OBJECTIVE: To study the effect of basic fibroblast growth factor (bFGF) and hyaluronic acid gel (HAG) combined with freeze-dried bone allograft in repairing segmental bone defect and to explore their mechanism. METHODS: The 15 mm segmental bone/periosteum defects were created on bilateral radius in 50 New Zealand rabbits and were treated with four different kinds of implants on 25 radius respectively (group A: bFGF and HAG combined with freeze-dried bone; group B: bFGF combined with freeze-dried bone; group C: HAG combined with freeze-dried bone; group D: simple freeze-dried bone as a control). The repair of defect was observed radiologically and histologically and were analyzed by radionuclide bone imaging and measurement of calcium contents at different periods. RESULTS: The new bone formation, bone metabolic activity and calcium contents of defects were higher in group A than in group B (P lt; 0.05), and were higher in group B than in groups C and D (P lt; 0.05). There were no significant difference between groups C and D. The bone defects healed in the 8th week in group A, in the 10th week in group B, but did not healed in the 10th week in groups C and D. CONCLUSION: As an osteogenetic factor, bFGF promotes the new bone formation; as a slow-release carrier, HAG enhances the effectiveness of bFGF. The combination of bFGF, HAG and freeze-dried bone allograft can repair the segmental bone defect more effectively.
OBJECTIVE: To review the recent advances of hyaluronic acid and its derivatives in medical application. METHODS: Recent original articles related to hyaluronan derivatives and their medical applications were retrieved extensively. RESULTS: Hyaluronic acid and its derivatives play important roles in visosurgery, arthritis therapy, prevention of adhesion, drug delivery, soft-tissue dilation, and percutaneous embolization. CONCLUSION: Development of hyaluronan derivatives may widen their medical application.
Purpose To study inhibition effects of retinal pigment epithelial (RPE) cells by hyaluronic acid-stimulating activity(HASA)． Methods The cultured human RPE cells added with a series of HASA was measured with cell counting,tetrazolium(MTT)colorimetric assay and tritium labelled thymidine deoxyribosｅ(3H-TdR)incorporation assay.Flow cytometry(FCM)analysis was used to examine RPE cells cycles. Results HASA at concentrations of 12.5～200 mu;g/ml and within 48 hours inhibited RPE cells proliferation with a dose-dependant and time dependant manners.The maximal inhibition rate of RPE cells by HASF was about 48.0%.FCM revealed that the cells in G1 phase increased 7.2% and cells in S phase decreased 9.7%,compared to controls. Conclusion HASA at a certain dose range and period can inhibit RPE cells proliferation. (Chin J Ocul Fundus Dis,1999,15:72-74)
Objective To systematically evaluate the effectiveness of intra-articular hyaluronic acid injection after arthroscopic debridement for knee osteoarthritis (KOA). Methods Databases including The Cochrane Library, SCI, MEDLINE, EMbase, CBM and WanFang Data were searched from inception to 2012, so as to collect randomized controlled trials (RCTs) on intra-articular hyaluronic acid injection after arthroscopic debridement (combined therapy) vs. monotherapy in treating KOA. Two reviewers independently screened literature according to inclusion and exclusion criteria, evaluated quality, and extracted data. Then the meta-analysis was conducted using RevMan5.0 software. Results A total of 7 RCTs involving 526 patients were included. The results of meta-analyses showed that: there was no significant difference in the excellent-good rate between the combined therapy group and the monotherapy groups including either the intra-articular hyaluronic acid injection group (RR=1.40, 95%CI 0.99 to 1.98, P=0.06) or the arthroscopic debridement group (RR=1.09, 95%CI 0.93 to 1.26, P=0.29). But the intra-articular hyaluronic acid injection group was inferior to the combined therapy group in improving Lysholm score, with a significant difference (MD=–14.81, 95%CI –17.55 to –12.08, Plt;0.000 01). Conclusion Arthroscopic debridement combined with intra-articular hyaluronic acid injection for KOA shows no significant difference in the excellent-good rate compared with the monotherapy, but it is superior to the monotherapy of hyaluronic acid injection in improving Lysholm score, so it is believed the combined therapy group is superior to the control groups in therapeutic effects. Due to the limited quantity and quality of the included studies, this conclusion needs to be proved by performing more high quality RCTs
Objective To review the recent advances of cross-linking reagent for producing hyaluronic acid(HA) derivative so as to provide more advice for thedevelopment of HA reagent. Methods Recent original articles related to the species, characteristic, cross-linking methodology and mechanism of the cross-linking reagent to producing HA derivative were summarized and systematically analyzed. Results The derivatives after special kinds of reagents modification would remain their own good biocompatibility and change their original rheololgical characterization and obtain relative long organism residence time. Conclusion Development of hyaluronic derivatives may widen their medical application.
ObjectiveTo discuss whether human amniotic mesenchymal stem cells (hAMSCs) possesses the characteristic of mesenchymal stem cells, and could differentiate into ligament cells in vitro after induction. MethodsThe hAMSCs were separated through enzyme digestion, and the phenotypic characteristics of hAMSCs were tested through flow cytometry. The cells at passage 3 were cultured with L-DMEM/F12 medium containing transforming growth factor β1 (TGF-β1)+basic fibroblast growth factor (bFGF) (group A), containing hyaluronic acid (HA) (group B), containing TGF-β1+bFGF+HA (group C), and simple L-DMEM/F12 medium (group D) as control group. The morphology changes of cells in each group were observed by inverted phase contrast microscope at 21 days after induction; the cellular activities and proliferation were examined by sulforhodamine (SRB) colorimetric method; and specific mRNA and protein expressions of ligament including collagen type I, collagen type III, and tenascin C (TNC) were measured by real-time fluorescence quantitative PCR and immunohistochemical staining. ResultsThe flow cytometry result indicated that hAMSCs expressed mesenchymal stem cell phenotype. After 21 days of induction, the cells in groups A, B, and C grew like spindle-shaped fibroblasts under inverted phase contrast microscope, and cells showed single shape, obvious directivity, and compact arrangement in group C. The SRB result indicated that the cells in each group reached the peak of growth curve at 6 days; the cellular activities of groups A, B, and C were significantly higher than that of group D at 6 days after induction. Also, the immunohistochemical staining results showed that no expressions of TNC were detected in 4 groups at 7 days; expressions of collagen type I in groups A, B, and C were significantly higher than that in group D at 7, 14, and 21 days (P<0.001); the expressions of collagen type III in groups A, B, and C were significantly higher than that in group D at 14 and 21 days (P<0.001). There was an increasing tendency with time in collagen type I of group B, in collagen type III and TNC of groups A and C, showing significant difference among different time points (P<0.001). The real-time fluorescence quantitative PCR results revealed that the mRNA expressions of collagen type I and TNC in group C were significantly higher than those in groups A and B (P<0.05), and the mRNA expression of collagen type III in group B were significantly higher than that in groups A and C at 21 days (P<0.05). The mRNA expressions of collagen type I and TNC in groups A and C and mRNA expression of collagen type III in group C had an increasing tendency with time, showing significant difference among different time points (P<0.001). ConclusionThe hAMSCs possesses the characteristics of mesenchymal stem cells and excellent proliferation capacity. After in vitro induction, the expressions of ligament specific genes can be up-regulated and the synthesis of ligament specific proteins can be also strengthened. As a result, it can be used as one of ligament tissue engineering seed cell sources.
OBJECTIVE: To observe the effects of hyaluronic acid (HA) and basic fibroblast growth factor (bFGF) on the proliferation of the cells from medial collateral ligament (MCL) and anterior cruciate ligament (ACL) cells. METHODS: The MCL cells and ACL cells of mature New Zealand white rabbit were cultured, while HA, bFGF or HA and bFGF were added to the cell culture media, the cellular proliferation was assayed by MTT method. RESULTS: HA only had no effect on the preoliferation of ACL cells, but had a small stimulatory effect on the proliferation of MCL cells. The addition of 1 ng/ml bFGF enhanced the proliferation of both MCL and ACL cells significantly, and this enhancement was maximal in the concentration of 50 ng/ml. However, the enhancement of proliferation of MCL and ACL cells could be achieved when the combination of HA in concentration of 100 micrograms/ml and bFGF in concentration of 100 ng/ml. CONCLUSION: It is evident that bFGF can enhance the proliferation of the ligament cells. HA can maintain the normal growth of ACL cells with no effect on the proliferation of the cells, while HA has a small stimulatory effect on the proliferation of MCL cells. However, when bFGF is coordinated with HA, more improvement of cellular proliferation can be achieved. HA can be used as a potential carrier for bFGF to enhance the healing of ligamentous tissue injuries.
Objective To observe and explore the effects of adipose-derived stem cells (ADSCs)-hyaluronic acid (HA) composite on heal ing of wound combined with radiation injury. Methods The ADSCs were harvested from the fat tissue in groin of 10 inbred Sprague Dawley (SD) rats and were isolated and cultured by enzyme digestion. The ADSCs-HA composite wasprepared with ADSCs (5 × 106 cells/mL) at passage 6 and HA (10 mg/mL). Thirty inbred SD rats, 15 males and 15 females, were randomly divided into groups A (n=10), B (n=10), and C (n=10). A 2 cm × 2 cm full-thickness skin defect was made on the rat back before 20 Gy 60Co radiation exposure. One week after debridement, wounds were treated by petrolatum gauze in group A as the control group, by HA (0.4 mL) and petrolatum gauze in group B, and by ADSCs-HA composite (0.4 mL) and petrolatum gauze in group C. The microvessel density (MVD) and the distribution of CD90 positive cells were observed at 1st, 2nd, 3rd, and 4th weeks. Results The wound heal ing was slower, and wound did not heal at 4th week and still filled with granulation tissue in group A; the wound heal ing of group B was faster than that of group A, and the wound did not heal completely with depression in the center at 4th week; the wound healed completely with epidermil izated surface and no obvious depression at 4th week in group C. The histological observation showed that MVD was significantly higher in group C than in groups A and B at the 1st, 2nd, and 3rd weeks (P lt; 0.05), and in group B than in group A at the 3rd week (P lt; 0.05); MVD was significantly higher in groups B and C than in group A (P lt; 0.05), but no significant difference was found between groups B and C (P gt; 0.05) at 4th week. No CD90 positive cell was found in groups A and B; CD 90 positive cells were observed in group C and gradually decreased with time. Conclusion ADSCs-HA composite can accelerate heal ing of wound combined with radiation injury by promoting and controll ing wound angiogenesis.
Objective To fabricate in situ crosslinking hyaluronic acid hydrogel and evaluate its biocompatibility in vitro. Methods The acrylic acid chloride and polyethylene glycol were added to prepare crosslinking agent polyethylene glycol acrylate (PEGDA), and the molecular structure of PEGDA was analyzed by Flourier transformation infrared spectroscopy and 1H nuclear magnetic resonance spectroscopy. Hyaluronic acid hydrogel was chemically modified to prepare hyaluronic acid thiolation (HA-SH). And the degree of HA-SH was analyzed qualitatively and quantitatively by Ellman method. HA-SH solution in concentrations (W/V) of 0.5%, 1.0%, and 1.5% and PEGDA solution in concentrations (W/V) of 2%, 4%, and 6% were prepared with PBS. The two solutions were mixed in different ratios, and in situ crosslinking hyaluronic acid hydrogel was obtained; the crosslinking time was recorded. The cellular toxicity of in situ crosslinking hyaluronic acid hydrogel (1.5% HA-SH and 4% PEGDA mixed) was tested by L929 cells. Meanwhile, the biocompatibility of hydrogel was tested by co-cultured with human bone mesenchymal stem cells (hBMSCs). Results Flourier transformation infrared spectroscopy showed that most hydroxyl groups were replaced by acrylate groups; 1H nuclear magnetic resonance spectroscopy showed 3 characteristic peaks of hydrogen representing acrylate and olefinic bond at 5-7 ppm. The thiolation yield of HA-SH was 65.4%. In situ crosslinking time of hyaluronic acid hydrogel was 2 to 70 minutes in the PEGDA concentrations of 2%-6% and HA-SH concentrations of 0.5%-1.5%. The hyaluronic acid hydrogel appeared to be transparent. The toxicity grade of leaching solution of hydrogel was grade 1. hBMSCs grew well and distributed evenly in hydrogel with a very high viability. Conclusion In situ crosslinking hyaluronic acid hydrogel has low cytotoxicity, good biocompatibility, and controllable crosslinking time, so it could be used as a potential tissue engineered scaffold or repairing material for tissue regeneration.
Objective To explore the possibility of constructing tissue engineered cartilage complex three-dimensional nano-scaffold with collagen type II and hyaluronic acid (HA) by electrospinning. Methods The three-dimensional porous nano-scaffolds were prepared by electrospinning techniques with collagen type II and HA (8 ∶ 1, W ∶ W), which was dissolved in mixed solvent of 3-trifluoroethanol and water (1 ∶ 1, V ∶ V). The morphology were observed by light microscope and scanning electron microscope (SEM). And the porosity, water absorption rate, contact angle, and degradation rate were detected. Chondrocytes were harvested from 1-week-old Japanese white rabbit, which was disgested by 0.25% trypsin 30 minutes and 1% collagenase overlight. The passage 2 chondrocytes were seeded on the nano-scaffold. The cell adhesion and proliferation were evaluated by cell counting kit 8 (CCK-8). The cell-scaffold composites were cultured for 2 weeks in vitro, and the biological morphology and extracelluar matrix (ECM) secretion were observed by histological analysis. Results The optimal electrospinning condition of nano-scaffold was 10% electrospinning solution concentration, 10 cm receiver distance, 5 mL/ h spinning injection speed. The scaffold had uniform diameter and good porosity through the light microscope and SEM. The diameter was 300-600 nm, and the porosity was 89.5% ± 25.0%. The contact angle was (35.6 ± 3.4)°, and the water absorption was 1 120% ± 34% at 24 hours, which indicated excellent hydrophilicity. The degradation rate was 42.24% ± 1.51% at 48 days. CCK-8 results showed that the adhesive rate of cells with scaffold was 169.14% ± 11.26% at 12 hours, and the cell survival rate was 126.03% ± 4.54% at 7 days. The histological and immunohistochemical staining results showed that the chondrocytes could grow well on the scaffold and secreted ECM. And the similar cartilage lacuma structure could be found at 2 weeks after co-culture, which suggested that hyaline cartilage formed. Conclusion The collage type II and HA complex three-dimensional nano-scaffold has good physicochemical properties and excellent biocompatibility, so it can be used as a tissue engineered cartilage scaffold.