Three-dimensional macroporous calcium phosphate bioceramics with nested chitosan sponges for load-bearing bone implants

Three-dimensional macroporous calcium phosphate bioceramics embedded with porous chitosan sponges were synthesized to produce composite scaffolds with high mechanical strength and a large surface/volume ratio for load-bearing bone repairing and substitutes. The macroporous calcium phosphate bioceramics with pore diameters of 300 microm to 600 microm were developed using a porogen burnout technique, and the chitosan sponges were formed inside the pores of the bioceramics by first introducing chiosan solution into the pores followed by a freeze-drying process. Our scanning electron microscopy results showed that the pore size of chitosan sponges formed inside the macroporous structure of bioceramics was approximately 100 microm, a structure favorable for bone tissue in-growth. The compressive modulus and yield stress of the composite scaffolds were both greatly improved in comparison with that of HA/beta-TCP scaffolds. The simulated body fluid (SBF) and cell culture experiments were conducted to assess the bioactivity and biocompatibility of the scaffolds. In the SBF tests, a layer of randomly oriented needle-like apatite crystals formed on the scaffold surface after sample immersion in SBF, which suggested that the composite material has good bioactivity. The cell culture experiments showed that MG63 osteoblast cells attached to the composite scaffolds, proliferated on the scaffold surface, and migrated onto the pore walls, indicating good cell biocompatibility of the scaffold. The cell differentiation on the composite scaffolds was evaluated by alkaline phosphatase (ALP) assay. Compared with the control in tissue culture dishes, the cells had almost the same ALP activity on the composite scaffolds during the first 11 days of culture.     

Preparation of Open Porous Hyaluronic Acid Scaffolds for Tissue Engineering Using the Ice Particulate Template Method

A novel method to fabricate highly interconnected porous hyaluronic acid (HA) scaffolds with open surface pore structures was developed by using embossed ice particulates as a template. HA sponges were cross-linked by water-soluble carbodiimide (WSC) and the optimal cross-linking condition was analyzed by infrared spectroscopy. Cross-linking with 50 mM WSC in a 90% (v/v) ethanol/water solvent mixture assured the highest degree of cross-linking and most stable structure and, therefore, was used to cross-link the HA sponges. Observation with a scanning electron microscope showed that the HA scaffolds had funnel-like porous structures. There were large, open pores on the top surfaces and inner bulk pores under the top surface of the funnel-like HA sponges. The inner bulk pores were interconnected with the large, top surface pores and extended into the whole sponge. The pore morphology and density of the large, top surface pores were dependent on the dimension and density of the ice particulates. The size of the inner bulk pores was dependent on the freezing temperature. The funnel-like pore structures of the HA sponges facilitated cell penetration into the inner pores of the sponges and resulted in homogenous cell distribution in the sponges.     

Fabrication and characterization of PLLA-chitosan hybrid scaffolds with improved cell compatibility

To combine the individual advantages of synthetic and natural polymers, poly(L-lactic acid) (PLLA)-chitosan hybrid scaffolds were fabricated. PLLA sponges were prepared by particulate-leaching, and then PLLA-chitosan hybrid scaffolds were obtained by dipping the PLLA sponges in chitosan solution and subsequently freeze-drying. Physicochemical properties of the scaffolds were characterized by scanning electron microscopy (SEM), water uptake test, and mechanical strength measurement. Moreover, cell adhesion, cell proliferation, and cell viability on the scaffolds were evaluated through osteoblast-like cell culture. The experimental results indicated that, PLLA sponges exhibited macroporous structure and the interconnected microporous structure of chitosan was formed within the macropores of PLLA sponges. The incorporation of chitosan reinforced PLLA sponges in dependence on chitosan content. The hybrid scaffolds had higher water uptake ability compared with PLLA sponges. Particularly, the hybrid scaffolds exhibited excellent cell attachment efficiency, cell proliferation, and cell viability. This study suggests that the hybrid scaffolds obtain good mechanical strength from PLLA and excellent cell compatibility from chitosan.     

Chitosan scaffolds for in vitro buffalo embryonic stem-like cell culture: an approach to tissue engineering

Three-dimensional (3D) porous chitosan scaffolds are attractive candidates for tissue engineering applications. Chitosan scaffolds of 70, 88, and 95% degree of deacetylation (% DD) with the same molecular weight were developed and their properties with buffalo embryonic stem-like (ES-like) cells were investigated in vitro. Scaffolds were fabricated by freezing and lyophilization. They showed open pore structure with interconnecting pores under scanning electron microscopy (SEM). Higher % DD chitosan scaffolds had greater mechanical strength, slower degradation rate, lower water uptake ability, but similar water retention ability, when compared to lower % DD chitosan. As a strategy to tissue engineering, buffalo ES-like cells were cultured on scaffolds for 28 days. It appeared that chitosan was cytocompatible and cells proliferated well on 88 and 95% DD scaffolds. In addition, the buffalo ES-like cells maintained their pluripotency during the culture period. Furthermore, the SEM and histological study showed that the polygonal buffalo ES-like cells proliferated well and attached to the pores. This study proved that 3D biodegradable highly deacetylated chitosan scaffolds are promising candidates for ES-like cell based tissue engineering and this chitosan scaffold and ES cell based system can be used as in vitro model for subsequent clinical applications.     

Single-step mineralization of woodpile chitosan scaffolds with improved cell compatibility

A facile and efficient single-step mineralization approach was exploited for achieving nanoscopic hydroxyapatite (HAP) crystal layer in chitosan porous matrix, wherein a mixed water-ethanol solvent was used to control the growth of minerals. The crystallographic structure, morphology, and mechanical properties of the scaffold were analyzed with XRD, FTIR, environmental scanning electric microscopy (ESEM), TEM, and compression tests. The behaviors and responses of MC3T3-E1 pre-osteoblast cells on the scaffolds were studied as well. The results showed that the scaffolds kept woodpile structure with predefined and controlled hierarchical structure after mineralization. The inorganic phase in the mineralized chitosan scaffolds was determined as pure rod-like HAP, which settled densely on the matrix. The compression strength and compressive modulus of the scaffolds increased dramatically to 0.54 ± 0.005 MPa and 5.47 ± 0.65 MPa, respectively. During a culture period of 2 and 3 weeks, cell proliferation and in-growth were observed by phase contrast light microscopy and SEM. The alkaline phosphatase (ALP) activity increased after 1 week. Cell viability and cell proliferation index (PI) obtained higher values than that of the chitosan scaffolds. The novel single-step mineralization approach and the porous hybrid scaffolds would be a promising method for designing hybrid bone graft.     

Engineering endochondral bone: in vitro studies

Chitosan scaffolds have been shown to possess biological and mechanical properties suitable for tissue engineering and clinical applications. In the present work, chitosan sponges were evaluated regarding their ability to support cartilage cell proliferation and maturation, which are the first steps in endochondral bone formation. Chitosan sponges were seeded with chondrocytes isolated from chicken embryo sterna. Chondrocyte/chitosan constructs were cultured for 20 days, and treated with retinoic acid (RA) to induce chondrocyte maturation and matrix synthesis. At different time points, samples were collected for microscopic, histological, biochemical, and mechanical analyses. Results show chondrocyte attachment, proliferation, and abundant matrix synthesis, completely obliterating the pores of the sponges. RA treatment caused chondrocyte hypertrophy, characterized by the presence of type X collagen in the extracellular matrix and increased alkaline phosphatase activity. In addition, hypertrophy markedly changed the mechanical properties of the chondrocyte/chitosan constructs. In conclusion, we have developed chitosan sponges with adequate pore structure and mechanical properties to serve as a support for hypertrophic chondrocytes. In parallel studies, we have evaluated the ability of this mature cartilage scaffold to induce endochondral ossification.     

Fabrication and characterization of gelatin-based biocompatible porous composite scaffold for bone tissue engineering

In this study, composite scaffolds were prepared with polyethylene oxide (PEO)-linked gelatin and tricalcium phosphate (TCP). Chitosan, a positively charged polysaccharide, was introduced into the scaffolds to improve the properties of the artificial bone matrix. The chemical and thermal properties of composite scaffolds were investigated by Fourier transform infrared spectroscopy, thermogravimetric analyzer, differential thermal analyzer. In vitro cytotoxicity of the composite scaffold was also evaluated and the sample showed no cytotoxic effect. The morphology was studied by SEM and light microscopy. It was observed that the prepared scaffold had an open interconnected porous structure with pore size of 230-354 μm, which is suitable for osteoblast cell proliferation. The mechanical properties were assessed and it was found that the composite had compressive modulus of 1200 MPa with a strength of 5.2 MPa and bending modulus of 250 MPa having strength of 12.3 MPa. The porosity and apparent density were calculated and it was found that the incorporation of TCP can reduce the porosity and water absorption. It was revealed from the study that the composite had a 3D porous microstructure and TCP particles were dispersed evenly among the crosslinked gelatin/chitosan scaffold. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3020-3028, 2012.     

三维多孔电磁复合支架构建与理化表征

模拟心肌细胞外基质(ECM)的组织结构和电生理特性,构建具有导电性和超顺磁响应性的三维多孔纳米纤维复合材料支架,为细胞黏附、增殖和分化提供有利的微环境。以明胶为壳层、聚乳酸为芯层,并在各层中引入四氧化三铁纳米颗粒,采用共轴静电纺丝技术,制备纳米纤维薄膜;将其粉碎并与碳纤维混合,经冷冻干燥和交联处理,得到三维多孔支架。用扫描电镜和透射电镜观察支架形貌及结构,用四探针仪测定支架的电导率,用振动样品磁强计测量支架磁滞回线,用万能材料试验机检测支架材料的压缩应力-应变曲线;根据质量和体积计算支架密度,根据支架吸水前后质量计算吸水率。用CCK-8和Western Blot,分析细胞在支架上的活性及功能。支架内部呈多孔蜂窝结构,孔隙间相互贯通;当碳纤维含量为0、1、3、5 mg/mL时,支架密度分别为73.07、72.56、65.88、63.34 mg/cm³,吸水率分别为1 164.60%、1 186.48%、1 284.84%、1 323.66%;电导率分别为0、0.008 8、0.246 7、2.662 5 s/m,最大磁饱和强度分别为3.68、3.15、2.45、2.90 emu/g,抗压缩能力也相应提高。上述复合材料支架能够显著促进心肌细胞成熟相关蛋白Cx43和RhoA的表达,诱导心肌细胞向成熟分化。三维多孔电磁复合支架同时具有超顺磁性和导电性,微观上具有纳米纤维网络和多孔结构,并通过碳纤维的复合,使力学性能得到显著提高,支持心肌细胞生长并促进其成熟。     

聚乳酸-羟基乙酸共聚物/硅酸钙三维多孔骨组织工程支架的构建与性能

BACKGROUND: Some disadvantages exsist in commonly used poly(lactic-co-glycolic acid) (PLGA) scaffolds, including acidic degradation products, suboptimal mechanical properties, low pore size, poor porosity and pore connectivity rate and uncontrollable shape. OBJECTIVE: To construct a scaffold with three-dimensional (3D) pores by adding calcium silicate to improve the properties of PLGA, and then detect its degradability, mechanical properties and biocompatibility. METHODS: PLGA/calcium silicate porous composite microspheres were prepared by the emulsion-solvent evaporation method, and PLGA 3D porous scaffold was established by 3D-Bioplotter, and then PLGA/calcium silicate composite porous scaffolds were constructed by combining the microspheres with the scaffold using low temperature fusion technology. The compositions, morphology and degradability of the PLGA/calcium silicate porous composite microspheres and PLGA microspheres, as well as the morphology, pore properties and compression strength of the PLGA 3D scaffolds and PLGA/calcium silicate composite porous scaffolds were measured, respectively. Mouse bone marrow mesenchymal stem cells were respectively cultivated in the extracts of PLGA/calcium silicate porous composite microspheres and PLGA microspheres, and then were respectively seeded onto the PLGA 3D scaffolds and PLGA/calcium silicate composite porous scaffolds. Thereafter, the cell proliferation activity was detected at 1, 3 and 5 days. RESULTS AND CONCLUSION: Regular pores on the PLGA microspheres and internal cavities were formed, and the PH values of the degradation products were improved after adding calcium silicate. The fiber diameter, pore, porosity and average pore size of the composite porous scaffolds were all smaller than those of the PLGA scaffolds. The compression strength and elasticity modulus of the composite porous scaffolds were both higher than those of the PLGA scaffolds (P < 0.05). Bone marrow mesenchymal stem cells grew well in above microsphere extracts and scaffolds. These results indicate that PLGA/calcium silicate composite porous scaffolds exhibit good degradability in vitro, mechanical properties and biocompatibility.     

Preparation of Interconnected Porous Chitosan Scaffolds by Sodium Acetate Particulate Leaching

For tissue-engineering applications, a 3D porous chitosan scaffold was simply prepared from a mixture of acidic chitosan solution and sodium acetate particles as the porogen by a salt-leaching method. Differences in the porous structure in terms of pore morphology and interconnectivity between the salt-leached chitosan scaffold and phase-separated scaffold as the control were examined by using scanning electron microscopy, protein release and enzymatic degradation tests. A fibroblast (NIH-3T3) cell culture was performed for cell affinity evaluation. The chitosan scaffold prepared by salt-leaching showed good interconnectivity and improved mechanical properties. Furthermore, the chitosan scaffolds showed a high initial cell adhesion after 4 h cell culture and increased cell proliferation than the control. Thus, salt-leached chitosan scaffolds can be used for various tissue-engineering applications.     

Structure-process-property relationship of the polar graphene oxide-mediated cellular response and stimulated growth of osteoblasts on hybrid chitosan network structure nanocomposite scaffolds

We here describe the structure-process-property relationship of graphene oxide-mediated proliferation and growth of osteoblasts in conjunction with the physico-chemical, mechanical, and structural properties. Chitosan-graphene network structure scaffolds were synthesized by covalent linkage of the carboxyl groups of graphene oxide with the amine groups of chitosan. The negatively charged graphene oxide in chitosan scaffolds was an important physico-chemical factor influencing cell-scaffold interactions. Furthermore, it was advantageous in enhancing the biocompatibility of the scaffolds and the degradation products of the scaffolds. The high water retention ability, hydrophilic nature, and high degree of interconnectivity of the porous structure of chitosan-graphene oxide scaffolds facilitated cell attachment and proliferation and improved the stability against enzymatic degradation. The cells infiltrated and colonized the pores of the scaffolds and established cell-cell interactions. The interconnectivity of the porous structure of the scaffolds helps the flow of medium throughout the scaffold for even cell adhesion. Moreover, the seeded cells were able to infiltrate inside the pores of chitosan-graphene oxide scaffolds, suggesting that the incorporation of polar graphene oxide in scaffolds is promising for bone tissue engineering.     

Chitosan scaffolds: interconnective pore size and cartilage engineering

This study was designed to determine the effect of interconnective pore size on chondrocyte proliferation and function within chitosan sponges, and compare the potential of chitosan and polyglycolic acid (PGA) matrices for chondrogenesis. Six million porcine chondrocytes were seeded on each of 52 prewetted scaffolds consisting of chitosan sponges with (1) pores 10 microm in diameter (n=10, where n is the number of samples); (2) pores measuring 10-50 microm in diameter (n=10); and (3) pores measuring 70-120 microm in diameter (n=10), versus (4) polyglycolic acid mesh (n=22), as a positive control. Constructs were cultured for 28 days in a rotating bioreactor prior to scanning electron microscopy (SEM), histology, and determination of their water, DNA, glycosaminoglycan (GAG) and collagen II contents. Parametric data was compared (p=0.05) with an ANOVA and Tukey's Studentized range test. PGA constructs consisted essentially of a matrix containing more cells than normal cartilage. Whereas very few remnants of PGA remained, chitosan scaffolds appeared intact. DNA and GAG concentrations were greater in PGA scaffolds than in any of the chitosan groups. However, chitosan sponges with the largest pores contained more chondrocytes, collagen II and GAG than the matrix with the smallest pores. Constructs produced with PGA contained less water and more GAG than all chitosan groups. Chondrocyte proliferation and metabolic activity improved with increasing interconnective pore size of chitosan matrices. In vitro chondrogenesis is possible with chitosan but the composition of constructs produced on PGA more closely approaches that of natural cartilage.     

Chitosan–gelatin scaffolds for tissue engineering: Physico-chemical properties and biological response of buffalo embryonic stem cells and transfectant of GFP–buffalo embryonic stem cells

The favorable cellular response of newly developed cell line, buffalo embryonic stem (ES) cells to three-dimensional biodegradable chitosan–gelatin composite scaffolds with regard to stem-cell-based tissue engineering is described. Chitosan–gelatin composites were characterized by a highly porous structure with interconnected pores, and the mechanical properties were significantly enhanced. Furthermore, X-ray diffraction study indicated increased amorphous content in the scaffold on the addition of gelatin to chitosan. To develop a transfectant of green fluorescence protein (GFP)–buffalo ES cell, transfection of GFP plasmid to the cell was carried out via the electroporation procedure. In comparison with pure chitosan, cell spreading and proliferation were greater in highly visualized GFP-expressing cell–chitosan–gelatin scaffold constructs. The relative comparison of biological response involving cell proliferation and viability on the scaffolds suggests that blending of gelatin in chitosan improved cellular efficiency. Studies involving scanning electron and fluorescence microscopy, histological observations and flow cytometer analysis of the constructs implied that the polygonal cells attached to and penetrated the pores, and proliferated well, while maintaining their pluripotency during the culture period for 28 days. Chitosan–gelatin scaffolds were cytocompatible with respect to buffalo ES cells. The study underscores for the first time that chitosan–gelatin scaffolds are promising candidates for ES-cell-based tissue engineering.     

A microfabricated porous collagen-based scaffold as prototype for skin substitutes

An important element of artificial skin is a tissue scaffold that allows for fast host regeneration. We present a microfabrication strategy, based on gelling collagen-based components inside a microfluidic device, that produces well-controlled pore sizes inside the scaffold. This strategy can produce finely patterned tissue scaffolds of clinically relevant dimensions suitable for surgical handling. Compared to porous collagen-based sponges produced by lyophilization, microfabricated tissue scaffolds preserve the fibrous structure and ligand density of natural occurring collagen. A fibroblast migration assay revealed fast cellular migration through the pores, which is desired for rapid tissue ingrowth. Finally, we also demonstrate a strategy to use this microfabrication technique to build anatomically accurate, multi-component skin substitutes in a cost-effective manner.     

Multinozzle low-temperature deposition system for construction of gradient tissue engineering scaffolds

Tissue engineering is a technology that enables us to construct complicated hominine organs composed of many different types of cells. One of the key points to achieve this goal is to control the material composition and porous structure of the scaffold accurately. A disposable syringe based volume-driven injecting (VDI) nozzle was proposed and designed to extrude both natural derived and synthetic polymers. A multinozzle low-temperature deposition and manufacturing (M-LDM) system is proposed to fabricate scaffolds with heterogeneous materials and gradient hierarchical porous structures. PLGA, collagen, gelatin, chitosan can be extruded without leaking to form hierarchical porous scaffolds for primary study. Composite scaffolds with two kinds of materials were fabricated via two different nozzles to get both hydrophilic and mechanical properties. The results from scanning electron microscopy (SEM) demonstrated that the natural-derived biomaterials were strongly absorbed onto the synthetic biomaterials to form a stable network. Several gradient PLGA/TCP scaffolds were also fabricated to supply several samples.     

In vitro evaluation of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds for bone tissue engineering

A three-dimensional (3-D) scaffold is one of the major components in many tissue engineering approaches. We developed novel 3-D chitosan/poly(lactic acid-glycolic acid) (PLAGA) composite porous scaffolds by sintering together composite chitosan/PLAGA microspheres for bone tissue engineering applications. Pore sizes, pore volume, and mechanical properties of the scaffolds can be manipulated by controlling fabrication parameters, including sintering temperature and sintering time. The sintered microsphere scaffolds had a total pore volume between 28% and 37% with median pore size in the range 170-200microm. The compressive modulus and compressive strength of the scaffolds are in the range of trabecular bone making them suitable as scaffolds for load-bearing bone tissue engineering. In addition, MC3T3-E1 osteoblast-like cells proliferated well on the composite scaffolds as compared to PLAGA scaffolds. It was also shown that the presence of chitosan on microsphere surfaces increased the alkaline phosphatase activity of the cells cultured on the composite scaffolds and up-regulated gene expression of alkaline phosphatase, osteopontin, and bone sialoprotein.     

Porosity and biological properties of polyethylene glycol-conjugated collagen materials

Collagen-based materials can be designed for use as scaffolds for connective tissue reconstruction. The goal of the present study was to evaluate the behavior of collagen materials as well as cell and tissue reactions after the conjugation of activated polyethylene glycols (PEGs) with collagen. It is known that proteins conjugated with PEGs exhibit a decrease in their biodegradation rate and their immunogenicity. Different concentrations and molecular weights of activated PEGs (PEG-750 and PEG-5000) were conjugated to collagen materials (films or sponges) which were then investigated by collagenase assay, fibroblast cell culture, and subcutaneous implantation. PEG-conjugated collagen sponge degradation by collagenase was delayed in comparison to untreated sponges. In culture, fibroblasts with a normal morphology reached confluency on PEG-conjugated collagen films. In vivo, the porous structure of non-modified sponges collapsed by day 15 with a few observable fibroblasts between the collagen fibers. In PEG-modified collagen sponges, the porous structure remained stable for 30 days. Cell infiltration was particularly enhanced in PEG-750-conjugated collagen sponges. In conclusion, PEGs conjugated onto collagen sponges stabilize the porous structure without deactivating the biological properties of collagen. These porous composite materials could function as a scaffold to organize tissue ingrowth.     

Chitosan scaffolds containing hyaluronic acid for cartilage tissue engineering

Scaffolds derived from natural polysaccharides are very promising in tissue engineering applications and regenerative medicine, as they resemble glycosaminoglycans in the extracellular matrix (ECM). In this study, we have prepared freeze-dried composite scaffolds of chitosan (CHT) and hyaluronic acid (HA) in different weight ratios containing either no HA (control) or 1%, 5%, or 10% of HA. We hypothesized that HA could enhance structural and biological properties of CHT scaffolds. To test this hypothesis, physicochemical and biological properties of CHT/HA scaffolds were evaluated. Scanning electron microscopy micrographs, mechanical properties, swelling tests, enzymatic degradation, and Fourier transform infrared (FTIR) chemical maps were performed. To test the ability of the CHT/HA scaffolds to support chondrocyte adhesion and proliferation, live-dead and MTT assays were performed. Results showed that CHT/HA composite scaffolds are noncytotoxic and promote cell adhesion. ECM formation was further evaluated with safranin-O and alcian blue staining methods, and glycosaminoglycan and DNA quantifications were performed. The incorporation of HA enhanced cartilage ECM production. CHT/5HA had a better pore network configuration and exhibited enhanced ECM cartilage formation. On the basis of our results, we believe that CHT/HA composite matrixes have potential use in cartilage repair.     

Fabrication of porous chitosan-polyvinyl pyrrolidone scaffolds from a quaternary system via phase separation

Three-dimensional porous chitosan-polyvinyl pyrrolidone (PVP) scaffolds were fabricated for tissue engineering applications via liquid–liquid or liquid–solid phase separation. A mixture of an acidic aqueous solution with butanol as a non-solvent and a chitosan-PVP quaternary system were freeze-dried. We then studied the homogenous open pore structure and the minute pore distribution in order to improve the mass transfer and cell seeding efficiency while also obtaining the optimal ratio of PVP to provide high interconnectivity and to improve the open-pore structure. The properties of the porous chitosan-PVP scaffolds – including the microstructure, chemical release, water absorption properties, and cell proliferation tests were studied – and the results were compared against those obtained from conventional scaffolds. chitosan-PVP scaffolds with a porosity of over 70% were obtained, and the pore morphology on the surface and within the porous scaffolds showed the presence of homogenous open pores with excellent interconnectivity. As the PVP content increased, main pores (50–100 μm) and minute pores (4–10 μm) could be clearly observed. Also, the porous scaffold showed an improved efficiency for cell adhesion after the cells were cultured for 4 h. After 72 h, the cultured cells presented an increase in the cell proliferation and on the porous scaffolds. These results strongly suggest that the porous chitosan-PVP scaffolds can be widely used in tissue engineering, including for biopatches and artificial skin applications.     

Porous alginate/hydroxyapatite composite scaffolds for bone tissue engineering: preparation, characterization, and in vitro studies

In this study a series of alginate/hydroxyapatite (HAP) composite scaffolds was prepared by phase separation. HAP was incorporated into the alginate gel solution to improve both the mechanical and cell-attachment properties of the scaffolds. These scaffolds had a well-interconnected porous structure with an average pore size of 150 microm and over 82% porosity. The alginate/HAP scaffold prepared at -40 degrees C with a 50% HAP content showed the best mechanical properties. The morphology of scaffolds could be manipulated by tuning the quenching temperature during the preparation. The dissolution of alginate/HAP composite scaffolds could be slowed by the pretreating them by immersion in 1.0 M CaCl(2) solution. The rat osteosarcoma UMR106 cells, an osteoblastic cell line, seeded in the scaffolds, displayed better cell attachment to the 75/25 and 50/50 alginate/HAP composite scaffolds than to the pure alginate scaffold. The natural polymeric sponges that fabricated in this study may be a promising approach for tissue-engineering applications.