Effect of hydroxyapatite content on physical properties and connective tissue reactions to a chitosan-hydroxyapatite composite membrane.

The present study investigated the effect on certain physical properties of adding various amounts of hydroxyapatite (HAP) to chitosan sol. Also investigated were connective tissue reactions to a composite membrane that is being developed for possible use in guided tissue regeneration and for the limitation of HA particle migration at sites of implantation. The physical properties evaluated were shrinkage, tensile strength, hardness, calcium ion release, and morphology. Assessment of physical properties indicated that a ratio of HA to chitosan sol of 4/11 by weight is optimal in the preparation of the composite membrane. Subperiosteal implantation of the membranes over rat calvaria revealed that the membranes were well tolerated, with fibrous encapsulation and occasional areas of osteogenesis. Increasing the hydroxyapatite content seems to enhance membrane degradation.     

Behavior of MG-63 cells on nylon/chitosan-blended membranes

In this work, the properties of nylon, chitosan, and their blended membranes were investigated by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and wide-angle X-ray diffraction analysis. The SEM photographs show that the undulating surface of the nylon membrane became less obvious by blending with chitosan. The DSC and X-ray diffraction analysis show that constitutionally different features in the combination of two polymer chains were revealed, suggesting that nylon and chitosan are immiscible at the microscopic level in the blended membranes. Furthermore, an attempt was made to understand whether the two components contribute independently to the adhesion, growth, and activation of MG-63 osteoblastlike cells. The cell adhesion increased with increasing chitosan content, indicating that the affinity between the cells and the membranes increased with increasing chitosan content. Although the blended membranes with higher nylon content exerted an inhibitory effect on cell adhesion, cells cultured on the nylon membrane proliferated at higher rates and the nylon membrane was the least stimulating of MG-63 cell cytokine production over a 4-day period when compared with all the other membranes. Combined with the result of cell growth and cell activation, the chitosan content in the blended membrane did not proportionally influence the behavior of MG-63 cells. It is proposed that cell's size was larger than the scale of nylon or chitosan domain in the blended membranes because of the incomplete miscibility between them. Therefore, even if the composition of the blended membranes is systematically changed, every cell covers a multiphase surface that is considered a totally new material for cells. Consequently, cell growth and cell activation on a blended membrane are not simply proportional to their composition. In contrast, cell adhesion is a simpler process, like a physical adsorption process, which is related to the bulk property of a blended membrane.     

Fabrication of zein/hyaluronic acid fibrous membranes by electrospinning

Electrospinning of biopolymers, such as proteins and polysaccharides, has recently attracted much attention for the fabrication of scaffolds for tissue engineering. In this report, zein/hyaluronic acid (HA) blend fibrous membranes were electrospun and characterized. To facilitate the compatibility of zein and HA, poly(vinyl pyrrolidone) (PVP) was introduced into aqueous ethanol solutions of the blend. A series of zein/HA/PVP blend fibrous membranes with different volume ratios were successfully electrospun. The effect of blend composition on the morphology of electrospun fibrous membranes was investigated by scanning electron microscopy. The average diameter of blend fibers increased with increasing the content of zein component. The electrospun zein/HA/PVP fibrous membranes were then cross-linked by methylene diphenyl diisocyanate (MDI). The morphology of the cross-linked zein/HA/PVP fibrous membranes changed slightly compared with un-cross-linked membranes. Tensile tests demonstrated that the mechanical properties of the zein/HA/PVP fibrous membranes were improved by cross-linking.     

Fabrication of zein/hyaluronic acid fibrous membranes by electrospinning

Electrospinning of biopolymers, such as proteins and polysaccharides, has recently attracted much attention for the fabrication of scaffolds for tissue engineering. In this report, zein/hyaluronic acid (HA) blend fibrous membranes were electrospun and characterized. To facilitate the compatibility of zein and HA, poly(vinyl pyrrolidone) (PVP) was introduced into aqueous ethanol solutions of the blend. A series of zein/HA/PVP blend fibrous membranes with different volume ratios were successfully electrospun. The effect of blend composition on the morphology of electrospun fibrous membranes was investigated by scanning electron microscopy. The average diameter of blend fibers increased with increasing the content of zein component. The electrospun zein/HA/PVP fibrous membranes were then cross-linked by methylene diphenyl diisocyanate (MDI). The morphology of the cross-linked zein/HA/PVP fibrous membranes changed slightly compared with un-cross-linked membranes. Tensile tests demonstrated that the mechanical properties of the zein/HA/PVP fibrous membranes were improved by cross-linking.     

Effects of chitosan solution concentration and incorporation of chitin and glycerol on dense chitosan membrane properties

The aim of this work was to perform a systematic study about the effects induced by chitosan solution concentration and by chitin or glycerol incorporation on dense chitosan membranes with potential use as burn dressings. The membrane properties analyzed were total raw material cost, thickness, morphology, swelling ratio, tensile strength, percentage of strain at break, crystallinity, in vitro enzymatic degradation with lysozyme, and in vitro Vero cells adhesion. While the use of the most concentrated chitosan solution (2.5% w/w) increased membrane cost, it also improved the biomaterial mechanical resistance and ductility, as well as reduced membrane degradation when exposed for 2 months to lysozyme. The remaining evaluated properties were not affected by initial chitosan solution concentration. Chitin incorporation, on the other hand, reduced the membranes cost, swelling ratio, mechanical properties, and crystallinity, resulting in thicker biomaterials with irregular surface more easily degradable when exposed to lysozyme. Glycerol incorporation also reduced the membranes cost and crystallinity and increased membranes degradability after exposure to lysozyme. Strong Vero cells adhesion was not observed in any of the tested membrane formulations. The overall results indicate that the majority of the prepared membranes meet the performance requirements of temporary nonbiodegradable burn dressings (e.g. adequate values of mechanical resistance and ductility, low values of in vitro cellular adhesion on their surfaces, low extent of degradation when exposed to lysozyme solution, and high stability in aqueous solutions).     

Characterization of chitosan-polycaprolactone blends for tissue engineering applications

The objective of this work was to study the effect of blending chitosan with poly(epsilon-caprolactone) (PCL) on their biomechanical properties. After testing the effect of molecular weight (MW), temperature, and humidity on the tensile properties in dry, wet at 25 degrees C and wet at 37 degrees C conditions, chitosan with a MW>310 kD was selected for use in the blend. Homogeneous blends of 25%, 50% and 75% PCL compositions were formed by dissolving chitosan and 80 kD PCL in a common solvent of approximately 77% aqueous acetic acid. Taking advantage of the low melting point of PCL, blend membranes were processed at 25, 37, 55 degrees C water bath or 55 degrees C oven into films. Also, membranes were solvent annealed using chloroform vapors. Tensile properties were analyzed in wet conditions at 25 degrees C. Support for cell viability and distribution of cytoskeletal actin were analyzed by in vitro cell culture of mouse embryonic fibroblasts (MEFs). Differential scanning calorimetry studies indicated the miscibility of the two components when approximated using Nishi-Wang equation. Drying the films at 55 degrees C in an oven formed membranes without separation of two phases. However, the analyzed tensile properties showed no significant alterations relative to chitosan. On the contrary, significant improvements were observed after solvent annealing. Interestingly, increased viability and redistribution of actin fibers was observed on blends formed with 50% PCL and 75% PCL relative to individual polymers. In summary, 50:50 blends when processed at 55 degrees C in an oven showed significant improvement in mechanical properties as well as support for cellular activity relative to chitosan.     

Chitosan-poly(acrylic acid) polyelectrolyte complex membranes: preparation, characterization and permeability studies

Polyelectrolyte complex (PEC) membranes were obtained by mixing solutions of two polymers of opposite charges, chitosan (Chi) and poly(acrylic acid) PAA. Three membranes were obtained: one made of pure chitosan and two membranes with chitosan mixed with PAA at a ratio of 95:5 (one prepared using PAA solution in 3.5% formic acid, named ChiPAA3.5, and another one using a PAA solution in 10% formic acid, named ChiPAA10). The membranes were characterized by swelling experiments, FT-IR spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), mechanical properties and permeability studies in relation to a drug model (sodium sulphamerazine). The calculation of degree of ionization showed that the lower the formic acid concentration was, the higher the PAA dissociation degree. Polyelectrolyte complex formation was characterized by FT-IR. Water uptake results showed that PEC membranes were more hydrophilic than pure chitosan, ChiPAA3.5 being the most. Morphological analysis by SEM and AFM showed that PAA addition changed the membranes morphology, especially for ChiPAA3.5. Mechanical properties indicated that PEC membranes were more rigid than pure chitosan membranes and that the morphology has an influence on tensile strength values. Permeability values decreased with complex formation and were lower for ChiPAA10 than ChiPAA3.5. However, as drug concentration was increased, the difference between the two complex membranes disappeared. The results were discussed considering the drug-membrane interactions. Diffusion coefficient values indicated that ChiPAA3.5 had a higher drug retention capacity than ChiPAA10.     

Chitosan-poly(acrylic acid) polyelectrolyte complex membranes: preparation,characterization and permeability studies

Polyelectrolyte complex (PEC) membranes were obtained by mixing solutions of two polymers of opposite charges, chitosan (Chi) and poly(acrylic acid) PAA. Three membranes were obtained: one made of pure chitosan and two membranes with chitosan mixed with PAA at a ratio of 95:5 (one prepared using PAA solution in 3.5% formic acid, named ChiPAA3.5, and another one using a PAA solution in 10% formic acid, named ChiPAA10). The membranes were characterized by swelling experiments, FT-IR spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), mechanical properties and permeability studies in relation to a drug model (sodium sulphamerazine). The calculation of degree of ionization showed that the lower the formic acid concentration was, the higher the PAA dissociation degree. Polyelectrolyte complex formation was characterized by FT-IR. Water uptake results showed that PEC membranes were more hydrophilic than pure chitosan, ChiPAA3.5 being the most. Morphological analysis by SEM and AFM showed that PAA addition changed the membranes morphology, especially for ChiPAA3.5. Mechanical properties indicated that PEC membranes were more rigid than pure chitosan membranes and that the morphology has an influence on tensile strength values. Permeability values decreased with complex formation and were lower for ChiPAA10 than ChiPAA3.5. However, as drug concentration was increased, the difference between the two complex membranes disappeared. The results were discussed considering the drug–membrane interactions. Diffusion coefficient values indicated that ChiPAA3.5 had a higher drug retention capacity than ChiPAA10.     

Water soluble complexes of chitosan-g-MPEG and hyaluronic acid

Novel water soluble, biocompatible, and highly viscoelastic polyelectrolyte complexes were prepared by mixing of positively charged chitosan grafted with poly (ethylene glycol) monomethyl ether (CS-g-MPEG) and negatively charged hyaluronic acid (HA). CS-g-MPEGs having different degrees of substitution were synthesized by reacting chitosan with MPEG-aldehyde. The molecular structure, thermal and rheological properties, as well as biocompatibility of CS-g-MPEG/HA complexes were characterized. Rheological results showed that a small amount of HA could greatly enhance the viscosity of CS-g-MPEG solution. The highest viscosity was obtained when the charge ratio of CS-g-MPEG/HA was close to 1.0. Small-angle X-ray scattering measurements provided some insights into the lamellar structure of the CS-g-MPEG/HA complex. The CS-g-MPEG/HA complex system offers promising potentials in pharmaceutical, cosmetic, and biotechnology applications (e.g., cell scaffold, artificial synovial fluid, and drug/gene delivery medium).     

壳聚糖改性丝素合金膜的制备及其相关性质研究

以无毒氧化葡萄糖醛作交联剂 ,采用溶液共混交联法制备壳聚糖改性丝素合金膜。用 FTIR、DSC表征其结构 ,测定其等电点、力学性能、不同 p H条件下的溶胀率和对模型药物 5 - Fu的渗透性。结果表明 :改性丝素合金膜中丝素和壳聚糖分子间存在着强烈的氢键相互作用及良好的相容性。改性膜的等电点对应的 p H值是 5 .35 ,而丝素膜的等电点是 4 .5。改性膜的力学性能优于单组分膜 ,当壳聚糖含量为 4 0 %～ 6 0 %时 ,具有最大的抗张强度和拉伸率 ,分别为 71.4～ 72 .7MPa和 2 .96～ 3.82 %。改性丝素合金膜对 5 - Fu的渗透量与壳聚糖的含量和时间成正相关关系 ,渗透系数随 p H值增大 (5→ 9)先逐渐减小然后略有增大 ,在 p H=7时最小。     

Preparation and characterization of bionic bone structure chitosan/hydroxyapatite scaffold for bone tissue engineering

Three-dimensional oriented chitosan (CS)/hydroxyapatite (HA) scaffolds were prepared via in situ precipitation method in this research. Scanning electron microscopy (SEM) images indicated that the scaffolds with acicular nano-HA had the spoke-like, multilayer and porous structure. The SEM of osteoblasts which were polygonal or spindle-shaped on the composite scaffolds after seven-day cell culture showed that the cells grew, adhered, and spread well. The results of X-ray powder diffractometer and Fourier transform infrared spectrometer showed that the mineral particles deposited in the scaffold had phase structure similar to natural bone and confirmed that particles were exactly HA. In vitro biocompatibility evaluation indicated the composite scaffolds showed a higher degree of proliferation of MC3T3-E1 cell compared with the pure CS scaffolds and the CS/HA10 scaffold was the highest one. The CS/HA scaffold also had a higher ratio of adhesion and alkaline phosphate activity value of osteoblasts compared with the pure CS scaffold, and the ratio increased with the increase of HA content. The ALP activity value of composite scaffolds was at least six times of the pure CS scaffolds. The results suggested that the composite scaffolds possessed good biocompatibility. The compressive strength of CS/HA15 increased by 33.07% compared with the pure CS scaffold. This novel porous scaffold with three-dimensional oriented structure might have a potential application in bone tissue engineering.     

低浓度N-三甲基壳聚糖对依托泊苷经肠黏膜透过性的影响

背景：N-三甲基壳聚糖作为透皮吸收促进剂,可以影响口服药物在体内经肠黏膜的透过性。 目的：考察低浓度N-三甲基壳聚糖对依托泊苷经肠黏膜透过性的影响。 方法：使用体外扩散池法评价依托泊苷经肠黏膜经时吸收方向和分泌方向的透过量和透过系数,并测定10%N-三甲基壳聚糖对依托泊苷经肠黏膜透过性的影响。依托泊苷在接受室中的浓度用高效液相色谱法测定。 结果与结论：10%N-三甲基壳聚糖具有增加依托泊苷经吸收方向的透过性,减少经分泌方向的透过性。说明低浓度的N-三甲基壳聚糖可用于改善受P-糖蛋白介导药物的吸收,有望提高此类药物的口服生物利用度。关键词：依托泊苷;P-糖蛋白; N-三甲基壳聚糖;扩散池;透过性 doi:10.3969/j.issn.1673-8225.2012.16.012     

Three-dimensional nanohydroxyapatite/chitosan scaffolds as potential tissue engineered periodontal tissue

The development of suitable three-dimensional scaffold for the maintenance of cellular viability and differentiation is critical for applications in periodontal tissue engineering. In this work, different ratios of porous nanohydroxyapatite/chitosan (HA/chitosan) scaffolds are prepared through a freeze-drying process. These scaffolds are evaluated in vitro by the analysis of microscopic structure, porosity, and cytocompatibility. The expression of type I collagen and alkaline phosphatase (ALP) activity are detected with real-time polymerase chain reaction (RT-PCR). Human periodontal ligament cells (HPLCs) transfected with enhanced green fluorescence protein (EGFP) are seeded onto the scaffolds, and then these scaffolds are implanted subcutaneously into athymic mice. The results indicated that the porosity and pore diameter of the HA/chitosan scaffolds are lower than those of pure chitosan scaffold. The HA/chitosan scaffold containing 1% HA exhibited better cytocompatibility than the pure chitosan scaffold. The expression of type I collagen and ALP are up-regulated in 1% HA/chitosan scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferate but also recruit surrounding tissue to grow in the scaffold. The degradation of the scaffold significantly decreased in the presence of HA. This study demonstrated the potential of HA/ chitosan scaffold as a good substrate candidate in periodontal tissue engineering.     

Hybrid nanofibrous membranes of PLGA/chitosan fabricated via an electrospinning array

Hybrid nanofibrous membranes of poly(lactic-co-glycolic acid) (PLGA) and chitosan with different chitosan amounts (32.3, 62.7, and 86.5%) were fabricated via a specially designed electrospinning setup consisting of two sets of separate syringe pumps and power supplies. After soaking in chloroform overnight to dissolve PLGA, the amount of chitosan in the hybrid membranes was determined. The structure, mechanical properties, water uptake, and cytocompatibilities of the nanofibrous membranes were investigated by scanning electron microscopy, tensile testing, incubation in phosphate buffer solution, and human embryo skin fibroblasts culturing. Results showed that the chitosan amount in PLGA/chitosan membranes could be well controlled by adjusting the number of syringe for electrospinning of PLGA or chitosan, respectively. Because of the introduction of chitosan, which is a naturally hydrophilic polymer, the hybrid PLGA/chitosan membranes after chitosan crosslinking exhibited good mechanical and water absorption properties. The cytocompatibility of hybrid PLGA/chitosan membranes was better than that of the electrospun PLGA membrane. The electrospun hybrid nanofibrous membranes of PLGA and chitosan appear to be promising for skin tissue engineering. The concept of using an electrospinning array to form multicomponent nanofibrous membranes will lead to the creation of novel scaffolds for tissue engineering applications.     

Vascular cell viability on polyvinyl alcohol hydrogels modified with water-soluble and -insoluble chitosan

Polyvinyl alcohol (PVA) hydrogels blended with chitosan or other biological macromolecules have shown promise for cell culture and tissue engineering. This study investigates the attachment and growth of bovine aortic endothelial (BAEC) and smooth muscle cells (BASMC) on the PVA hydrogels modified with water soluble and water insoluble chitosan. Cell adhesion on the surface of the membranes was examined by phase contrast microscopy while cell morphologies were studied using immunocytochemistry staining with EC and SMC specific biomarkers (F-actin and alpha actin respectively). Cells cultured on 6% PVA, 0.4% chitosan (water soluble and insoluble) hydrogel membranes displayed excellent adhesion and spreading characteristics, in addition to negligible cell structural morphological changes in comparison to a polystyrene control. Similar vascular cell adhesion features were apparent on PVA membranes blended with water-soluble and -insoluble chitosan. Fluorescent activated cell sorter (FACS) analysis was used to determine BAEC and BASMC proliferation and cell viability. Apoptotic levels in BAEC after 7 days were 12.8% +/- 2.5% on the PVA- chitosan WS-1 membrane and 10.1% +/- 1.5% on the control well (n = 3) while comparable results were also noted for BASMC. Equivalent proliferative activity was apparent for BAEC on the control and PVA-chitosan membrane after 7 days, while BASMC showed increased proliferative activity on the membranes. These results indicate that the PVA-chitosan blended hydrogel membranes show promise for cell culture and tissue engineering applications.     

Protein adsorption,fibroblast activity and antibacterial properties of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) grafted with chitosan and chitooligosaccharide after immobilized with hyaluronic acid

Poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) membrane was treated with ozone and grafted with acrylic acid. The resulting membranes were further grafted with chitosan (CS) or chitooligosaccharide (COS) via esterification. Afterward hyaluronic acid (HA) was immobilized onto CS- or COS-grafting membranes. The antibacterial activity of CS and COS against Staphylococus aureus, Escherichia coli, and Pseudomonas aeruginosa was preserved after HA immobilization. Among them, CS-grafted PHBV membrane showed higher antibacterial activity than COS-grafted PHBV membrane. In addition, after CS- or COS-grafting, the L929 fibroblasts attachment and protein adsorption were improved, while the cell number was decrease. After immobilizing HA, the cell proliferation was promoted, the protein adsorption was decreased, and the cell attachment was slightly lower than CS- or COS-grafting PHBV.     

Effect of argon-plasma treatment on proliferation of human-skin-derived fibroblast on chitosan membrane in vitro

Chitosan is not only a nontoxic, biocompatible, and biodegradable polymer, but has also a chemical structure similar to glycosaminoglycans (GAGs), which promote scarless wound healing of skin. In this study, chitosan membranes were treated with argon plasma to improve their surface hydrophilicity. The results showed that the water contact angles of these surface-treated membranes were significantly reduced from 60.76 to 11.57 degrees . The total surface energy was increased from 41.06 to 67.31 mJ/m(2), with 60-86.95% improvement in the gamma-negative component and a 20% difference in the nonpolar component. Argon-plasma-treated chitosan membranes exhibited excellent attachment, migration, and proliferation of the human-skin-derived fibroblasts (hSFs) compared to the untreated ones. It was found that the duration of argon-plasma treatment influenced the cell proliferation, and the optical densities in MTT assay were enhanced. Argon-plasma treatment improved the surface hydrophilicity of chitosan membranes and promoted the attachment and proliferation of hSFs.     

透明质酸修饰的壳聚糖复合支架用于神经组织工程可行性研究

目的探索壳聚糖复合支架经透明质酸（HA）及多聚赖氨酸（PLL）修饰后,神经元在体外复合支架黏附情况,以及将不同复合支架植入鼠脑,观察支架对大鼠脑组织炎症反应、胶质纤维表达的差异。方法孔隙为（70.32±15.33）μm壳聚糖复合支架分别用质量浓度0.05mg/mLHA和质量浓度2mg/mLPLL进行表面涂镀法修饰,继而在上述两组及未修饰复合支架上进行小鼠神经细胞培养,1d后对支架细胞固定行扫描电子显微镜形态学观察,记录两组各30例支架高倍视野下细胞黏附数量;另将复合支架植入大鼠脑皮层损伤区,术后不同时间点分别取脑组织支架行苏木精-伊红染色、免疫组织化学观察炎性细胞及胶质纤维的表达情况。数据用SAS9.1.3统计软件处理,P〈0.05为差异有统计学意义。结果 HA修饰的壳聚糖复合支架上细胞黏附较多[扫描电子显微镜,×1700,（24.9±4.5）/高倍镜],而经PLL修饰的壳聚糖复合支架上细胞黏附相对较少[扫描电子显微镜,×1700,（19.2±3.2）/高倍镜],未修饰支架上细胞碎片较多,并且细胞聚集,细胞总数少;在体内用HA修饰的壳聚糖复合支架组与其他组相比,支架周围炎症反应轻、支架周围胶质纤维酸性蛋白（GFAP）阳性细胞数少。所获得数据分析后差异均有显著统计学意义（P〈0.01）。结论 HA修饰的壳聚糖复合支架能够提高体外神经元黏附,并且在体内观察炎性细胞,胶质纤维表达均优越于PLL修饰的壳聚糖复合支架、单纯壳聚糖复合支架。HA修饰的壳聚糖复合支架在神经组织工程应用中更具有研发前景。     

Hyaluronic acid as an internal wetting agent in model DMAA/TRIS contact lenses

Model silicone hydrogel contact lenses, comprised of N,N-dimethylacrylamide and methacryloxypropyltris (trimethylsiloxy) silane, were fabricated and hyaluronic acid (HA) was incorporated as an internal wetting agent using a dendrimer-based method. HA and dendrimers were loaded into the silicone hydrogels and cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide chemistry. The presence and location of HA in the hydrogels was confirmed using X-ray photoelectron spectroscopy and confocal laser scanning microscopy, respectively. The effects of the presence of HA on the silicone hydrogels on hydrophilicity, swelling behavior, transparency, and lysozyme sorption and denaturation were evaluated. The results showed that HA increased the hydrophilicity and the equilibrium water content of the hydrogels without affecting transparency. HA also significantly decreased the amount of lysozyme sorption (p?相似文献   

Rheological, microstructural, and in vitro characterization of hybrid chitosan-polylactic acid/hydroxyapatite composites

In this work, hybrid chitosan/hydroxyapatite composites material were developed and characterized. The polymer matrix was first dissolved in polylactic acid, and then hydroxyapatite (HA) was added as filler material. The effects of the added amounts of a crosslinking agent (genipin) and of the concentrations of lactic acid, and of the presence of HA powder on the evolution of rheological properties were evaluated. A significant decrease of gelation time with increasing amounts of crosslinking agent was observed, the effect being even more pronounced in the presence of HA. The chitosan matrix and the composites with a chitosan/HA weight ratio of 2/5 were characterized using microstructural analysis and in vitro tests. The formation of large pore sizes in the chitosan-based scaffolds was favored by low concentrations of lactic acid and genipin. The in vitro tests in synthetic body fluid revealed an extensive formation of an apatitic layer onto the surface of the chitosan/HA composite scaffolds crosslinked with genipin.