Decellularization and genipin crosslinking of amniotic membrane suitable for tissue engineering applications

Abstract

Amniotic membrane has the potential to be used as scaffold in various tissue engineering applications. However, increasing its biostability at the same time maintaining its biocompatibility is important to enhance its usage as a scaffold. This studied characteristics genipin-crosslinked amniotic membrane as a bioscaffold. Redundant human amniotic membranes (HAM) divided into native (nAM), decellularized (dAM) and genipin-crosslinked (clAM) groups. The dAM and clAM group were decellularized using thermolysin (TL) and sodium hydroxide (NaOH) solution. Next, clAM group was crosslinked with 0.5% and 1.0% (w/v) genipin. The HAM was then studied for in vitro degradation, percentage of swelling, optical clarity, ultrastructure and mechanical strength. Meanwhile, fibroblasts isolated from nasal turbinates were then seeded onto nAM, dAM and clAM for biocompatibility studies. clAM had the slowest degradation rate and were still morphologically intact after 30 days of incubation in 0.01% collagenase type 1 solution. The dAM had a significantly highest percentage of swelling than other groups (p?<?0.05). Besides, the dAM retained the collagen content at similar level of nAM. Although the dAM had highest mechanical strength compared to the rest of the groups, the differences were statistically insignificant. Cell attachment on dAM and 0.5% clAM was higher compared to that on nAM and 1.0% clAM. In conclusion, clAM have better biostability and biocompatibility compared to the nAM and dAM. Together with other suitable characteristics of the clAM such as percentage of swelling, structural integrity and ECM content, clAM is suitable as scaffold for various tissue engineering applications.     

Genipin-crosslinked polyvinyl alcohol/silk fibroin/nano-hydroxyapatite hydrogel for fabrication of artificial cornea scaffolds—a novel approach to corneal tissue engineering

Abstract

Design of artificial corneal scaffolds substitute is crucial for replacement of impaired cornea. In this paper, porous polyvinyl alcohol/silk fibroin/nano-hydroxyapatite (PVA/SF/n-HA) composite hydrogel was prepared via the genipin (GP) cross-linking, the pore diameter of the hydrogel ranged from 8.138?nm and 90.269?nm, and the physical and physiological function of hydrogel were investigated. The resulting hydrogel exhibited favourable physical properties. With the GP content increasing, the structural regularity of PVA/SF/n-HA composite hydrogel was enhanced and the thermal stability was improved. The moisture content was slightly decreased and generally maintained at approximately 70%. The tensile strength was heightened up to 0.64?MPa, while the breaking elongation was decreased slightly. Moreover, the biofunction was investigated. The in vitro degradation test demonstrated that with the addition of GP, the stability of the composite hydrogels in protease XIV solution was promoted and the three-dimensional porosity structure of composite hydrogels was maintained as ever. And the human corneal fibroblasts (HCFs) were employed to examine the cells cytotoxicity of the PVA/SF/n-HA composite hydrogels with different GP content by CCK-8 assay. Based on confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM), HCFs had individually commendable adhesion and proliferation on PVA/n-HA/SF composite hydrogel. HCFs proliferated and grew into the pores of composite hydrogel. The results of biocompatibility experiments of composite hydrogel suggested that it was no acute toxicity, in vitro cytotoxicity was 0 or 1 grade. Overall, results from this paper, PVA/n-HA/SF composite hydrogel was a qualified medical material which conformed to the national standard, could be a promising alternative for artificial cornea scaffold material—a novel approach to corneal tissue engineering.     

Is quercetin an alternative natural crosslinking agent to genipin for long‐term dermal scaffolds implantation?

As biocompatible matrices, porcine dermal scaffolds have limited application in tissue engineering due to rapid degradation following implantation. This study compared the physical, chemical and biomechanical changes that occurred when genipin and quercetin were used to crosslink dermal scaffolds and to determine whether quercetin could be used as an alternative to genipin. Physicochemical changes in the collagen were assessed using spectroscopic methods [X‐ray diffraction analysis (XRD) and nuclear magnetic resonance (NMR) analysis]. The crosslinking reaction was evaluated by quantification of amino acids and the degree of this reaction by ninhydrin assay. Because the mechanical behaviour of the collagen matrices is highly influenced by crosslinking, the tensile strength of both sets of scaffolds was evaluated. The highest mechanical strength, stiffness, degree of crosslinking and changes in the packing features of collagen (measured by XRD) were achieved using genipin. Some of the results found in the quercetin‐crosslinked scaffolds were possibly due to hydration and dehydration effects elicited by the solvents (phosphate‐buffered saline or ethanol), as seen in the NMR results. In the quercetin‐ethanol‐crosslinked scaffolds, possible reorientation of the amino groups of the collagen molecule may have taken place. Therefore, depending on their proximity to the crosslinking reagent, different types and numbers of interactions may have occurred, inducing a higher crosslinking degree (as evidenced by the ninhydrin assay) and reduction in the free amino acids after reaction. Both crosslinking agents and solvents interfere in the physicochemical properties of collagen thereby inducing variations in the matrix structure. Quercetin‐crosslinked scaffolds may have broader clinical application where a lower degree of crosslinking and stiffness is required. Copyright © 2016 John Wiley & Sons, Ltd.     

Genipin-Cross-linked Electrospun Collagen Fibers

The fabrication of a fibrous collagen scaffold using electrospinning is desirable for tissue-engineering applications. Previously, electrospun collagen fibers were shown to be unstable in aqueous environments and, therefore, cross-linking is essential to stabilize these fibers. In this study genipin, a significantly less cytotoxic cross-linking agent compared to glutaraldehyde, was used to cross-link electrospun collagen fibers. The significance of this research lies in the use of four alcohol/water solvent systems to carry out the crosslinking reaction to maintain fibrous morphology during cross-linking. The four cross-linking conditions established were: (1) ethanol, 5% water and 3 days, (2) ethanol, 3% water and 5 days, (3) ethanol, 5% water and 5 days, and (4) isopropanol, 5% water and 5 days at a genipin concentration of 0.03 M. Results illustrated that genipin-cross-linking was effective in maintaining collagen fiber integrity in aqueous and cell culture media environments for up to 7 days. In addition, it was shown that fiber swelling could be controlled by using different cross-linking conditions. Swelling of cross-linked fibers immersed in Dulbecco's modified eagle medium for 7 days ranged from 0 to 59 ± 4%. The cross-linked fibers were analyzed using scanning electron microscopy, Fourier transform infrared spectroscopy and ninhydrin assay. Finally, studies using primary human fibroblasts indicated good cell adhesion to these scaffolds. Overall, our data suggest that these stabilized fibrous collagen scaffolds provide a promising environment for tissue-regeneration applications.     

Genipin‐crosslinked decellularized annulus fibrosus hydrogels induces tissue‐specific differentiation of bone mesenchymal stem cells and intervertebral disc regeneration

Biomaterial‐based therapy that can restore annulus fibrosus (AF) function in early stage and promote endogenous repair of AF tissues is a promising approach for AF tissue repair. In this study, we established a genipin‐crosslinked decellularized AF hydrogels (g‐DAF‐G) that are injectable and could manifest better in situ formability than noncrosslinked decellularized AF hydrogel, while preserving the capacity of directing differentiation of human bone mesenchymal stem cells (hBMSCs) towards AF cells. Hematoxylin and eosin staining, 4',6‐diamidino‐2‐phenylindole staining, and so forth showed that the majority of cellular components were removed, whereas extracellular matrix and microstructure were largely preserved. The storage modulus increased from 465.5 ± 9.4 Pa to 3.29 ± 0.24 MPa after 0.02% genipin crosslinking of decellularized AF hydrogels (DAF‐G) to form g‐DAF‐G. AF‐specific genes (COL1A1, COL5A1, TNMD, IBSP, FBLN1) were significantly higher in DAF‐G and g‐DAF‐G groups than that in control group after 21 days of culturing. g‐DAF‐G significantly restored nucleus pulposus water content and preserved intervertebral structure in vivo. Summarily, we produced a novel AF regeneration biomaterial, g‐DAF‐G, which exhibited well biocompatibility, great bioactivity, and much higher mechanical strength than DAF‐G. This study will provide an easy and fast therapeutic alternative to repair AF injuries or tears.     

Effect of genipin on the biliary excretion of cholephilic compounds in rats.

Aim: Genipin, a metabolite of geniposide, is reported to stimulate the insertion of multidrug resistance protein 2 (Mrp2) in the bile canalicular membrane, and to cause choleresis by increasing the biliary excretion of glutathione, which has been considered to be a substrate of Mrp2. In the present study, the effect of colchicine on the choleretic effect of genipin was investigated. The effect of genipin on the biliary excretion of the substrates of bile salt export pump and Mrp2 was also studied. Methods: After bile duct cannulation into rats, genipin was administered at the rate of 0.2 mumol/min/100 g, and the effect of colchicine pretreatment (0.2 mg/100 g) was examined. Metabolites of genipin in the bile were examined by a thin layer chromatography. Taurocholate (TC), sulfobromophthalein (BSP), and pravastatin were infused at the rate of 1.0, 0.2 and 0.3 mumol/min/100 g, respectively, and the effect of genipin co-administration was examined. Results: Genipin increased bile flow and the biliary glutathione excretion, and those increases were not inhibited by colchicine. The biliary excretion of genipin glucuronide was less than 10% of the genipin excreted into bile. The biliary excretion of TC, BSP, and pravastatin was unchanged by genipin co-administration. Conclusion: It was indicated that colchicine-sensitive vesicular transport has no role on the genipin-induced insertion of Mrp2 to the canalicular membrane. Choleresis of genipin is considered to be mainly due to the increased biliary glutathione excretion by genipin, not by the biliary excretion of glucuronide. TC had no effect on the biliary glutathione excretion.     

Genipin‐crosslinked gelatin–silk fibroin hydrogels for modulating the behaviour of pluripotent cells

Different hydrogel materials have been prepared to investigate the effects of culture substrate on the behaviour of pluripotent cells. In particular, genipin‐crosslinked gelatin–silk fibroin hydrogels of different compositions have been prepared, physically characterized and used as substrates for the culture of pluripotent cells. Pluripotent cells cultured on hydrogels remained viable and proliferated. Gelatin and silk fibroin promoted the proliferation of cells in the short and long term, respectively. Moreover, cells cultured on genipin‐crosslinked gelatin–silk fibroin blended hydrogels were induced to an epithelial ectodermal differentiation fate, instead of the neural ectodermal fate obtained by culturing on tissue culture plates. This work confirms that specific culture substrates can be used to modulate the behaviour of pluripotent cells and that our genipin‐crosslinked gelatin–silk fibroin blended hydrogels can induce pluripotent cells differentiation to an epithelial ectodermal fate. Copyright © 2014 John Wiley & Sons, Ltd.     

10批栀子中环烯醚萜苷类成分含量测定

目的 测定不同产地的10批栀子药材中栀子苷、京尼平龙胆二糖苷及总环烯醚萜苷含量。方法 采用HPLC测定栀子苷、京尼平龙胆二糖苷含量;采用紫外分光光度法测定总环烯醚萜苷含量。结果 江西产栀子中环烯醚萜苷类成分含量高。结论 不同产地栀子药材中环烯醚萜苷类成分含量有所差别。     

二次通用旋转试验设计优化栀子苷的酶解工艺

目的 研究苦杏仁酶水解栀子苷制备栀子苷元的工艺条件。方法 以产物得率为指标,通过二次通用旋转试验设计考察缓冲液pH值、反应温度、反应时间和酶加入量4个主要因素对工艺的影响,得出苦杏仁酶水解栀子苷的最合适工艺条件;采用理化性质及NMR谱鉴定产物结构,HPLC峰面积归一化法检测产物纯度。结果 酶加入量、缓冲液pH值和反应时间对产物得率有显著影响,所确定的最合适工艺条件为缓冲液pH值5.25,反应温度50.5 ℃,反应时间90 min,酶加入量1∶10;产物栀子苷元得率＞34.5%,质量分数为99.6%。结论 用苦杏仁酶水解栀子苷制备栀子苷元的方法是可行的。     

Crosslinking of an oesophagus acellular matrix tissue scaffold

The oesophagus acellular matrix (EAM) tissue-scaffold has the potential to serve as the foundation for a tissue-engineered oesophagus for repair of ablative defects. Similar to all collagen-based biomaterials, the EAM is subject to enzymatic degradation in vivo. The introduction of exogenous crosslinks to collagen molecules via glutaraldehyde (Glu) is the most accepted method of stabilizing collagen biomaterials, but fixation with Glu incurs adverse effects. Genipin (Gp), a naturally occurring crosslinking agent, has shown to be effective at improving the stability of collagen-based biomaterials with less cytotoxicity and reduced in vivo inflammatory responses than Glu. The aim of this study was to show that crosslinking with Gp improves the stability of the EAM while maintaining minimal biological reactivity and preserving EAM regeneration potential in a rat model. EAMs were crosslinked with Gp and Glu. Uncrosslinked EAMs served as controls. Denaturation temperature measurement and burst-pressure measurement after enzymatic degradation assays were used to determine the effectiveness of crosslinking on in vitro stability. Subcutaneous allograft implantation and oesophageal epithelial cell-seeding studies assessed the crosslinking effects on biological reactivity and regeneration potential, respectively. Both Gp and Glu improved EAM stability. After 30 days of implantation, the EAM elicited a minimal inflammatory response and crosslinking did not increase inflammation. Gp-crosslinked EAMs supported epithelial adhesion and proliferation while Glu-crosslinked EAMs did not. Gp improves the stability of the EAM while maintaining minimal biological reactivity and preserving EAM epithelial proliferation capacity, yielding a tissue scaffold that may form the basis of a durable and biocompatible tissue-engineered oesophagus.