beta-TCP/MCPM-based premixed calcium phosphate cements.

Novel premixed calcium phosphate cements (CPCs) were prepared by combining cement liquids comprised of glycerol or polyethylene glycol with CPC powders that consisted of beta-tricalcium phosphate (beta-TCP) and monocalcium phosphate monohydrate (MCPM). Changing the powder to liquid mass ratio enabled the setting time to be regulated, and improved the compressive strength of the CPCs. Although some ratios of the new premixed CPCs had long setting times, these ranged from 12.4 to 27.8 min which is much shorter than the hour or more reported previously for a premixed CPC. The premixed CPCs had tolerable washout resistance before final setting, and the cements had strengths matching that of cancellous bone (5-10 MPa); their maximum compressive strength was up to 12 MPa. The inflammatory response around the premixed CPCs implanted in the subcutaneous tissue in rabbits was more prominent than that of apatite cement. These differences might be due to the much faster resorption rate of the premixed CPCs.

[An experimental study on rabbit's radial bone defect healed by application of mimetic periosteum with tissue-engineered bone].

OBJECTIVE: The purpose of this study is to investigate the osteogenic potential and possibility of combination application of mimetic osteoinductive periosteum with tissue-engineered bone. METHODS: The three-dimensional construction of tissue-engineered bone was made by implantation of adipose derived stromal cells (ADSCs) into rhBMP-2 mediated bio-derived carrier, and mimetic periosteum was constructed by loading ADSCs into Cs-Col-beta3-TCP with rhBMP-2. 10 mm defects of right radiuses were established in adult New Zealand rabbits, group A was transplanted by tissue-engineered bone with mimetic periosteum, group B was implanted by tissue-engineered bone, and group C was implanted by mimetic periosteum, group D was transplanted by bio-derived compound bone as blank scaffold. X-ray, histology, immunohistochemistry stain, dural energy X-ray absorptiometry (DEXA) and transmission electron microscopy (TEM) examinations were performed at different periods. RESULTS: Group A played a predominant role in process of new tissue regeneration and mature bone reconstitution, defect completely healed at 12 weeks. Group B showed primary repair, group C also existed in modeling stage. While, group D displayed retard regeneration with poor osteogenic capacity. DEXA result showed that group A had statistical significance over control group according to data of BMC and BMD ( P < 0.05). CONCLUSIONS: Enhanced osteogenic potential can be obtained by using tissue-engineered bone with mimetic osteoinductive periosteum. Defect can be healed with concord pattern of osteoinductive and osteopromotive and osteoconductive effects.

Synthesis and characterization of microparticles made of carboxymethyloxysuccinic-acid-modified PLA-PEG co-polymer.

A novel tri-block co-polymer, HO2C-PLA-PEG-PLA-CO2H (co-polymer II), with polybasic carboxylic acids as the end-groups was synthesized and characterized by IR and 1H-NMR. Based on the successful synthesis of co-polymer II, water-soluble sodium salicylate and oil-soluble tetrandrine, used as model drugs, were loaded in the co-polymer microparticles prepared through a modified multiple emulsion method. The encapsulation efficiency and drug-releasing behaviour were also investigated. It is found that the microparticles of about 10 microm in size are porous and can be produced from co-polymer II in better encapsulation efficiency (up to 86.65% and 55.94%) compared to those made from PLA/PEG/PLA (co-polymer I) (77.50% and 44.01%). The drug-release behaviour of co-polymer II exhibits an extended continuous release behaviour and the initial burst was reduced obviously. The results show that such functionalised PLA/PEG/PLA carrier provides higher drug encapsulation efficiency and better profiles.

[Cellular uptake and cytotoxicity of modified chitosans as gene carriers].

OBJECTIVE: To evaluate the effects of arginine modified chitosan or hexadecylated modified chitosan as gene carriers on the cellular uptake by vascular smooth muscle cells and its in vitro cytotoxicity. METHODS Plasmid DNA was labeled with alpha-32P-dATP and complexed with the modified chitosans or unmodified chitosan to form nanoparticle complexes by complex coacervation method. Uptake of all kinds of chitosan/ DNA nanoparticle complexes (CNC) by A10 cells was measured by beta-liquid scintillation counting. The in vitro cytotoxicity of the CNC was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The diameters of the CNC ranged from 55.9-174.9 nm and the zeta potentials were from 10. 8 mV for the arginine modified chitosan/DNA nanoparticle complexes (ACNC) to 1.8 mV for the hexadecylated chitosan/DNA nanoparticle complexes (HCNC). The cellular uptake of the modified chitosan/ DNA nanoparticle complexes (MCNC) by A10 cells increased significantly when compared with the unmodified chitosan/DNA nanoparticle complexes (UCNC) (P < 0.05), with the HCNC at N/P ratio of 1:1 and the ACNC at ratio of 8:1 showing the highest cellular uptake (1.3 fold higher than UCNC, P < 0.05). MCNC were much less cytotoxic when compared with Lipofectamine 2000-DNA nanoparticles. CONCLUSION: DNA nanoparticle complexes prepared with either arginine or hexadecylated modified chitosan can improve the cellular uptake of the DNA, while the in vitro cytotoxicity of both of the modified chitosan is much less than that of Lipofectamine 2000.

A study on biomineralization behavior of N-methylene phosphochitosan scaffolds.

Biomimetic growth of calcium phosphate over natural polymer may be an effective approach to constituting an organic/inorganic composite scaffold for bone tissue engineering. In this work, N-methylene phosphochitosan (NMPCS) was prepared via formaldehyde addition and condensation with phosphoric acid in a step that allowed homogeneous modification without obvious deterioration in chitosan (CS) properties. The NMPCS obtained was characterized by using FT-IR and elemental analysis. The macroporous scaffolds were fabricated through a freeze-drying technique. A comparative study on NMPCS and CS scaffold biomimetic mineralization was carried out in different media, i.e, a simulated body fluid (SBF) or alternative CaCl(2) and Na(2)HPO(4) solutions respectively. Apatite formation within NMPCS and CS scaffolds was identified with FT-IR, scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) and X-ray diffractometery (XRD). The results revealed alternate soaking of the scaffolds in CaCl(2) and Na(2)HPO(4) solutions was better than soaking in SBF solution alone in relation to apatite deposition on the scaffold pore walls. Biomineralization provides an approach to improve nature derived materials, e.g., chitosan derivative NMPCS properties e.g., compressive modulus, etc. SEM image of a NMPCS/apatite composite scaffold.

Self-aggregation behavior of alkylated chitosan and its effect on the release of a hydrophobic drug.

Chitosan (CS) was hydrophobically modified with butyl bromide and dodecyl bromide. The self-aggregation in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light-scattering method. The results indicate that introducing butyl and dodecyl moieties leads to the formation of self-aggregates. Along with the enhancement in the hydrophobicity of chitosan the self-association occurs at a lower concentration, and the mean size of self-aggregates increases. The loading capacity of butylated chitosan (4-CS) and dodecylated chitosan (12-CS) for vitamin B2 are markedly increased compared to that of chitosan, and the release of drug from alkylated chitosans is somewhat hindered due to its increased affinity for hydrophobic carriers.

N-alkylated chitosan as a potential nonviral vector for gene transfection.

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The self-aggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the self-aggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (+/-) of 1/1; ACS/DNA complexes were formed at a lower charge ratio (+/-) of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C(2)C(12) cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.

Biomimetic surface modification of poly(L-lactic acid) with chitosan and its effects on articular chondrocytes in vitro.

The objective of this study was to investigate the efficiency of two treatments for poly(L-lactic acid) (PLLA) surface modification with chitosan, via entrapment and coupling by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. The properties of original PLLA films, chitosan-entrapped and coupled PLLA films were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). The contact angle indicated the change in hydrophilicity and the ESCA data suggested that the modified PLLA films became enriched with nitrogen atoms. The cytocompatibility of modified PLLA films might be improved. Therefore, the attachment and proliferation of bovine articular chondrocyte seeded on modified PLLA films and control one were examined. A whole cell enzyme-linked immunosorbent assay (Cell ELISA) that detects the BrdU incorporation during DNA synthesis and collagen type II secretion was applied to evaluate the chondrocytes on different PLLA films and tissue culture plates. Cell viability was estimated by the MTT assay and cell function were assessed by measuring sulfated glycosaminoglycan secreted by chondrocytes. These results implied that chitosan used to modify PLLA surface through entrapment and coupling could enhance the chondrocyte adhesion, proliferation and function.

The properties of chitosan-gelatin membranes and scaffolds modified with hyaluronic acid by different methods.

The objective of the present study was to investigate the properties of chitosan-gelatin membranes or scaffolds, which were modified by incorporation of hyaluronic acid in the surface or bulk phase through co-crosslinking with N,N-(3-dimethylamino-propyl)-N'-ethyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in 2-morpholinoethane sulfonic acid (MES) buffer. The comparative study on properties of surface modification (HA(S)) and polyblend membranes (HA(C)) revealed that gelatin was enriched on the surface of HA(C), while hyaluronic acid was enriched on the surface of the HA(S). The HA(S) membranes made by surface modification method had a characteristic surface morphology. The corresponding scaffolds were prepared through freeze-drying. The incorporation of hyaluronic acid improved flexibility and fibroblasts adhesion, while slowing down the rate of biodegradation of chitosan-gelatin scaffold. Human fibroblasts adhered and proliferated well on the membranes or scaffolds in vitro.

Structure and properties of bilayer chitosan-gelatin scaffolds.

Chitosan-gelatin hybrid polymer network scaffolds were prepared via the freeze-drying technique by using the ice microparticle as a porogen. Monolayer and bilayer scaffolds were obtained by using different pre-freezing methods. The novel bilayer scaffolds were prepared via contact with -56 degrees C lyophilizing plate directly, then lyophilized. The properties of chitosan-gelatin scaffolds, such as microstructure, physical and mechanical and degradable properties, were studied. These results suggested that the porosity and pore size of the scaffolds could be modulated with thermodynamic and kinetic parameters of ice formation. The scaffolds prepared from chitosan and gelatin can be utilized as a promising matrix for tissue engineering.

Effects of baicalin-modified poly(D,L-lactic acid) surface on the behavior of osteoblasts.

In the present study, the functions of rat calvaria osteoblasts on baicalin-modified poly(D,L-lactic acid) (PDLLA) films were investigated in vitro. The surface characteristics of surfaces (both modified and control) were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). Cell morphologies on these surfaces were examined by scanning electron microscopy (SEM). Cell adhesion and proliferation were used to assess cell growth on the modified and control surfaces. The MTT assay was used to determine cell viability and alkaline phosphatase (ALP) activity was performed to evaluate differentiated cell function. Compared to control films, cell attachment of osteoblasts on baicalin-modified PDLLA film was significantly higher (P<0.05 and P<0.01) after 6 h and 8 h culture, and cell proliferation was also significantly greater (P<0.05 and P<0.01) at the end of 4th and 7th day, respectively. The MTT assay suggested that the cell viability of osteoblasts cultured on baicalin-modified PDLLA film was significantly higher (P<0.05) than that seeded on the control. Meanwhile, the ALP activity of osteoblasts cultured on modified films was also considerably enhanced (P<0.01) compared to that found on control. These results revealed that the biocompatibility PDLLA could be improved by surface modification with baicalin.

[Tissue engineering study on chitosan-gelatin/hydroxyapatite composite scaffolds--osteoblasts culture].

OBJECTIVE: To investigate the behavior of rat calvarial osteoblasts cultured on chitosan-gelatin/hydroxyapatite (CS-Gel/HA) composite scaffolds. METHODS: The rat calvarial osteoblasts (the 3rd passage) were seeded at a density of 1.01 x 10(6) cells/ml onto the CS-Gel/HA composite scaffolds having porosity 85.20%, 90.40% and 95.80%. Cell number was counted after cultured for 3 days, 1 week, 2 weeks and 3 weeks. Cell proliferation, bone-like tissue formation, and mineralization were separately detected by HE, von Kossa histological staining techniques. RESULTS: The CS-Gel/HA composite scaffolds supported the attachment of seeded rat calvarial osteoblasts. Cells proliferated faster in scaffold with higher porosity 90.40% and 95.80% than scaffold with lower porosity 85.20%. The osteoblasts/scaffold constructs were feasible for mineral deposition, and bone-like tissue formation in 3 weeks. CONCLUSION: This study suggests the feasibility of using CS-Gel/HA composite scaffolds for bone tissue engineering.

Preparation of oxidized glucose-crosslinked N-alkylated chitosan membrane and in vitro studies of pH-sensitive drug delivery behaviour.

Glucose was oxidized to generate a glucose dialdehyde and chitosan (CS) was hydrophobicallly modified with butyl bromide, octyl bromide and dexyl bromide. The analysis of IR and X-ray diffraction results of CS derivatives confirms that the hydrogen bonds and crystallinity were weakened by the incorporation of pendant alkyl. The permeability coefficient P and diffusion coefficient D for model drug vitamin B2 through oxidized glucose-crosslinked alkylated CS membrane were determined under different pH media. The results show that for the same alkylated CS in different pH media, P and D decrease with an increase in pH; for different alkxylated CSs in acidic media, P and D diminish with the increase in the length of alkyl side, which is supposedly originated from the enhancement of hydrophobicity. In alkali medium, P and D show a rising trend with the increase in the length of alkyl chain, which might be related to the loose stacking of network as it occurs to shrink in alkali medium. The preliminary cytotoxicity assay indicates that oxidized glucose-crosslinked alkylate CS membrane is non-toxic in vitro.

[Advances in research and development of tissue engineering].

OBJECTIVE: From the point of view of material science, the methods of tissue repair and defect reconstruct were discussed, including mesenchymal stem cells (MSCs), growth factors, gene therapy and tissue engineered tissue. METHODS: The advances in tissue engineering technologies were introduced based on the recent literature. RESULTS: Tissue engineering should solve the design and preparation of molecular scaffold, tissue vascularization and dynamic culture of cell on the scaffolds in vitro. CONCLUSION: Biomaterials play an important role in the tissue engineering. They can be used as the matrices of MSCs, the delivery carrier of growth factor, the culture scaffold of cell in bioreactors and delivery carrier of gene encoding growth factors.