solution, and chitosan/IO4-heparin hydrogel did not show a significant neovascularizations in db/db mice, co-addition of FGF-2 into the chitosan/IO4-heparin hydrogel resulted in a significant enhanced vascularization effect [16, 17].The capillary number per microphotograph (X 100) in tissues near the injected sites in db/db mice was determined in Fig. 5. Numerous mature vessels containing erythrocytes were observed around the injected FGF-2-incorporated chi-tosan/IO4-heparin hydrogels at 1 and 2 weeks. The neovas-cularization was however not observed on day 2 after injec-tion of the FGF-2-incorporated chitosan/IO4-heparin hydro-gel.The oxygen saturation (ankle) and limb blood flows (be-low knee and midportion of calf) were measured (Fig. 6) on day 3, 7, 14, 21, and 28 after the ligation of femoral artery in rats [17]. The blood flows at below knee and midportion of calf decreased to 40 - 60% of normal tissues (without the ligation of femoral artery), respectively, just after the ligation of femoral artery and the reductions were almost constant until 4 weeks. The administrations of FGF-2-incorporated chitosan/IO4-heparin hydrogel recovered the blood flows at below knee and midportion of calf to normal level from 1 week after the injection and the recoveries were maintained until at least 4 weeks. The oxygen saturation of ankle was also decreased to 76 - 87% of normal tissues after the liga-tion of femoral artery and the injection of FGF-2-incorporated chitosan/IO4-heparin hydrogel gradually recov-ered the reduction [17]. Thus, the FGF-2-incorporated chi-tosa/IO4-heparin hydrogel has great potential as an angio-genic therapy in medical use.Fig. (5). Effect of FGF-2-incorporated chitosan/IO4-heparin hydro-gel-injection on the vascularization in vivo. The number of capil-laries in FGF-2-incorporated chitosan/IO4-heparin hydrogel injected (black bars), chitosan/IO4-heparin hydrogel injected (gray bars), and chitosan (CH-LA) injected (white bars) db/db mice was counted using the microphotograph of each section (n=6) consid-ered to show the largest mature capillary density.SAFETY OF CHITOSAN HYDROGELNeither Az-CH-LA nor its photocrosslinked hydrogel showed any cytotoxicity in cell culture tests of human skin fibroblasts, endothelial cells, or smooth muscle cells. No reduced survival rate was observed in mice to which up to 1 ml of a 30 mg/ml Az-CH-LA aqueous solution was ad-minidtered intraperitoneally or implanted with the pho-tocrosslinked chitosan hydrogel prepared from 200 _l of 30 mg/ml Az-CH-LA aqueous solution. In addition, toxicity tests toward organisms including those for mutagenecity and cytotoxicity have shown the safety of both the Az-CH-LA solution and its photocrosslinked hydrogel (data not shown). These results suggest the safe use of the chitosan hydrogel in medical applications, although more detailed toxicity tests using appropriate animal models remain to be evaluated. Furthermore, detailed safety tests for the UV-irradiation to produce the photocrosslinked chitosan hydrogel also remain to be evaluated.CONCLUSIONThe photocrosslinked chitosan hydrogel could effectivelystop bleeding from carotid artery and air leakage from lung。