Vitreous cryopreservation of tenocytes co-cultured with porous polydimethylsiloxane scaffolds / 中国组织工程研究

BACKGROUND:Accumulative evidence supports that vitreous cryopreservation can improve the cel survival rate. OBJECTIVE:To investigate the effect of vitreous cryopreservation on the tenocytes co-cultured with the porous polydimethylsiloxane (PDMS) scaffold. METHODS:Tenocytes were co-cultured with the porous PDMS scaffold for 9-14 days, and then preserved and resuscitated in the 10%dimethyl sulfoxide (DMSO), 21%DMSO and VS55, respectively. One hour later, the survival rate of post-resuscitated tenocytes versus pre-resusciated tenocytes was analyzed by live/dead double color fluorescent staining and flow cytometry. RESULTS AND CONCLUSION:Live/dead double color fluorescent staining revealed that tenocytes in the 10%DMSO group appeared to be irregular and double stained, and a large number of cel s shedding from the scaffold. The VS55 and 21%DMSO groups showed some spindle and hemispherical cel s single stained for green fluorescence and few double stained irregular cel s. Additional y, the cel density in the two groups was significantly lower than that in the control group. Flow cytometry results found that there were homogenous cel s in the control group;the number of cel s in the 10%DMSO group was too low to undergo flow cytometry;smal cel particles were visible in the VS55 group;in the 21%DMSO group, the cel volume was similar with the control group, and smal particles also existed. The survival rate in the VS55 group (64.9%) was significantly lower than that in the 21%DMSO group (76.2%;P<0.05). Conversely, the survived cel s were rare in the 10%DMSO group. To conclude, 21%DMSO vitreous cryopreservation improves the cel survival rate and is beneficial for tenocyte adherence to the scaffold.

Research on influence of repair with tissue engineered tendon of vitreous cryopreservation on ultrastructure of Achilles tendon defect / 生物医学工程学杂志

By observations of the features of ultrastructure changes in the tissue engineered artificial tendon of vitreous cryopreservation, we investigated the repairing effect of tendon after an in-vivo implantation and hence we provided an important theoretical and experimental basis for the vitreous cryopreservation and application of tissue engineered artificial tendon. After vitreous cryopreservation, the implantation materials of tissue engineered artificial tendon dynamically constructed in vitro were implanted in rats for reparation of the tendon defect. A scanning electron microscope was used. At the 2nd week, the materials presented a reticular formation and there were juvenile tendon cells among materials. At the 6th week, materials were already degraded and absorbed, and then were substituted by neonatal tendon cells and collagen fibers. At the 8th week, dense tendon tissues containing uniform tendon cells and collagen fibers were found already formed; the density of collagen fibers significantly increased with time. Using a transmission electron microscope at the 2nd week, we found active proliferation of tendon cells; most of them were immature cells with a complete nuclear membrane, clear nucleolus and little collagen fibers. At the 6th week, tendon cells were more mature with a little-sized, deep-stained nucleolus surrounded by plenty of collagen fibers with complete fiber structure and clear cross striation. There was no significant difference between the two groups. Using an electron microscope, we found a very good agreement in observation of the tissue engineered artificial tendon after the in-vivo implantation in two groups. Neonatal tendon cells and collagen fiber tissues grew well and are in a similar form and order when compared versus normal tendon tissues. This proved that vitreous cryopreservation has no significant influence on the function of tendon cells. The neonatal tissue-engineered tendon exerts good function of growth and repair.

Study on cryopreservation of tissue engineered tendon by vitrification / 生物医学工程学杂志

In search of a practical method for the cryopreservation of tissue engineered tendon (TET) by vitrification, we adopted 3 kinds of different cryoprotective agents (CPA)(21% DMSO, DP6 and VS55) in studying the freeze-stored effect of different CPA. The cellular morphology and post-thaw viability of the TET were examined by scanning electron microscopy (SEM), flow cytometry, and confocal laser microscopy (CLM). The results showed that there existed statistically significant difference in respect to the post-thaw viability between 21% DMSO and DP6, VS55; The cells specially adhered to the surface of scaffold both before or after cryopreservation by use of 21% DMSO. It was suggested that 21% DMSO as a CPA for TET cryopreservation was better than DP6 and VS55 in the current study.

Rat tail tendon cell as a model seed cell for tendon tissue engineering study / 中国组织工程研究

BACKGROUND:The low output of seed cells and long cycle of traditional ceils culture methods in tendon animal models(rabbits and chicken) restrict the researches of tendon tissue engineering study.OBJECTIVE:To establish an ideal culture protocol of tail tendon in SD rats,to get more seed cells within less time for subsequent engineered tendon construction research.DESIGN,TIME AND SETTING:Controlled observation was performed in the National Key Laboratory of Biotherapy,Department of Stem Cells and Tissue Engineering,Sichuan University between February and November in 2006.MATERIALS:Rat tail tendon cells were harvested from 2 SD rats,aged 7-10 days;human prepuce fibroblasts were offered by National Key Laboratory of Biotherapy,Department of Stem Cells and Tissue Engineering,Sichuan University.METHODS:Tail tendon of SD rats was draw off and cut into pieces,which were then cultured in 10% fetal bovine serum+DFculture medium for getting primary tendoncyte by using suspension tissue culture method. The third generation cells were processed into immuocytochemistry stain with collagen type Ⅰ and Ⅲ,while human prepuce fibroblasts served as controls.Absorbance of stain result was measured by image-pro plus 5.02 for statistical analysis.MAIN OUTCOME MEASURES:Immuocytochemistry stain and absorbance measurement of SD rat tail tendon cells.RESULTS:The second generation of SD rat tail tendon cells were positive for type Ⅰ collagen stain,and negative for type Ⅲ collagen stain;human fibroblast were positive for both Ⅰ and Ⅲ collagen. In the rat tail tendon cells and human flbroblasts,the absorbance value of type Ⅰ collagen expression was dramatically higher than of type Ⅲ collagen(P＜0.05). There was no significant differences addressing the absorbance of type Ⅲ collagen expression between type Ⅰ and Ⅲ collagen of SD rat tail tendon cells and blank control group (P＞0.05).CONCLUSION:Cells cultured from SD rat tail tendon have biological characteristic of tendon cells. Tissue piece suspensionculture can obtain a quantity of primary or subcuitured cells of rat tail tendon within a short time.

Effects of micropatterned surfaces coated with type I collagen on the orientation and growth of tenocytes / 生物医学工程学杂志

It is crucial to improve the orientation and growth of cells on substrates in tissue engineering. In this study, we investigated the effects of micropatterned surfaces coated with type I collagen (CNI) on the orientation and growth of SD rat tenocytes. Using the technique of microcontact printing and microfluidic channels, we prepared micropatterned microgrooves with a 10 microm width and 4 microm depth on silicone membrane substrates. The microgrooves were coated with CNI at concentrations 0.25, 0.5, 0.75, 1.0, and 1.25 mg/ml, respectively. The rat tenocytes at 1 x 10(5)/ml were seeded onto the CNI-coated substrates and the control substrates (without CNI coating), and then cultured in a humidified 37 degrees C/5% CO2 incubator for 48 hours. Cell proliferation was measured by MTT method. After 1, 12, 24, 48 hrs of incubation, the tenocytes' alignment and morphology were observed by means of inverted phase microscope, scanning electron microscope and fluorescent microscope. The results showed there was obvious orientation of tenocytes in CNI-modified grooves, and most of the tenocytes spread along the grooves. The tenocyte orientation became more obvious with the increasing CNI concentration over a range from 0.25 to 1.25 mg/ml. This method could find important application in the construct of engineered tendons which need precise spatial organization of cells.

Experimental study on cryopreservation of seeding cells of tissue engineered tendons / 生物医学工程学杂志

This study sought to find out a good way for the cryopreservation of tendon seeding cells so as to facilitate the preparation of tissue engineering tendons as products. The related questions are how different factors affect cell survival rate at the procedure of preservation and whether cryopreservation affects seeding cells' biological characters as well as collagen secretive function. The results of experiment indicate that DMSO is a more effective cryoprotectant in cryopreservation of tissue engineered tendon seeding cells. Blood serum nourishment is very important in cell culture, preservation and treatment. The same sustenance after cryopreservation increases cell survival rate. In the process of cryopreservation, the concentration of cells is important to cell survival rate; cell survival rate will decrease when it is less than 1.0 x 10(6)/ml. In the process of cryopreservation, the cooling speed is also important to cell survival rate, slow cooling method achieves higher cell survival rate than does the rapid cooling method. Cryopreservation by use of 10%DMSO+15%FCS+75%DMEM does not affect seeding cells' collagen secretive function greatly and does not affect seeding cells' growth curve, cell cycle and chromosome mode obviously. The prescription of 10%DMSO +15%FCS+75%DMEM is suited for the cryopreservation of tendon seeding cells.

Progress in vitreous cryopreservation of living bioproducts / 生物医学工程学杂志

In this brief review, some key issues related to vitreous cryopreservation of living tissues (natural or engineered), including cells, embryos, tissues, organs, and engineered tissues, are outlined. The principle of vitreous cryopreservation for the biological activity and functionality is demonstrated. The procedures of cooling/ rewarming, composition and function of optimal cryoprotectants, and their effects on bioproducts are described. Vitrification could, therefore, prove to be a useful and effective method of bioproduct cryopreservation for a long period of time, particularly for organized tissues and organs. However, the toxicity of the cryoprotective agents and the devitrification occurring during the rewarming process need additional investigations. Several key areas of research on vitrification are also addressed.

Recent researches on the responses of fibroblasts exposed to cyclic mechanical stretching in vitro / 生物医学工程学杂志

Recently, in vitro dynamical cell-culture has been drawing more and more attention from researchers in the areas of tissue engineer, and a series of researches have demonstrated that cyclic mechanical stretching has significant effects on the cell proliferation, differentiation, and on the cell alignment on scaffold, as well as on the synthesis of extracellular matrix, cytokines, and matrix metalloproteinases (MMPs). By focusing on reviewing the culture of several kinds of fibroblasts in vitro, we learned that these cellular responses mentioned above induced by cyclic mechanical stretching were tested by many precisely designed experiments, and assumed that cyclic mechanical stretching, if applied properly, would contribute significantly to our purpose of constructing more sophisticated tissue-engineered tendon and ligament.

Bio-tribological properties of dental prosthesis made of nitriding titanium alloy material / 生物医学工程学杂志

Titanium alloy material (TC4) samples were treated with nitriding technique. The dynamic friction and wear behavior of the modified layer were examined on a reciprocating sliding rig in artificial saliva. Microhardness, depth profile and wear mechanisms were investigated by means of MVK-H12, TALYSURF6, XPS and microscopy. The results demonstrate that after being treated with nitriding technique the titanium alloy material (TC4) has better tribological behavior and up-graded wear resistance. The wear mechanism involves adhesion.

Basic researches on cell adhesion and its application in tendon tissue engineering / 生物医学工程学杂志

Cell adhesion is a basic and very important tissue in the field of tissue engineering. Fibronectin and integrins are the most important elements to cell adhesion. Some surface receptors of fibroblast can also conjugate with type I collagen in extracellular matrix (ECM) directly. Laminin receptors on the surface of fibroblast bound to laminin also play a role in cell adhesion. In this paper are reviewed a number of related articles. The structures and function of fibronectin and integrins are discussed in detail; the tendon cell's adhesion structures are also discussed. Yet, there was scarcely any paper on the effects which the preservation of tissue engineered products may have on cells' adhesion fo ECM. Therefore, researching on cell adhesion and finding a way of preservation that has no or very little adverse effect on cell adhesion is an important topic. Results from expected advanced researches on cell adhesion may probably find promising applications in the field of tissue engineering.

Histologic pattern and mechanical properties of tissue-engineered tendon implants for tendon defects / 生物医学工程学杂志

This is a study on the histologic pattern and mechanical properties of tissue-engineered tendon implanted for treatment of tendon defects. Tendons were resected from Roman chickens. Tendon cells were isolated from the tendons and cultured in vitro. The 2nd-4th passages of tendon cells were seeded on the degradable polyglycolic acid mesh to form cell-scaffold composites, which were further cultured for 7-10 days to construct tissue-engineered tendons. The tendon defects, 0.5 cm-0.8 cm in length, were made in the second digit flexor tendon bilaterally in 20 Roman chickens and then bridged with the constructed tissue-engineered tendons. At 2 weeks, 4 weeks, 6 weeks, and 8 weeks post-operation, the samples of regenerated tendons were collected for gross examination, histologic staining and biomechanical test. After implantation of the tissue-engineered tendons, the wounds healed well. The gross appearance, the cells and collagen fibers arrangement of the regenerated tendons were similar to those of natural tendons, but there were relatively not many closely packed collagen fiber bundles organized in parallel with the tendons ("remodel"), so the maximum tensile force increased slowly and its value was 15.40+/-10.63 N at 8 weeks after surgery, reaching only 23% of that of natural tendon. The maximum strain was 22.49%+/-10.21% at 8 weeks, being 10% higher than that of natural tendons. Polyglycolic acid scaffolds are degraded in vivo so rapidly that the regenerated tendons lose the normal biomechanical stimulus and then are unable to be remodeled. As a result, the mechanical strength of regenerated tendons is much lower than that of natural tendons. These results suggest that the normal biomechanical stimulus may be an important factor for the regenerated tendons to remodel.

The cryopreservation technology in research and development of tissue engineered products / 生物医学工程学杂志

In this brief review, some key issues related to cryopreservation of seeding cells, scaffolds, and engineered tissues are outlined. The importance of cryopreservation technology to the research and development of tissue engineered products is demonstrated. The biological or biochemical reaction rate must be reduced or completely shut off in order to preserve the tissue engineered products for a long period of time. Cryopreservation may be one of the possible approaches to the fulfillment of this requirement. Seeding cells are stored at low temperature. Tissue engineered scaffold products are usually lyophilized. Engineered tissues are preserved by vitreous cryopreservation technology.

Biomechanical properties of tissue-engineered tendons after repairing digital flexor tendons in chickens / 生物医学工程学杂志

Experiments have been performed to investigate why the biomechanical strength of repaired tendons is lower than that of the normal tendon when the engineered tendons are implanted in vivo to replace the tendon defects. We seeded the primary culture tendon cells derived from Roman chickens' digital flexor tendons on the degradable polyglycolic acid meshes to construct tissue-engineered tendons. The flexor tendon defects (0.5 cm-0.8 cm) excised in second digit bilaterally in 20 Roman chickens, had been repaired with the constructed tissue-engineered tendons. The samples of repaired tendons were collected at 2, 4, 6 and 8 weeks after operation. Tests for scaffold weight, hydroxyproline content, and mechanical strength of the samples were performed. We found that from 2 weeks to 8 weeks afteroperation, the weight of the scaffolds decreased significantly, almost disappearing at 8 weeks; the hydroxyproline content determining the total collagen content increased gradually without significance; mechanically, both energy at break and tensile strength showed a tendency of drastic decrease at first 4 weeks afteroperation and a gradual increase afterwards, but the tensile strength at 8 weeks afteroperation was only 23% of that of the normal tendon. We conclude that the lower biomechanical strength of repaired tendons is owing to the serious mismatch between scaffold degradation and collagen synthesis.

The related research of tissue-engineered tendon in vivo / 生物医学工程学杂志

Using tissue-engineered tendons to repair tendons and ligaments as well as functional reconstruction is the focus of nowadays researches. The scaffolds must be not only unharmful to health, but also easy for cells attachment, and be able to induce collagen deposition to form a neotendon with mechanic properties similar to those of normal tendon. In recent researches, it has been found that the mechanic properties of the implants change with the degrading and femdonizing of scaffolds. The relationships between collagen deposition, scaffolds degradation and mechanic properties of neotendon need to be defined more clearly.

Experimental studies on procedures and properties of natural derived scaffold materials of tissue engineered bone / 生物医学工程学杂志

To detect the properties of natural xenogeneic bone derived materials which were processed with different physical and chemical treatments, we made fully deproteinized bone(FDB), partially deproteinized bone (PDPB), partially decalcified bone(PDCB) from pig ribs. Their morphological features, constitute components and mechanical properties were examined by scanning electron microscopy, x-rays diffraction analysis, mechanical assay and so on. The results showed that FDB, PDPB and PDCB maintained natural network pore system. The ratios of calcium to phosphorus were 1.81, 1.74 and 1.50, and the protein contents were 0.01% +/- 0.02%, 22.41% +/- 0.83% and 35.75% +/- 2.12% respectively. The sequence of their mechanic strength was PDCB > PDPB > FDB. These data indicate that FDB, PDPB and PDCB possess natural network pore system. Their organic and inorganic component ratios and contents are different, so their mechanic properties are not alike. Additionally, more investigations will be necessary to detect the biocompatibility of the three different scaffold materials of natural derived bone.

A tissue-engineered strain scaffold for three-dimensional cell cultures / 生物医学工程学杂志

This article introduces a three-dimensional scaffold which is used to perform three-dimensional cell culture under mechanical stretch from the point of construction of tissue-engineered tissue. The composition, structure, surface characteristics, mechanical property, and cell compatibility of the scaffold have been studied by using surface chemistry and material mechanics testing methods. The results indicate that the polyvinyl alcohol (PVA) sponge, which is water-tolerant, coated with Poly-DL-lactic-co-glycolic acid (PLGA) possesses a good nature in appropriate surface feature, porosity, elastic recoil, and cell compatibility. These features provide wide options for using this scaffold to study the effects of mechanical stretch on cells maintained in three-dimensional culture to provide a three-dimensional matrix.

Characteristics of tenocyte adhesion to biologically-modified surface of polymer / 生物医学工程学杂志

In this study we examined the in vitro characteristics of tenocyte adhesion to biologically-modified surface of polymer. Polylactic-co-glycolic acid (PLGA) 85/15 films were prepared by a solvent-casting technique. Each film was adhered onto the bottom of a chamber. The film was precoated with poly-D-lysine (PDL), and then coated with serum-free F12 medium containing various concentrations of fibronectin (FN), type I collagen (CN I), and insulin-like growth factor1 (IGF-1). The monoclonal antibodies (to FN and to CN I) with various dilutions were used to inhibit attachment of tenocytes to surface precoated with FN or CN I. Human embryonic tendon cells (HETCs) and transformed human embryonic tendon cells (THETCs) were used as the seeding cells. The system used for the measurement of adhesion force was the micropipette aspiration experiment system. The micropipette was manipulated to aspirate a small portion of the tenocyte body by using a small aspiration pressure. Then the pipette was pulled away from the adhesion area by micromanipulation. The minimum force required to detach the tenocyte from the substrate was defined as the adhesion force. The results showed that modification of FN or CN I by precoating significantly enhanced attachment of tenocytes to surface of polymer (P < 0.05). As antibodies to FN or CN I were added to a polymer film precoated with FN or CN I, the adhesion force decreased significantly (P < 0.05). We concluded that the specific adhesion forces of tenocytes to extracellular matrix adhesion proteins (FN and CN I) had coordinated action and showed good dependence on their precoating concentrations, and were inhibited by the antibodies to these adhesion proteins. Films precoated with IGF-1 strongly accelerated the adhesion of tenocytes to polymer. These results indicate that the specific adhesion of tenocytes to polymer can be promoted by coating extracellular matrix adhesive proteins and insulin-like growth factor1. It is of great importance to construct tissue-engineered tendon.

Experimental studies on histocompatibility of three bio-derived bones / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study the histocompatibility of three bio-derived bones.</p><p><b>METHODS</b>After treatment with different physical and chemical method, three bio-derived bones, the composite fully deproteinized bone (CFDB), partially deproteinized bone (PDPB) and partially decalcified bone (PDCB) were implanted into rabbits. The toxicity, immune response and subperiosteum osteogenesis of CFDB, PDPB and PDCB were studied through gross observation, serum antibody measurement, evaluation of local cellular immune response and HE staining.</p><p><b>RESULTS</b>The study showed that CFDB, PDPB and PDCB had no toxicity. They could conduct peripheral tissue to grow into them and had no harmful effect on subperiosteum osteogenesis. They could also promote cartilage and osteoid tissue derived from periosteum to calcify to new bone, and combine with the peripheral bone. The degree of immune response caused by them was in the sequence of PDCB > PDPB > CFDB.</p><p><b>CONCLUSIONS</b>The three bio-derived bones, CFDB, PDPB and PDCB have good histocompatibility.</p>

Ethical Anglysis for Tissue Engineering / 中国医学伦理学

Objective To explore the medical ethical problens in the research of tissue engineering and their clinical application.Methods According to the technical route of tissue engineering ,including seeding cells.scaffold materials,implantation in body,ethical problems and their disposal were dissussed.Results Patient's rights to know the facts of test,efficacy and security of clinical application must be fully ensured during implantation of seeding cells and scaffold materials to human body.Conclusion In needs to formulate related standard of tissue engineered products and perfect politics and regulations.