Regeneration of full-thickness abdominal wall defects in rats using collagen scaffolds loaded with collagen-binding basic fibroblast growth factor

Biomaterials are increasingly used in the repair of tissue defects. The aim of the present study was to evaluate a new composite biomaterial for reconstruction of a 2 × 2.5 cm full-thickness abdominal wall defect. In this study, the collagen membrane was activated with the engineered human basic fibroblast growth factor (bFGF). To enhance the binding of bFGF to collagen membranes, a specific peptide of collagen-binding domain (CBD) was fused to the N-terminal of bFGF. After implantation, little adhesion was caused in collagen/CBD-bFGF, collagen/NAT-bFGF and collagen/PBS groups. Moreover, collagen/CBD-bFGF group could effectively promote the vascularization at 30 d after surgery and significantly accelerate the integration of myofibers into the collagen material at 90 d after surgery compared to the other two groups. Due to the replacement of the myofibers in materials, the mechanical strength of implanted biomaterials in collagen/CBD-bFGF group was also greater than the other two groups at 90 d after surgery. Thus, the collagen/CBD-bFGF composite biomaterial was promising for the treatment of full-thickness abdominal wall defect.     

Extrahepatic bile duct regeneration in pigs using collagen scaffolds loaded with human collagen-binding bFGF

Extrahepatic bile duct defects and their complications are benign lesions but with malignant outcomes. Extrahepatic bile duct regeneration at the injury site could be important for the repair. In our previous work, a human basic fibroblast growth factor (bFGF) fused with a collagen-binding domain (CBD) was produced to activate the collagen membrane to obtain targeted tissue regeneration. This collagen/growth factor functional biomaterial could promote the regeneration of skin, bladder and full-thickness abdominal wall by accelerating vascularization and cellularization of autologous tissues. We speculate that the functional biomaterial could also provide the repairing effect on extrahepatic bile duct injuries. Using a pig extrahepatic bile duct injury model, we found that the collagen/CBD-bFGF composite biomaterial could significantly promote the extrahepatic bile duct regeneration at the injury site without causing structure deformation or hepatic dysfunction during both short- and long-time observations.     

Promotion of skin regeneration in diabetic rats by electrospun core-sheath fibers loaded with basic fibroblast growth factor

Diabetic skin ulcer is difficult to heal due to the lack of cellular and molecular signals required for normal wound repair. Emulsion electrospinning was adopted to imbed basic fibroblast growth factor (bFGF) into ultrafine fibers with a core-sheath structure to promote the wound healing process. An initially burst release as low as 14.0 ± 2.2% was achieved, followed by gradual release for around 4 wk. In vitro investigations on mouse embryo fibroblasts indicated that bFGF-loaded fibrous mats enhanced cell adhesion, proliferation, and secretion of extracellular matrix (ECM). Skin wounds were created in the dorsal area of diabetic rats for in vivo evaluation of skin regeneration after covered with bFGF-loaded fibrous mats. Compared with fibrous mats infiltrated with free bFGF, bFGF-loaded scaffolds revealed significantly higher wound recovery rate with complete re-epithelialization and regeneration of skin appendages. Higher density and mature capillary vessels were generated during 2 wk after treatment with bFGF-loaded fibers, and there was no fiber fragment observed in the histological sections at week 4 after operation. The gradual release of bFGF from fibrous mats enhanced collagen deposition and ECM remodeling, and the arrangement and component of collagen fibers were similar to normal tissues. The above results demonstrate the potential use of bFGF-loaded electrospun fibrous mats to rapidly restore the structural and functional properties of wounded skin for patients with diabetic mellitus.     

Sustained release and activation of the growth factor basic fibroblast growth factor from loaded scaffolds in heart valve tissue engineering

Objectives. Loading of biological matrices offers an opportunity to induce specific cell behaviour. We previously reported the use of growth factors to promote cell invasion and proliferation in tissue valve engineering.

We investigated biological matrices preloaded with heparin as an ionically attractive template for the binding, activation and sustained release of basic fibroblast growth factor (bFGF).

Methods. Heparin loading concentrations were evaluated and different incubation times were tested. Heparin and heparin-bound bFGF uptake and release were evaluated by ¹²³I radio-labelling. Biological activity of bFGF was evaluated in vitro.

Results. Maximum heparin uptake was observed for 2000 μg/ml at 2 h and stabilized thereafter. bFGF-loaded matrices showed an initial burst release of 15% within 4 h and thereafter sustained release reaching 21% at 24 h. Released bFGF was bioactive.

Conclusions. This model would be useful in tissue engineering using porcine aortic matrices and could be applied using other growth factors or combinations.     

Vascularization and cellularization of collagen scaffolds incorporated with two different collagen-targeting human basic fibroblast growth factors

To develop a collagen-based wound targeting repair system, we introduced two collagen-binding domains (CBDs) into the human basic fibroblast growth factor (bFGF). Three expression vectors were constructed: the first one (named V-bFGF) contained bFGF and the CBD WREPSFCALS derived from von Willeband's factor (vWF); the second (named C-bFGF) contained bFGF and the CBD TKKTLRT derived from collagenase; the third (named bFGF) was bFGF as a control. The recombinant proteins of V-bFGF and C-bFGF were demonstrated to retain both growth factor activity and collagen-binding activity. We found that C-bFGF possessed higher collagen-binding ability than V-bFGF. The targeted repair systems consisting of collagen scaffolds and CBD-bFGFs were assembled in vitro and then implanted subcutaneously. Results showed that C-bFGF promoted vascularization at the implanted sites more effectively than V-bFGF. Histological analysis showed more cells migrated into collagen scaffolds incorporated with C-bFGF than those with V-bFGF. These data suggested that the higher collagen-binding ability the CBD-bFGF possessed, the more significant vascularization, and cellularization were observed. In summary, CBD-bFGF/collagen system could be used as a targeted repair system with beneficial effects of the restriction of bFGF diffusion, the prolonging of bFGF activity, and the targeted promotion of vascularization and cellularization.     

Promotion of peripheral nerve growth by collagen scaffolds loaded with collagen-targeting human nerve growth factor-beta

Nerve growth factor (NGF) plays a critical role in neuronal development and regeneration. However, the lack of efficient NGF delivery system limits its clinical application. We reported that a peptide deduced from collagenase, TKKTLRT, fused with NGF-beta could develop a collagen based NGF targeting delivery system. Our results showed that this peptide could allow fused NGF-beta bind to collagen specifically. In addition, we found that the polypeptide could result in a 2.3-fold increase in the expression level and a significant improvement of bioactivity of fused NGF-beta. In the in vivo function study, collagen membranes loaded with the collagen binding NGF enhanced the nerve growth. Thus, the targeting wound repair system could be important for the repair of peripheral nerve injury.     

Regeneration of anterior cruciate ligament by biodegradable scaffold combined with local controlled release of basic fibroblast growth factor and collagen wrapping

The objective of this study was to increase the therapeutic efficacy of anterior cruciate ligament (ACL) surgery using an artificial ligament material developed through a combination of tissue engineering technologies. A poly-L-lactic acid (PLLA) scaffold of plain-woven braid was incorporated with a gelatin hydrogel for controlled release of basic fibroblast growth factor (bFGF) and wrapped with a collagen membrane to allow space for ligament regeneration. For the ACL reconstruction surgery, the PLLA braid scaffold combined with the gelatin hydrogel incorporating bFGF and the collagen wrapping was applied to a tunnel prepared in the femur and tibia of rabbits. The hydrogel was placed in the bone, whereas the portion of the braid inside the joint cavity was wrapped with the membrane. As controls, the PLLA scaffold was applied with the hydrogel or the membrane, or without either material. Bone regeneration in the tunnel and ACL tissue regeneration in the joint cavity were histologically evaluated, and the mechanical strength and collagen content of the regenerated ACL were assessed. When the PLLA scaffold was integrated with both the hydrogel and the membrane, bone and ACL tissues were regenerated in the corresponding sites, in marked contrast to the control groups. Combination of bFGF-controlled release resulted in enhanced mechanical strength of the regenerated ACL tissue. In the joint cavity, it is possible that the local bFGF release inside the membrane enhanced the cell migration and collagen production, and that the surrounding PLLA scaffold results in the biological regeneration of ligament-like tissue. Additionally, significant bone regeneration around the scaffold was observed in the bone tunnel. It is therefore possible that the local controlled release of bFGF near the PLLA braid induced both osseointegration and intrascaffold cell migration in the bone tunnel and joint cavity, respectively, resulting in an overall increase in the mechanical strength of the regenerated ACL.     

In vivo cellular repopulation of tubular elastin scaffolds mediated by basic fibroblast growth factor

In vivo tissue engineering has been explored as a method to repopulate scaffolds with autologous cells to create a functional, living, and non-immunogenic tissue substitute. In this study, we describe an approach to in vivo cellular repopulation of a tissue-derived tubular elastin scaffold. Pure elastin scaffolds were prepared from porcine carotid arteries (elastin tubes). Elastin tubes were filled with agarose gel containing basic fibroblast growth factor (bFGF) to allow sustained release of growth factor. These tubes were implanted in subdermal pouches in adult rats. The elastin tubes with growth factor had significantly more cell infiltration at 28 days than those without growth factor. Immunohistochemical staining indicated that most of these cells were fibroblasts, of which a few were activated fibroblasts (myofibroblasts). Microvasculature was also observed within the scaffolds. Macrophage infiltration was seen at 7 days, which diminished by 28 days of implantation. None of the elastin tubes with bFGF calcified. These results demonstrated that the sustained release of bFGF brings about repopulation of elastin scaffolds in vivo while inhibiting calcification. Results showing myofibroblast infiltration and vascularization are encouraging since such an in vivo implantation technique could be used for autologous cell repopulation of elastin scaffolds for vascular graft applications.     

A myocardial patch made of collagen membranes loaded with collagen-binding human vascular endothelial growth factor accelerates healing of the injured rabbit heart

Tissue-engineered myocardial patches could be useful in the repair of myocardial injuries. The aim of the present study was to evaluate a collagen targeting delivery system for myocardial repair. A specific peptide collagen-binding domain (CBD) was fused to human vascular endothelial growth factor (VEGF) to enhance the binding of VEGF to collagen. In this study, collagen membranes loaded with CBD-VEGF, natural VEGF, or phosphate-buffered saline are used as cardiac patches to repair the infarcted myocardium in a rabbit model. CBD-VEGF/collagen group could effectively induce more cells to penetrate into the collagen membrane after 4 weeks and promote more vascularization in infarcted myocardium after 12 weeks compared with the other two control groups. Echocardiography and hemodynamic studies both show cardiac function improvement in the CBD-VEGF/collagen group. These results reveal that implantation of CBD-VEGF collagen membrane patch into the infarcted myocardium could effectively improve left ventricle cardiac function and increase the vascular density.     

Monoclonal antibodies against human basic fibroblast growth factor

Recombinant human basic fibroblast growth factor (hbFGF) was used as an antigen to develop, by a somatic cell fusion technique, four monoclonal antibodies (MAbs), that recognize the complete and amino-terminal truncated form of hbFGF. Isotype identification showed that MAbs designated MAb12 and MAb98 were IgG1; and those designated MAb52 and MAb78 were IgG2b. All these MAbs bound the complete form of hbFGF produced in E.coli. Competition with synthetic polypeptides, a replication of 1-9 aa and of 141-146 aa of hbFGF, and truncated forms of hbFGF by 13 and 40 amino acid residues in its amino-terminal produced in E. coli by recombinant technique, revealed at least two epitopes recognized by the four IgG type MAbs. MAb12 and MAb78 recognized the epitope located within the first 9 amino acid residues at the amino terminal of the complete hbFGF. MAb52 and MAb98 recognized the one located between the amino acid residue no. 14 and 40. None of MAbs bound bovine acidic FGF (aFGF). Using MAb52 or MAb98 and MAb78, a two-site EIA has been developed. This EIA is sensitive enough to detect 0.5 ng/ml of hbFGF. Furthermore, MAb78 was used as a ligand for affinity chromatography to purify hbFGF mutein CS4, which binds weakly to a heparin affinity column.     

Expression of basic fibroblast growth factor in normal human tissues

The distribution of basic fibroblast growth factor (bFGF) was studied immunohistochemically in fresh frozen sections of normal human tissues. Immunodetection was performed with a specific anti-bFGF mouse monoclonal antibody that was found to react with recombinant human bFGF in Western blot analysis, and to specifically neutralize the mitogenic activity of bFGF on bovine vascular endothelial cells. Expression of bFGF on normal human tissues was ubiquitously detected in the basement membranes of all size blood vessels, but was not found in epidermal or epithelial basement membranes of a variety of tissues tested. Intensity and patterns of localization in blood vessels was consistent in various tissues, but varied among different regions of the vascular bed. Whereas homogeneous and intense immunoreactivity were observed in large and intermediate size blood vessels, heterogeneity of expression was found in capillaries. The most intense immunoreactivity was observed in branching capillaries. Endothelial cell staining was heterogeneous and varied in different regions. Strong staining for bFGF was also found in cardiac muscle fibers, smooth muscle cells of mid-size blood vessels, the gut and the myometrium, in central nervous system neurons and cerebellar Purkinje cells, and on epithelial cells of the bronchi, colon, endometrium, and sweat gland ducts of the skin. The presence of bFGF in the extracellular compartment of a diverse variety of organs may play a role in angiogenesis. However, the function of bFGF in parenchymal cells remains to be determined.     

Delivery of basic fibroblast growth factor (bFGF) from photoresponsive hydrogel scaffolds

Exogenous growth factor therapy has shown a notable promise in accelerating the healing of acute and chronic wounds. However, their susceptibility to enzymatic degradation and short contact time with the wound bed warrant the use of sophisticated delivery vehicles that stabilize the encapsulated peptides and control their rate of release. Herein, we describe the synthesis of a nitrocinnamate-derived polyethylene glycol (PEG-NC) hydrogel system and study the release kinetics of basic fibroblast growth factor (bFGF) as a function of hydrogel properties. Long-wave ultraviolet irradiation (365 nm) was used to alter the physical properties of the gel scaffold (i.e. degree of swelling) and consequently control the release rates of the encapsulated bFGF. The degree of swelling (DS) decreased from 10.7 to 8 as the length of irradiation increased from 5 to 30 min. Similarly, the DS decreased from 17.5 to 11.5 by increasing the initial PEG-NC concentration from 10 to 30 w/v% while keeping the crosslinking irradiation at 10 min. Radiolabeled I(125) studies were used to monitor the release of bFGF from PEG-NC hydrogels with variable swellabilities. By increasing the length of irradiation from 2 to 10 min the rate of bFGF release from PEG-NC gel scaffolds was decreased by 29% due to the enhanced crosslinking density. The bFGF-releasing PEG-NC hydrogels were not cytotoxic to human neonatal fibroblast cells and the released growth factor maintained its activity and induced fibroblast proliferation and collagen production in vitro. The addition of heparin within the gel scaffolds further increased the growth factor's activity.     

Proliferation of endothelial cells on surface-immobilized albumin-heparin conjugate loaded with basic fibroblast growth factor.

Seeding of endothelial cells (ECs) on the luminal surface of small-diameter vascular grafts is a promising method to avoid occlusion of these prostheses. Immobilization of basic fibroblast growth factor (bFGF) to substrates used to coat or fill porous prostheses may enhance the formation of a confluent monolayer of ECs. Human umbilical vein endothelial cells (HUVECs) were grown on bFGF-loaded albumin-heparin conjugate bound to CO2 gas-plasma-treated polystyrene. In the order of 2-3 ng/cm2 bFGF had to be immobilized to form a confluent monolayer of HUVECs. The most prominent effect of surface-immobilized bFGF was stimulation of the proliferation shortly after seeding, resulting within 3 days in confluent cell monolayers with high density. In contrast, in cultures with 0.3 ng/mL bFGF in the medium instead of bFGF bound to the surface, it took almost a week before the cell layers reached confluency. Binding of bFGF to heparin and the biological activity of bFGF towards ECs were not influenced by the (radio-)labeling of bFGF with iodine. However, only a minor part of the bFGF used in this study displayed heparin affinity. Furthermore, degradation and multimerization of labeled bFGF in time occurred when the growth factor was stored at 20 degrees -37 degrees C. This limits the use of labeled bFGF to short-term (hours) experiments. In conclusion, bFGF loading of vascular graft surfaces through complexation of bFGF with a heparin-containing matrix probably will lead to more rapid formation of a confluent monolayer of ECs on graft surfaces upon seeding of the cells.     

Perlecan domain I promotes fibroblast growth factor 2 delivery in collagen I fibril scaffolds

Perlecan, a heparan sulfate proteoglycan, is widely distributed in developing and adult tissues and plays multiple, important physiological roles. Studies with knockout mouse models indicate that expression of perlecan and heparan sulfate is critical for proper skeletal morphogenesis. Heparan sulfate chains bind and potentiate the activities of various growth factors such as fibroblast growth factor 2 (FGF-2). Previous studies indicate that important biological activities are associated with the heparan sulfate-bearing domain I of perlecan (PlnDI; French et al. J. Bone Miner. Res. 17 , 48, 2002). In the present study, we have used recombinant, glycosaminoglycan-bearing PlnDI to reconstitute three-dimensional scaffolds of collagen I. Collagen I fibrils bound PlnDI much better than native collagen I monomers or heat-denatured collagen I preparations. Heparitinase digestion demonstrated that recombinant PlnDI was substituted with heparan sulfate and that these heparan sulfate chains were critically important not only for efficient integration of PlnDI into scaffolds, but also for FGF-2 binding and retention. PlnDI-containing collagen I scaffolds to which FGF-2 was bound sustained growth of both MG63, an osteoblastic cell line, and human bone marrow stromal cells (hBMSCs) significantly better than scaffolds lacking either PlnDI or FGF-2. Collectively, these studies demonstrate the utility of PlnDI in creating scaffolds that better mimic natural extracellular matrices and better support key biological activities.     

Cartilage tissue engineering PLLA scaffold with surface immobilized collagen and basic fibroblast growth factor

A previously reported "grafting and coating" method (J. Biomed. Mater. Res. (Appl. Biomater.) 63 (2002) 838) was modified and used to introduce stable collagen layer and incorporate basic fibroblast growth factor (bFGF) on PLLA scaffold surface to prepare tissue engineering scaffold with improved biocompatibility. To make the modification of the 3-D porous PLLA scaffold possible, grafting of polymethacrylic acid (PMAA) onto the PLLA surface was initiated by the -OOH/Fe2+ system instead of the UV light used in the former method. Water soluble carbodimmide chemistry was applied to graft collagen onto the PLLA scaffold surface, followed by physical coating of the collagen solution with or without basic fibroblast growth factor (bFGF). Surface modification of 2-D PLLA membrane was also done for fundamental understanding of the modification. The -COOH density on/in the PMAA grafted PLLA membrane/scaffold was measured by colorimetric method and the collagen content on/in the collagen immobilized PLLA membrane/scaffold was measured by ninhydrin method. Chondrocyte culturing on the collagen immobilized PLLA surfaces showed significantly improved cell spreading and growth. Incorporation of fibroblast growth factors in the collagen layer further enhanced the cell growth. This convenient and effective method can be used to prepare bioactive scaffolds with extra cellular matrix (ECM)-mimic composition for tissue engineering.     

构建缓慢释放碱性成纤维细胞生长因子的脱细胞羊膜人工活性真皮**☆

背景：目前人工皮肤替代品的种类较多,各有优缺点,仍然没有一种理想的产品应用于临床。 目的：探讨构建一种可以缓慢释放碱性成纤维细胞生长因子的新型人工活性真皮的可行性。 方法：组织块法培养幼儿包皮成纤维细胞;采用酶-去垢剂法制备人脱细胞羊膜;双相法制备碱性成纤维细胞生长因子-明胶-壳聚糖缓释微球;缓释微球黏附于脱细胞羊膜上;三四代成纤维细胞培养于负载缓释微球的脱细胞羊膜上。 结果与结论：制备的脱细胞羊膜为白色半透明状薄膜有较高的孔隙率,空隙不规则,孔径大小为10~100 nm,无细胞毒性;碱性成纤维细胞生长因子-明胶-壳聚糖缓释微球分散较均匀,呈球形,粒径均匀,球体表面比较光滑,载药率为20 ng/g,包封率为80.5%,体外药物缓释曲线显示药物控释效果良好;成纤维细胞在支架表面爬行生长良好,层粘连蛋白表达较对照组高。表明将成纤维细胞种植于负载碱性成纤维细胞生长因子-明胶-壳聚糖缓释微球的脱细胞羊膜上,缓释微球能较好地黏附于脱细胞羊膜表面。     

Regeneration of canine tracheal cartilage by slow release of basic fibroblast growth factor from gelatin sponge

We investigated the efficiency of basic fibroblast growth factor (b-FGF) released from a gelatin sponge in the regeneration of tracheal cartilage. A 1-cm gap was made in the midventral portion of each of 10 consecutive cervical tracheal cartilages (rings 4 to 13) in 15 experimental dogs. In the control group (n = 5), the resulting gap was left blank. In the gelatin group (n = 5), a gelatin sponge alone was implanted in the gap. In the b-FGF group (n = 5), a gelatin sponge containing 100 mug b-FGF solution was implanted in the gap. We euthanatized one of the five dogs in each group at 1 month after implantation and one at 3 months and examined the implant sites macroscopically and microscopically. In the control and gelatin groups, no regenerated cartilage was observed in the tracheal cartilage gap at 1 or 3 months. The distances between the cartilage stumps had shrunk. In the b-FGF group, fibrous cartilage had started to regenerate from both host cartilage stumps at 1 month. At 3 months, regenerated fibrous cartilage filled the gap and had connected each of the stumps. The regenerated cartilage was covered with regenerated perichondrium originating from the host perichondrium. Shrinkage of the distance between the host cartilage stumps was not observed in the b-FGF group. We succeeded in inducing cartilage regeneration in the gaps in canine tracheal cartilage rings by using the slow release of b-FGF from a gelatin sponge. The regenerated cartilage induced by b-FGF was fibrous cartilage.     

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.     

碱性成纤维细胞生长因子和表皮生长因子联合诱导骨髓基质细胞分化形成神经球

目的观察碱性成纤维细胞生长因子(bFGF)和表皮生长因子(EGF)联合诱导骨髓基质细胞(MSCs)能否分化形成神经球。方法采用全骨髓法培养MSCs,bFGF和EGF联合诱导MSCs,倒置显微镜下观察分化细胞的形态,免疫荧光法和免疫印迹法鉴定分化细胞。结果 bFGF和EGF联合应用,可有效地诱导MSCs形成神经球。此神经球能传代并分化成神经元样细胞。免疫荧光和免疫印迹法均证实神经球表达nestin蛋白,神经球分化的细胞表达神经元、胶质细胞和少突胶质细胞标志性蛋白。结论 bFGF和EGF联合应用,可有效地诱导MSCs形成神经球。此神经球能自我更新并分化为神经元、胶质细胞和少突胶质细胞。