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.

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.
Achilles Tendon ; injuries ; surgery ; ultrastructure ; Animals ; Cryopreservation ; Female ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tendons ; cytology ; transplantation ; Tissue Engineering ; methods ; Tissue Preservation ; methods ; Tissue Scaffolds ; Vitrification

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.
Animals ; Cell Differentiation ; Cells, Cultured ; Cryopreservation ; Cryoprotective Agents ; analysis ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Tendons ; cytology ; transplantation ; Tissue Engineering ; Tissue Preservation ; methods ; Tissue Scaffolds ; Vitrification

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.

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.
Animals ; Animals, Newborn ; Cell Culture Techniques ; methods ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coated Materials, Biocompatible ; chemistry ; pharmacology ; Collagen Type I ; pharmacology ; Guided Tissue Regeneration ; methods ; Rats ; Rats, Sprague-Dawley ; Surface Properties ; Tendons ; cytology ; Tissue Engineering ; methods

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.
Cell Count ; Cell Survival ; Cryopreservation ; methods ; Dimethyl Sulfoxide ; Humans ; Muscle, Skeletal ; cytology ; Tendons ; cytology ; Tissue Engineering ; Tissue Preservation ; methods

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.
Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Dimethyl Sulfoxide ; pharmacology ; Humans ; Organ Preservation ; methods ; Tissue Preservation ; methods

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.
Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Fibroblasts ; cytology ; Humans ; Matrix Metalloproteinases ; metabolism ; Periodicity ; Shear Strength ; Stress, Mechanical ; Tissue Engineering

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.
Cell Adhesion ; Extracellular Matrix ; metabolism ; Fibroblasts ; cytology ; Fibronectins ; metabolism ; Humans ; Integrins ; metabolism ; Laminin ; metabolism ; Tendons ; metabolism ; Tissue Engineering

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.
Adsorption ; Biocompatible Materials ; Dental Alloys ; chemistry ; Dental Materials ; Dental Prosthesis ; Electricity ; Friction ; Humans ; Materials Testing ; Surface Properties ; Titanium ; chemistry