[Study on the correlation between the content of bone morphogenetic protein 2 in demineralized bone matrix and its osteogenic activity in vitro and in vivo].

OBJECTIVE: To investigate the correlation between the content of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) and its osteogenic activity in vitro and in vivo, in order to choose a simple and convenient method to evaluate the osteogenic activity of DBM. METHODS: The left mid-femoral tissues of 9 donors were taken, and DBMs (S1-S9) were prepared by dynamic decalcification process, and inactivated DBM (control group) was prepared at the same time. Protease inhibitor method, collagenase method, guanidine hydrochloride/ethylene diamine tetraacetic acid (EDTA) method, and RIPA lysate method were used to extract BMP-2 in S1-S9 and inactivated DBMs. The BMP-2 content was measured and the differences between DBMs were compared. Then the S1-S9 and inactivated DBMs were co-cultured with mouse embryonic osteoblasts MC3T3-E1, respectively. The cell proliferation was detected by MTT method and fluorescence staining, and alkaline phosphatase (ALP) activity was detected at the same time. Thirty BALB/c male nude mice were divided into 10 groups, namely S1-S9 DBM groups (S1-S9 groups) and inactivated DBM group (control group), with 3 mice in each group. Muscle pockets of the middle thighs were prepared on both hindlimbs of mice in each group, and implanted corresponding DBM materials. At 4 weeks after operation, the samples were taken for HE staining observation and semi-quantitative evaluation, and the new bone formation score was calculated. RESULTS: The BMP-2 content of DBM derived from different donor bones was distinct. The BMP-2 content obtained by different extraction methods for DBM prepared from the same donor bone was also different, and the extraction efficiency of the guanidine hydrochloride/EDTA method was the highest. In vitro cell experiments, MTT test displayed that cell proliferations and ALP activity were significantly higher in S4 and S6 groups than in other groups at each time point after co-cultivation ( P<0.05). Moreover, the cell proliferation of S4 group was the most significant at 7 days ( P<0.05); fluorescence staining demonstrated that the osteoblasts of each group was in good condition, but the osteoblasts of S1, S2, S3, S4, and S6 groups were significantly more than other groups. In vivo ectopic osteogenesis experiments, the cartilage and new bone formation could be seen in the bone graft area of S1-S6 groups at 4 weeks after operation, and with the increase of BMP-2 content, the more new bone formation induced by the material, the higher the score of new bone formation of the material ( P<0.05). Among them, S4 and S6 groups contained a large number of chondrocytes and osteoblasts in the osteogenesis area. CONCLUSION: The osteogenic activity of DBM can be evaluated through BMP-2 quantitative detection combined with in vitro osteoblast proliferation and differentiation experiments.

In situ forming gelatin/hyaluronic acid hydrogel for tissue sealing and hemostasis.

Fibrin glue has been widely used as a surgical sealing and hemostatic agent. Its application is restricted due to poor tissue adhesion and low mechanical strength. To develop better tissue sealant and hemostatic agent, this study prepared the injectable hydrogels by chemically cross-linking gelatin (G) with or without hyaluronic acid (HA) in situ at a mild condition. The rheological analysis, Fourier transform infrared spectroscopy, swelling, proteolytic degradation, biocompatibility, tissue sealing, and hemostatic ability of the hydrogels were investigated. It was found that the chemical cross-linking rapidly formed in both self-crosslinking gelatin (sc-G) and gelatin/hyaluronate acid (G/HA) hydrogels. The hydrogels could be degraded by trypsin and had a desirable biocompatibility. The tissue sealing ability of the hydrogels was superior to fibrin glue. Furthermore, the G/HA hydrogel had similar hemostatic performance as fibrin glue, and was better than that of gelatin hydrogel. The results in the study indicated that the G/HA hydrogel could be used in clinic as a tissue sealant or surgical hemostat.

In situ injectable hyaluronic acid/gelatin hydrogel for hemorrhage control.

Tissue sealants are used for hemorrhage control which is imperative in many surgical procedures. It is a highly challenging task to obtain the ideal tissue sealant. Only a few commercially tissue sealants are available to be used for internal tissue or organ hemorrhage control. This study introduced two in situ injectable hydrogels for hemorrhage control: self-crosslinking gelatin (sc-G) hydrogel and hyaluronic acid/gelatin (HA/G) hydrogel. They were prepared on the tissue surface in situ and characterized by rheological analysis, stability, cytotoxicity, and bursting strength test. The hemostatic ability of the hydrogels was evaluated in a liver-bleeding rat model. The sc-G and HA/G hydrogels gelled around 90 s and 50 s, respectively. They were preferable for cell attachment and proliferation. The bursting strengths of both hydrogels exceeded that of fibrin glue. The hemostatic ability of HA/G hydrogel was better than that of sc-G hydrogel, and was same as that of fibrin glue. The HA/G hydrogel could be used as a tissue sealant for hemorrhage control in clinic.

Tendon-Derived Stem Cell Differentiation in the Degenerative Tendon Microenvironment.

Tendinopathy is prevalent in athletic and many occupational populations; nevertheless, the pathogenesis of tendinopathy remains unclear. Tendon-derived stem cells (TDSCs) were regarded as the key culprit for the development of tendinopathy. However, it is uncertain how TDSCs differentiate into adipocytes, chondrocytes, or osteocytes in the degenerative microenvironment of tendinopathy. So in this study, the regulating effects of the degenerative tendon microenvironment on differentiation of TDSCs were investigated. TDSCs were isolated from rat Achilles tendons and were grown on normal and degenerative (prepared by stress-deprived culture) decellularized tendon slices (DTSs). Immunofluorescence staining, H&E staining, real-time PCR, and Western blot were used to delineate the morphology, proliferation, and differentiation of TDSCs in the degenerative microenvironment. It was found that TDSCs were much more spread on the degenerative DTSs than those on normal DTSs. The tenocyte-related markers, COL1 and TNMD, were highly expressed on normal DTSs than the degenerative DTSs. The expression of chondrogenic and osteogenic markers, COL2, SOX9, Runx2, and ALP, was higher on the degenerative DTSs compared with TDSCs on normal DTSs. Furthermore, phosphorylated FAK and ERK1/2 were reduced on degenerative DTSs. In conclusion, this study found that the degenerative tendon microenvironment induced TDSCs to differentiate into chondrogenic and osteogenic lineages. It could be attributed to the cell morphology changes and reduced FAK and ERK1/2 activation in the degenerative microenvironment of tendinopathy.

Matrix stiffness regulates the differentiation of tendon-derived stem cells through FAK-ERK1/2 activation.

Tendon derived stem cells (TDSCs) were vital in tendon homeostasis. Nevertheless, the regulation of TDSCs differentiation in tendinopathy is unclear. Matrix stiffness modulated stem cells differentiation, and matrix stiffness of tendinopathic tissues decreased significantly. In order to clarify the role of matrix stiffness in TDSCs differentiation, they were cultured on the gelatin hydrogels with the stiffness from 2.34 ±â€¯1.48 kPa to 24.09 ±â€¯14.03 kPa. The effect of matrix stiffness on TDSCs proliferation and differentiation were investigated with CCK8 assay, immunofluorescences, real time PCR and western blot. It was found the proliferation of TDSCs increased and more stress fibers formed with increasing matrix stiffness. The differentiation of TDSCs into tenogenic, chondrogenic, and osteogenic lineages were inhibited on stiff hydrogel evidenced by reduced expression of tenocyte markers THBS4, TNMD, SCX, chondrocyte marker COL2, and osteocyte markers Runx2, Osterix, and ALP. Furthermore, the phosphorylation of FAK and ERK1/2 were enhanced when TDSCs grew on stiff hydrogel. After FAK or ERK1/2 was inhibited, the effect of matrix stiffness on differentiation of TDSCs was inhibited as well. The above results indicated matrix stiffness modulated the proliferation and differentiation of TDSCs, and the regulation effect could correlate to the activation of FAK or ERK1/2.

A method for systematically evaluating the hemostatic ability of hydrogels in vitro.

The bursting strength is a key parameter to assess hemostatic ability of tissue sealants. It is associated to mechanical property of the materials, the binding strength of the materials to the tissues as well as the applied conditions of the materials, such as temperature and wound size. Few works have systematically investigated the relationship of the hemostatic ability of hydrogels with the factors listed above. This study introduced a method to systematically investigate the effect of the thickness, covered area and components of hydrogels, and the applied conditions on the bursting strength of hydrogels. The gelatin hydrogel and fibrin glue were used in this study. The method quantitatively investigated the effect of material properties and applied conditions on the bursting strength of materials. It also suggested a minimum dosage of tissue sealant used in both animal study and clinical practice. This study proved that the method we proposed is reliable to assess the bursting strength of materials for the hemostatic application.

In situ cross-linking carbodiimide-modified chitosan hydrogel for postoperative adhesion prevention in a rat model.

BACKGROUND: Postoperative intra-abdominal adhesion often causes many complications. Chitosan fluid has been used in clinic to prevent intra-abdominal adhesion. However, fluid can be easily diluted and cannot stay on the wound site. As hydrogel is able to form stable physical barrier to separate injured tissues, we developed a chitosan hydrogel for better prevention of intra-abdominal adhesion in this study. METHODS: We synthesized a carbodiimide-derivatized chitosan gelatin (cd-CS-gelatin) hydrogel and investigated its rheological properties. A rat model was used to compare the anti-adhesion effect of chitosan hydrogel and fluid. The wounds were created with damage of the underlying muscle of the abdominal wall and the serosal layer of the cecum. They were coated with chitosan fluid or cd-CS-gelatin hydrogel. At day 14 after surgery, the animals were euthanized and intra-abdominal adhesion was assessed. RESULTS: The cd-CS-gelatin hydrogel solidified within 3min after the mixing of the reagents. The cecum-abdomen adhesion occurred in all rats without anti-adhesion treatment. The application of cd-CS-gelatin significantly reduced the adhesion rate from 100% to 50%, compared the chitosan fluid only to 88%. The decrease of adhesion breaking strength also manifested that cd-CS-gelatin was more effective than chitosan fluid to reduce postsurgical intra-abdominal adhesion formation. CONCLUSIONS: Chitosan hydrogel is more effective than chitosan fluid to prevent postoperative cecum-abdomen adhesion. It indicates that hydrogel could be a more promising state than liquid to prevent postoperative intra-abdominal adhesion.

Evaluation of surgical anti-adhesion products to reduce postsurgical intra-abdominal adhesion formation in a rat model.

BACKGROUND: Adhesions frequently occur after abdominal surgery. Many anti-adhesion products have been used in clinic. However, the evidences are short for surgeons to reasonably choose the suitable anti-adhesion produces in clinical practice. This study provided such evidence by comparing the efficiency of five products to prevent abdominal adhesion formation in a rat model. METHODS: Fifty-six Sprague-Dawley rats were randomly divided into seven groups: sham-operation group, adhesion group, and five product groups (n = 8). The abdomens of rats were opened. The injuries were created on abdominal wall and cecum in the adhesion and product groups. The wounds on abdominal wall and cecum of rats in the adhesion group were not treated before the abdomens were closed. The wounds on abdominal wall and cecum of rats in the product groups were covered with anti-adhesion product: polylactic acid (PLA) film, Seprafilm®, medical polyethylene glycol berberine liquid (PEG), medical sodium hyaluronate gel (HA), or medical chitosan (Chitosan). Fourteen days after surgery, the adhesions were evaluated by incidence, severity, adhesion area on abdominal wall and adhesion breaking strength. RESULTS: The application of PLA film and Seprafilm® significantly reduced the incidence, severity, adhesion area and breaking strength of cecum-abdomen adhesion (P<0.05). HA, PEG and Chitosan failed to significantly reduce the cecum-abdomen adhesion (P>0.05). The statistical significances in the incidence and severity of abdomen-adipose adhesion between adhesion group and the product groups were not achieved. However, Seprafilm® was more effective to reduce abdomen-adipose adhesion than PLA film. Furthermore, it was found that the products tested in this study did not effectively reduce cecum-adipose adhesion. The application of PEG could result in abdomen-small intestine adhesion. CONCLUSION: Based on the results of this study, the preference order of anti-adhesion products used to reduce postsurgical intra-abdominal adhesion formation is Seprafilm > PLA >> HA > Chitosan > PEG.

Silk fibroin nanoparticles prepared by electrospray as controlled release carriers of cisplatin.

To maintain the anti-tumor activity of cis-dichlorodiamminoplatinum (CDDP) while avoiding its cytotoxicity and negative influence on normal tissue, CDDP-loaded silk fibroin nanoparticles approximately 59 nm in diameter were successfully prepared by electrospray without using organic solvent. CDDP was incorporated into nanoparticles through metal-polymer coordination bond exchange. In vitro release tests showed that the cisplatin in the nanoparticles could be slowly and sustainably released for more than 15 days. In vitro anti-cancer experiments and intracellular Pt content testing indicated that CDDP-loaded silk fibroin nanoparticles were easily internalized by A549 lung cancer cells, transferring CDDP into cancer cells and then triggering their apoptosis. In contrast, the particles were not easily internalized by L929 mouse fibroblast cells and hence showed weaker cell growth inhibition. The CDDP-loaded silk fibroin nanoparticles showed sustained and efficient killing of tumor cells but weaker inhibition of normal cells. In general, this study provides not only a novel method for preparing CDDP-loaded silk fibroin nanoparticles but also a new carrier system for clinical therapeutic drugs against lung cancers and other tumors.