Degradation in vitro and in vivo of ß-TCP/MCPM-based premixed calcium phosphate cement.

Premixed calcium phosphate cements (CPCs) have been developed to shorten the surgical time of conventional CPCs. However, there is lack of investigation on degradation behavior of premixed CPCs in vitro and in vivo. In this study, the premixed CPCs are prepared by mixing glycerol or polyethylene glycol (PEG) with the CPC power (ß-tricalcium phosphate (ß-TCP) and monocalcium phosphate monohydrate (MCPM)), and their degradation performances including the microstructure, chemical composition and mechanical properties are systematically evaluated both in vitro and in vivo (subcutaneous implantations in rabbits). When the premixed CPCs aged in PBS or FBS, results show weight loss of the specimens, decreased pH value and increased calcium ion concentration of aging media. Meanwhile, the setting products convert from dicalcium phosphate dihydrate (DCPD) to dicalcium phosphate anhydrous (DCPA), and no hydroxyapatite deposit. The specimen size and the molecular weight of non-aqueous solvent can modulate the setting product of premixed CPCs. For the larger specimens, DCPA is the main setting product, for the smaller ones, the composite contained DCPD and DCPA. With the decrease of the molecular weight of the non-aqueous solvent PEG, the setting product change from both DCPD and DCPA to DCPA due to the quicker exchange rate of PEG with water. After a period of subcutaneous implantation, the surface of the grafts obviously disintegrated with the formation of porous structures, but their internal morphology do not obviously change.

High mechanical strength chitosan-based hydrogels cross-linked with poly(ethylene glycol)/polycaprolactone micelles for the controlled release of drugs/growth factors.

High mechanical strength hydrogels without burst release are known to be beneficial to deliver bioactive molecules including drugs and growth factors. Herein, chitosan-based hydrogels are fabricated by the covalent attachment of poly(ethylene glycol) diacrylate (PEGDA) to thiol groups of thiolated natural polymers via Michael addition reaction under physiological conditions. Poly(ethylene glycol-b-caprolactone-b-ethylene glycol) (PECL) micelles bearing double bonds act as both fillers and chemical cross-linkers to mechanically reinforce chitosan-based hydrogels, which is confirmed by the results of rheological behavior and compressive strength measurements. Indomethacin (IMC) and/or basic fibroblast growth factor (bFGF) are/is entrapped into the PECL micelle cross-linked hydrogel network primarily through hydrophobic interaction and specific affinity to thiolated heparin, respectively. After a relatively quick initial release, rather than an initial burst commonly occurring in conventional hydrogels based on drug delivery systems, IMC and/or bFGF are/is released from these PECL micelle cross-linked hydrogels at a slower rate until a steady state is reached. The release rate of IMC and/or bFGF could be readily tuned by varying the micelle amount and the thiolated heparin content in a polymer matrix.

Alginate/PEG based microcarriers with cleavable crosslinkage for expansion and non-invasive harvest of human umbilical cord blood mesenchymal stem cells.

Porous microcarriers are increasingly used to expand and harvest stem cells. Generally, the cells are harvested via proteolytic enzyme treatment, which always leads to damages to stem cells. To address this disadvantage, a series of alginate/PEG (AL/PEG) semi-interpenetrating network microcarriers are prepared in this study. In this AL/PEG system, the chemically cross-linked alginate networks are formed via the reaction between carboxylic acid group of alginate and di-terminated amine groups of cystamine. PEG is introduced to modulate the degradation of microcarriers, which does not participate in this cross-linked reaction, while it interpenetrates in alginate network via physical interactions. In addition, chitosan are coated on the surface of AL/PEG to improve the mechanical strength via the electrostatic interactions. Biocompatible fibronectin are also coated on these microcarriers to modulate the biological behaviors of cells seeded in microcarriers. Results suggest that the size of AL/PEG microcarriers can be modulated via adjusting the contents and molecular weight of PEG. Moreover, the microcarriers are designed to be degraded with cleavage of disulfide crosslinkage. By changing the type and concentration of reductant, the ratio of AL to PEG, and the magnitude of chitosan coating, the degradation ability of AL/PEG microcarriers can be well controlled. In addition, AL/PEG microcarriers can support the attachment and proliferation of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs). More importantly, the expanded hUCB-MSCs can be detached from microcarriers after addition of reductant, which indeed reduce the cell damage caused by proteolytic enzyme treatment. Therefore, it is convinced that AL/PEG based microcarriers will be a promising candidate for large-scale expansion of hUCB-MSCs.

Chemically crosslinked alginate porous microcarriers modified with bioactive molecule for expansion of human hepatocellular carcinoma cells.

Microcarrier is an essential matrix for the large-scale culture of anchorage-dependent cells. In this study, chemical cross-linked alginate porous microcarriers (AMC) were prepared using microemulsion and freeze-drying technology. Moreover, chitosan was coated on the surface of microcarriers (AMC-CS) via electrostatic interactions to improve the mechanical strength. The size of AMC can be modulated through adjusting the concentration of alginate, amount of dispersant and stirring rate. The surface chemical characteristics and morphology of AMC-CS were evaluated by Fourier transformed infrared, X-ray photoelectron spectroscopy, and scanning electron microscope. Fibronectin (Fn) or heparin/basic fibroblast growth factor (bFGF) was then immobilized on the surface of microcarriers via layer-by-layer technology to improve the cytocompatibility. Our data suggested that the size of AMC can be accurately modulated from 90 µm to 900 µm with a narrow size distribution. Micropore structures of AMC-CS were relatively disordered and the pore size ranged between 20 µm and 100 µm. Using AMC after modified with Fn or bFGF as the cell expansion microcarriers, we showed that the proliferation rates of HepG2 cells increased significantly, reaching to more than 30-fold of cell expansion after 10 days of culture, with minor cellular damage caused by the microcarriers. Moreover, the AMC microcarriers modified with Fn or bFGF can increase albumin secretion of HepG2. We suggest that our new modified AMC-based microcarriers will be an attractive candidate for the large-scale cell culture of therapeutic cells.

Modulation of mesenchymal stem cells behaviors by chitosan/gelatin/pectin network films.

In this article, the chitosan/gelatin/pectin (CGP) network films were prepared to build appropriate physicochemical and mechanical microenvironment for attachment, proliferation, and differentiation of mesenchymal stem cells (MSCs). Results suggested that the hydrophilicity and mechanical character of CGP composites films could be modulated via adjusting the pectin content in the composites. The investigations of attachment and proliferation behaviors of mesenchymal stem cells (MSCs) on the CGP films were carried out. The morphology of cells was observed with hematoxylin/eosin staining (HE) and scanning electron microscope (SEM). The osteogenic differentiation of MSCs was investigated via ALP and polymerase chain reaction (PCR). Results suggested that the CGP films have excellent biocompatibility. MSCs seeded on CGP (0.1) film show higher proliferation capacity compared with other samples. Moreover, osteogenic differentiation of MSCs also depends on the properties of the substrate. The MSCs seeded on CGP (0.5) expressed the highest ALP activity, osteogenic gene expression and mineral formation capacity. These results suggest that the composition of the CGP network films could effectively modulate their physicochemical and mechanical properties and further regulate the cell behaviors of MSCs.

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.

[Studies on the progress of premixed calcium phosphate cements].

Calcium phosphate cement (CPC) is considered as an important bone repairing materials due to its excellent biocompatibility, osteoconductivity and remodellability, the study about its performance is still a hot topic in the field of bone tissue engineering. Premixed calcium phosphate cements (PCPC) has advantages that can save operatiion time,be convenient to the operation and preservation compared with the traditional way of immediately mixing calcium phosphate cement. PCCP has overcome the shortcomings of uneven and inadequate mixing, and can be arbitrarily remodeled according to the shape of defect, thus researches on PCCP has also become more and more interested

[Advances in interaction of macrophages with tissue engineering related biomaterials].

The host inflammatory reaction is a normal response to injury and the presence of foreign substances. Macrophage is one of the principal cell types in controlling host inflammatory and immune processes; hence, its response to biomaterials has a direct impact on biocompatibility and stability of biomaterials in vivo. This review describes the interaction of macrophages with tissue engineering related biomaterials. The bulk physicochemical structure and surface performance of biomaterials could be designed to control macrophages behaviors (i. e. adhesion, activation, fusion, apoptosis) and host responses, resulting in improving biocompatibility of biomaterials.

Real-time PCR detection of telomerase activity using specific molecular beacon probes.

Telomerase is a potentially important biomarker and a prognostic indicator of cancer. Several techniques for assessing telomerase activity, including the telomeric repeat amplification protocol (TRAP) and its modified versions, have been developed. Of these methods, real-time quantitative TRAP (RTQ-TRAP) is considered the most promising. In this work, a novel RTQ-TRAP method is developed in which a telomeric repeats-specific molecular beacon is used. The use of the molecular beacon can improve the specificity of the RTQ-TRAP assay, making the method suitable for studying the overall processivity results and the turnover rate of telomerase. In addition, the real-time, closed-tube protocol used obviates the need for post-amplification procedures, reduces the risk of carryover contamination, and supports high throughput. Its performance in synthetic telomerase products and cell extracts suggests that the developed molecular beacon assay can further enhance the clinical utility of telomerase activity as a biomarker/indicator in cancer diagnosis and prognosis. The method also provides a novel approach to the specific detection of some particular gene sequences to which sequence-specific fluorogenic probes cannot be applied directly. Figure Real-time PCR detection of telomerase activity using specific molecular beacon probes.

Construction of chitosan-gelatin-hyaluronic acid artificial skin in vitro.

To further enhance the properties of chitosan (Cs)-gelatin (Gel) scaffolds for skin tissue engineering, hyaluronic acid (HA) is introduced to the Cs-gel complex. Porous scaffolds composed of Cs, Gel, and HA are prepared using the freeze-drying method. The scaffold has an interconnected pore structure with two different pore size layers. The water uptake ability, flexibility, and biocompatibility of the scaffold are greatly increased with the incorporation of HA. To construct an artificial skin in vitro, fibroblasts and keratinocytes are co-cultured in Cs-Gel-HA scaffolds at an air-liquid interface. After 2 weeks of co-culture, the epithelial layer becomes progressively stratiform, including cubic perpendicularly oriented cells and a superficial layer of flattened cells. Immunohistochemical analyses confirmed the presence of laminin and type IV collagen, typical molecules of the basement membrane. The results of this study suggest that it is possible to construct a functional artificial skin in vitro and the Cs-Gel-HA scaffold is a promising matrix for skin tissue engineering.

Modulation of nano-hydroxyapatite size via formation on chitosan-gelatin network film in situ.

Natural bone is actually an inorganic/organic composite mainly make up of nano-hydroxyapatite (Ca(10)(PO(4))(6)(OH)(2), nHA) and collagen fibers. It is most important to form nHA/polymer composites in order to provide good biocompatibility and integration with bone tissue. In this work, nHA was formed in-situ on the surface of chitosan-gelatin (CG) network films in tris-buffer solution containing Ca(NO(3))(2)-Na(3)PO(4). The interaction between CG network film and nHA crystalline were studided using the diffuse reflectance FT-IR (ATR-FTIR), thermal analysis (TGA) and X-ray diffraction analysis (XRD), and the influence the nHA size factors, e.g. the ratio of chitosan (CS) and gelatin (Gel), concentration of calcium ions and reaction temperature, were elucidated by XRD and transmission electron microscope (TEM). Results suggested that carboxyl groups, CO and amino groups play crucial roles for HA formatting on the surface of CG network films and the average size of nHA crystalline decreasing with enhancing Gel content and increase with the increasing calcium and phosphate concentration, and when the reaction temperature below 50 degrees C the nHA crystalline size is almost fixedness (range from 17.2 to 19.2 nm) but when the temperature arrived at 70 degrees C it increase to 52.3 nm.

Influence of the concentrations of hyaluronic acid on the properties and biocompatibility of Cs-Gel-HA membranes.

The object of this study was to investigate the relationship between the concentrations of HA solutions and the physicochemical properties and the biocompatibility of Cs-Gel-HA membranes. The addition of different concentrations of HA not only improved the wettability significantly and extended the degradation time of Cs-Gel-HA membranes, but also changed their mechanical properties. The concentration of HA had a significant influence on the biocompatibility of Cs-Gel-HA membranes. Results demonstrated that it was only the concentrations of HA in a certain range (0.01-0.1%), that could promote the cell adhesion, migration and proliferation, while the concentration of HA was above 0.1% it would either reduce or even inhibit these behaviors.

Preparation and characterization of macroporous chitosan-gelatin/beta-tricalcium phosphate composite scaffolds for bone tissue engineering.

A biodegradable composite scaffold was developed using beta-tricalcium phosphate (beta-TCP) with chitosan (CS) and gelatin (Gel) in the form of a hybrid polymer network (HPN) via co-crosslinking with glutaraldehyde. Various types of scaffolds were prepared by freezing and lyophilizing. These scaffolds were characterized by Fourier transform infrared, X-ray diffractometer, and scanning electron microscopy. The macroporous composite scaffolds exhibited different pore structures. Compressive properties were improved, especially compressive modulus from 3.9-10.9 MPa. Biocompatibility was evaluated subcutaneously on rabbits. A mild inflammatory response was observed over 12 weeks. The results suggest that the scaffolds can be utilized in nonloading bone regeneration.

Gelatin manipulation of latent macropores formation in brushite cement.

Macroporous brushite cement was prepared from a mixture of beta-tricalcium phosphate (beta-TCP) and monocalcium phosphate monohydrate (MCPM) using gelatin powder as a latent templates. In a setting reaction coexisting with gelatin, closed packed, open-pore structure with 100-200 microm macropores are obtained after immersion of the set cement into PBS buffer (pH 7.4) at 37 degrees C for 1-4 weeks. The macroporous brushite cement has compressive strength of 15 MPa originally, which reducing to 5.5 MPa with macropore formation gradually in comparison to that of cancellous bone (5-10 MPa).

Preparation and histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosan-gelatin network composite scaffolds.

A novel biodegradable hydroxyapatite/chitosan-gelatin network (HA/CS-Gel) composite of similar composition to that of normal human bone was prepared as a three-dimensional biomimetic scaffold by phase separation method for bone tissue engineering. Changing the solid content and the compositional variables of the original mixtures allowed control of the porosities and densities of the scaffolds. The HA granules were dispersed uniformly in the organic network with intimate interface contact via pulverizing and ultrasonically treating commercial available HA particles. Scaffolds of 90.6% porosity were used to examine the proliferation and functions of the cells in this three-dimensional microenvironment by culturing neonatal rat caldaria osteoblasts. Histological and immunohistochemical staining and scanning electron microscopy observation indicated that the osteoblasts attached to and proliferated on the scaffolds. Extracellular matrices including collagen I and proteoglycan-like substrate were synthesized, while osteoid and bone-like tissue formed during the culture period. Furthermore, the cell/scaffold constructs had good biomineralization effect after 3 weeks in culture.

[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.