阅读说明：本技术 一种力学性能可调的可注射自愈合水凝胶及其制备方法与应用 (Injectable self-healing hydrogel with adjustable mechanical properties and preparation method and application thereof ) 是由 陈宗刚 翁洪娟 贾维彬 顾国锋 郭忠武 于 2020-05-21 设计创作，主要内容包括：本发明涉及一种力学性能可调的可注射自愈合水凝胶及其制备方法与应用。本发明的水凝胶包括原料氧化透明质酸和琥珀酰壳聚糖,还包括钙离子、聚乙二醇衍生物、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺中的一种或多种。本发明通过在氧化透明质酸和琥珀酰壳聚糖的基础上,引入钙离子、聚乙二醇衍生物、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、N-羟基琥珀酰亚胺(NHS)中的一种或几种,通过调节水凝胶的组分和组分含量,制备得到可自愈合和可注射水凝胶,使其具备力学性能可调控的特性,提高其生物相容性、止血、促伤口愈合、促血管生成等性能。(The invention relates to an injectable self-healing hydrogel with adjustable mechanical properties, and a preparation method and application thereof. The hydrogel comprises raw materials of oxidized hyaluronic acid and succinyl chitosan, and also comprises one or more of calcium ions, polyethylene glycol derivatives, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. According to the invention, on the basis of oxidizing hyaluronic acid and succinyl chitosan, one or more of calcium ions, polyethylene glycol derivatives, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are introduced, and the self-healing and injectable hydrogel is prepared by adjusting the components and the component content of the hydrogel, so that the hydrogel has the characteristics of adjustable mechanical properties, and the biocompatibility, hemostasis, wound healing promotion, angiogenesis promotion and other properties of the hydrogel are improved.)

1. An injectable self-healing hydrogel with adjustable mechanical properties is characterized by comprising the following raw materials: oxidized hyaluronic acid and succinyl chitosan, and one or more of calcium ion, polyethylene glycol derivative, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

2. The injectable self-healing hydrogel according to claim 1, wherein said calcium ions are derived from calcium chloride or calcium carbonate;

preferably, the polyethylene glycol derivative is a four-arm polyethylene glycol amino group or an amino-terminated carboxyl-terminated polyethylene glycol, and the weight average molecular weight of the polyethylene glycol derivative is 5000-10000 Da.

3. The method for preparing the injectable self-healing hydrogel with adjustable mechanical properties according to claim 1, comprising the steps of:

(1) preparing oxidized hyaluronic acid solution, and adding one or more of calcium ion solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and N-hydroxysuccinimide solution into the oxidized hyaluronic acid solution to prepare oxidized hyaluronic acid composite solution;

(2) preparing a succinyl chitosan solution, and adding a polyethylene glycol derivative solution into the succinyl chitosan solution to prepare a succinyl chitosan composite solution;

(3) and (3) mixing the oxidized hyaluronic acid solution and the succinyl chitosan composite solution in the steps (1) and (2), or mixing the oxidized hyaluronic acid composite solution and the succinyl chitosan solution, or mixing the oxidized hyaluronic acid composite solution and the succinyl chitosan composite solution, and carrying out a crosslinking reaction to obtain the injectable self-healing hydrogel with adjustable mechanical properties.

4. The method according to claim 3, wherein the oxidized hyaluronic acid in the step (1) is prepared according to the following steps: dropwise adding sodium periodate aqueous solution into hyaluronic acid aqueous solution, and stirring and reacting for 2 hours in the dark; then adding excessive glycol, and continuing stirring for 1 h; dialyzing the obtained solution in deionized water for 3 days, and freeze-drying to obtain oxidized hyaluronic acid;

preferably, the weight average molecular weight of the hyaluronic acid is 870-1430KDa, and the concentration of the hyaluronic acid aqueous solution is 10 mg/mL; the concentration of the sodium periodate aqueous solution is 0.5 mol/L; wherein the hyaluronic acid repeating disaccharide unit: the molar ratio of sodium periodate is 1: (0.6-1); the cut-off molecular weight of dialysis is 8-14 KDa.

5. The method according to claim 3, wherein the concentration of the oxidized hyaluronic acid solution in step (1) is 40 to 80mg/mL, and the solvent is a phosphate buffer having a pH of 7.4.

6. The method according to claim 3, wherein the concentration of the calcium ion solution in the step (1) is 60 to 120 mg/mL; the concentrations of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and the N-hydroxysuccinimide solution are both 60-120mg/mL, and the solvent is phosphate buffer solution with the pH value of 7.4; in the oxidized hyaluronic acid complex solution, the ratio of oxidized hyaluronic acid: the mass ratio of the calcium ion compound is 1: (0.05-0.2); oxidized hyaluronic acid: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride salt: the mass ratio of the N-hydroxysuccinimide is 1: (0.1-0.2): (0.04-0.2).

7. The method of claim 3, wherein the succinylchitosan of step (2) is prepared by the steps of: dispersing chitosan in dimethyl sulfoxide containing succinic anhydride, stirring at 60 deg.C for reaction for 1.5-4h, and centrifuging; dispersing the precipitate in water, and adjusting pH to 10-12 with sodium hydroxide solution to obtain yellow solution; adding acetone to produce a precipitate; centrifuging, collecting the precipitate, washing with acetone, and drying to obtain succinyl chitosan;

preferably, the deacetylation degree of the chitosan is 80-90%, and the weight-average molecular weight is 10-50 KDa; the concentration of the chitosan in the reaction system is 40-60 mg/mL; the molar ratio of amino groups of the chitosan to succinic anhydride is 1: (1-2).

8. The method according to claim 3, wherein the concentration of the succinylchitosan solution in step (2) is 40-80mg/mL, and the solvent is phosphate buffer at pH 7.4;

preferably, the concentration of the polyethylene glycol derivative solution in the step (2) is 60-120mg/mL, and the solvent is phosphate buffer with pH 7.4; in the succinyl chitosan complex solution, the ratio of succinyl chitosan: the mass ratio of the polyethylene glycol derivatives is 1: (0.1-0.5).

9. The method according to claim 3, wherein the mass ratio of the oxidized hyaluronic acid solution and the succinyl chitosan complex solution when mixed in the step (3) is 1: (1-2.3); the mass ratio of the oxidized hyaluronic acid composite solution to the succinyl chitosan solution is 1: (1-2.3); when the oxidized hyaluronic acid composite solution and the succinyl chitosan composite solution are mixed, the mass ratio of the oxidized hyaluronic acid composite solution to the succinyl chitosan composite solution is 1: (1-2.3);

preferably, the temperature of the crosslinking reaction in the step (3) is 25 to 37 ℃ and the time is 30 to 60 seconds.

10. The injectable self-healing hydrogel with tunable mechanical properties according to claim 1, can be used for promoting hemostasis and wound healing.

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to an injectable self-healing hydrogel with adjustable mechanical properties, and a preparation method and application thereof.

Background

Tissue bleeding and trauma are quite common in daily life and clinically, and care should be taken to promote wound repair and reduce scar formation while stopping bleeding. The hydrogel is widely applied to biomedical materials, and has a soft and high-water-content 3D structure similar to an extracellular matrix structure, so that the hydrogel is beneficial to cell growth, adhesion, secretion of growth factors and the like. The hydrogel can absorb water in blood, enrich blood platelets and blood cells, and keep a wound moist, thereby achieving the effects of hemostasis and wound healing. However, most of currently studied hydrogels are prefabricated hydrogels, which are difficult to fill and cover irregular wounds, lack the characteristics of injectability and self-healing, and are easy to break and expose the wounds when being pressed by external force, thereby weakening the hemostatic effect.

Hyaluronic acid is one of the main components of extracellular matrix and has the effect of promoting wound healing, but hyaluronic acid lacks active groups capable of forming hydrogel and dynamic chemical bonds under physiological conditions, and the application of hyaluronic acid in hydrogel is limited. Chitosan is one of the commonly used hemostatic materials, because the amino group with positive charge can generate electrostatic interaction with the negative charge on the surface of erythrocyte, which is beneficial to accelerating blood coagulation and further hemostasis. However, chitosan is difficult to dissolve under physiological conditions, which prevents its wider application in biomedical fields. Hyaluronic acid and chitosan which are not chemically modified are difficult to form hydrogel, so that the hyaluronic acid and the chitosan need to be modified, and the hyaluronic acid and the chitosan can quickly form hydrogel under physiological conditions, so that the advantages of the hyaluronic acid and the chitosan are combined, and the application potential of the hyaluronic acid and the chitosan in hemostasis and wound healing promotion is explored. Chinese patent document CN105833344A (application No. 201610266469.9) discloses the use of an injectable hydrogel in intraocular tamponade, which is characterized by its use as an intraocular tamponade in vitrectomy surgery and as an intraocular drug carrier; the injectable hydrogel is composed of two agents, wherein the first agent is glue solution containing oxidized polysaccharide, the second agent is glue solution containing chitin derivatives and/or collagen, the two agents are respectively filled into two injection pipes of a duplex injector, the two agents of glue solution are injected through the duplex injector at the same time, and are mixed and crosslinked in the injection process, namely, the double aldehyde group of the oxidized polysaccharide and the amino group of the chitin derivatives and/or the collagen are subjected to crosslinking reaction to form the hydrogel with viscoelasticity. The injectable hydrogel prepared by the invention is an in-situ forming injectable hydrogel, two hydrogel precursor solutions are required to be filled into a duplex syringe, the hydrogel can be formed after injection, the process has high requirements on instruments and operation, and the operation is complicated. The gelation time needs to be strictly controlled, if the gelation is too slow, the hydrogel is not formed in vivo after being injected into the body and can be diluted by body fluid, the toxicity of the raw materials is released, and the gelation time is prolonged; if the gelation is too fast, the needle may be clogged and the injection may not be performed smoothly. In addition, the hydrogel prepared by the patent only forms a single reversible imine bond by oxidizing aldehyde groups of polysaccharide and primary amine groups of chitin derivatives and/or collagen, so that the hydrogel not only has lower mechanical strength, but also has higher cytotoxicity due to more residual free aldehyde groups. In addition, the hydrogel prepared by the patent is only applied to preparing intraocular fillers, and no further research is carried out on the aspects of hemostasis and wound healing promotion.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an injectable self-healing hydrogel with adjustable mechanical properties, and a preparation method and application thereof.

The technical scheme of the invention is as follows:

an injectable self-healing hydrogel with adjustable mechanical properties comprises the following raw materials: oxidized hyaluronic acid and succinyl chitosan, and one or more of calcium ion, polyethylene glycol derivative, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

According to a preferred embodiment of the present invention, the injectable self-healing hydrogel comprises the following raw materials: oxidized hyaluronic acid and succinyl chitosan, and calcium ions and/or polyethylene glycol derivatives.

According to a preferred embodiment of the present invention, the injectable self-healing hydrogel comprises the following raw materials: oxidized hyaluronic acid and succinyl chitosan, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide and/or polyethylene glycol derivatives.

Preferably, according to the invention, the calcium ions are derived from calcium chloride or calcium carbonate.

According to the invention, the polyethylene glycol derivative is preferably a four-arm polyethylene glycol amino group or an amino-terminated carboxyl-terminated polyethylene glycol, and the weight-average molecular weight of the polyethylene glycol derivative is 5000-10000 Da.

The preparation method of the injectable self-healing hydrogel with adjustable mechanical properties comprises the following steps:

(1) preparing oxidized hyaluronic acid solution, and adding one or more of calcium ion solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and N-hydroxysuccinimide solution into the oxidized hyaluronic acid solution to prepare oxidized hyaluronic acid composite solution;

(2) preparing a succinyl chitosan solution, and adding a polyethylene glycol derivative solution into the succinyl chitosan solution to prepare a succinyl chitosan composite solution;

(3) and (3) mixing the oxidized hyaluronic acid solution and the succinyl chitosan composite solution in the steps (1) and (2), or mixing the oxidized hyaluronic acid composite solution and the succinyl chitosan solution, or mixing the oxidized hyaluronic acid composite solution and the succinyl chitosan composite solution, and carrying out a crosslinking reaction to obtain the injectable self-healing hydrogel with adjustable mechanical properties.

Preferably, according to the present invention, the oxidized hyaluronic acid in step (1) is prepared according to the following steps: dropwise adding sodium periodate aqueous solution into hyaluronic acid aqueous solution, and stirring and reacting for 2 hours in the dark; then adding excessive glycol, and continuing stirring for 1 h; the resulting solution was dialyzed in deionized water for 3 days and then lyophilized to obtain oxidized hyaluronic acid.

Further preferably, the weight average molecular weight of the hyaluronic acid is 870-1430KDa, and the concentration of the hyaluronic acid aqueous solution is 10 mg/mL; the concentration of the sodium periodate aqueous solution is 0.5 mol/L; wherein the hyaluronic acid repeating disaccharide unit: the molar ratio of sodium periodate is 1: (0.6-1); the cut-off molecular weight of dialysis is 8-14 KDa.

Preferably, the concentration of the oxidized hyaluronic acid solution in the step (1) is 40-80mg/mL, and the solvent is phosphate buffer with the pH value of 7.4; further preferably 60 mg/mL.

Preferably, according to the invention, the concentration of the calcium ion solution in the step (1) is 60-120 mg/mL; the concentrations of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and the N-hydroxysuccinimide solution are both 60-120mg/mL, and the solvent is phosphate buffer solution with the pH value of 7.4; in the oxidized hyaluronic acid complex solution, the ratio of oxidized hyaluronic acid: the mass ratio of the calcium ion compound is 1: (0.05-0.2); oxidized hyaluronic acid: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride salt: the mass ratio of the N-hydroxysuccinimide is 1: (0.1-0.2): (0.04-0.2).

Preferably, according to the invention, the concentration of the calcium ion solution in the step (1) is 120 mg/mL; in the oxidized hyaluronic acid complex solution, the ratio of oxidized hyaluronic acid: the mass ratio of the calcium ion compound is 1: 0.1; oxidized hyaluronic acid: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride salt: the mass ratio of the N-hydroxysuccinimide is 1: 0.2: 0.05 or 1: 0.1: 0.04.

preferably according to the present invention, the succinylchitosan of step (2) is prepared according to the following steps: dispersing chitosan in dimethyl sulfoxide containing succinic anhydride, stirring at 60 deg.C for reaction for 1.5-4h, and centrifuging; dispersing the precipitate in water, and adjusting pH to 10-12 with sodium hydroxide solution to obtain yellow solution; adding acetone to produce a precipitate; centrifuging, collecting precipitate, washing with acetone, and drying to obtain succinyl chitosan.

Further preferably, the deacetylation degree of the chitosan is 80-90%, and the weight-average molecular weight is 10-50 KDa; the concentration of the chitosan in the reaction system is 40-60 mg/mL; the molar ratio of amino groups of the chitosan to succinic anhydride is 1: (1-2).

Preferably, the concentration of the succinylchitosan solution in step (2) is 40-80mg/mL, and the solvent is phosphate buffer at pH 7.4; further preferably 60 mg/mL.

Preferably, the concentration of the polyethylene glycol derivative solution in the step (2) is 60-120mg/mL, and the solvent is phosphate buffer with pH 7.4; in the succinyl chitosan complex solution, the ratio of succinyl chitosan: the mass ratio of the polyethylene glycol derivatives is 1: (0.1-0.5).

Preferably, according to the invention, the concentration of the polyethylene glycol derivative solution in the step (2) is 120 mg/mL; in the succinyl chitosan complex solution, the ratio of succinyl chitosan: the mass ratio of the polyethylene glycol derivatives is 1: 0.2.

according to the present invention, the ratio of the oxidized hyaluronic acid solution and the succinyl chitosan complex solution when mixed in step (3) is preferably 1: (1-2.3); the mass ratio of the oxidized hyaluronic acid composite solution to the succinyl chitosan solution is 1: (1-2.3); when the oxidized hyaluronic acid composite solution and the succinyl chitosan composite solution are mixed, the mass ratio of the oxidized hyaluronic acid composite solution to the succinyl chitosan composite solution is 1: (1-2.3).

Preferably, according to the invention, the temperature of the crosslinking reaction in step (3) is from 25 to 37 ℃ for from 30 to 60 seconds.

The injectable self-healing hydrogel with adjustable mechanical properties can be applied to the aspects of hemostasis promotion and wound healing.

The invention has the technical characteristics that:

according to the invention, succinyl chitosan and oxidized hyaluronic acid are used as main raw materials, one or more of calcium ions, polyethylene glycol derivatives, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added, a series of injectable self-healing hydrogel with adjustable mechanical properties based on succinyl chitosan-oxidized hyaluronic acid is prepared by adjusting the components and the content of the components of the hydrogel, and the influence of different hydrogel components and contents on the biocompatibility, the hemostatic property, the wound healing property and the regeneration of blood vessels, hair follicles, sebaceous glands and the like of the hydrogel is further researched.

The succinyl chitosan-oxidized hyaluronic acid hydrogel is formed by the reaction of primary amine groups on succinyl chitosan and aldehyde groups on oxidized hyaluronic acid through Schiff base, the reaction is simple and rapid, but the Schiff base reaction is reversible reaction, so that more uncrosslinked free succinyl chitosan and oxidized hyaluronic acid exist while the hydrogel is generated, which can cause greater cytotoxicity and is not beneficial to the wide application of the hydrogel in the biomedical field. Therefore, minimizing the content of free oxidized hyaluronic acid with high toxicity is an effective strategy for improving the biocompatibility of the hydrogel.

The polyethylene glycol derivative with primary amine groups is introduced into the succinyl chitosan-oxidized hyaluronic acid hydrogel, so that reversible imine bonds are formed between aldehyde groups on the oxidized hyaluronic acid and the primary amine groups on the succinyl chitosan and the polyethylene glycol derivative, the aldehyde group content in a hydrogel system is reduced, and the four-arm polyethylene glycol amino group and the amino-terminated carboxyl-terminated polyethylene glycol have excellent biocompatibility, so that the biocompatibility of the hydrogel can be remarkably improved.

Calcium ions are introduced into the succinyl chitosan-oxidized hyaluronic acid hydrogel, so that aldehyde groups on the oxidized hyaluronic acid and primary amine groups on the succinyl chitosan form reversible imine bonds, and carboxyl groups on the oxidized hyaluronic acid and the calcium ions form ionic bonds, so that the content of free oxidized hyaluronic acid can be reduced, and the biocompatibility of the hydrogel is improved. And because calcium ions are blood coagulation factor IV and intracellular second messenger, the calcium ion concentration in cells is increased, so that cytoskeleton calmodulin can be activated, and the performances of stopping bleeding, promoting wound healing, regenerating blood vessels, hair follicles and sebaceous glands and the like of the hydrogel are improved.

1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are introduced into the succinyl chitosan-oxidized hyaluronic acid hydrogel, so that aldehyde groups on the oxidized hyaluronic acid and primary amine groups on the succinyl chitosan form reversible imine bonds, and carboxyl groups on the oxidized hyaluronic acid and the primary amine groups form irreversible amide bonds, so that the content of free oxidized hyaluronic acid can be reduced, the compactness of a hydrogel polymer network is improved, and the mechanical strength of the hydrogel polymer network is increased.

The invention has the beneficial effects that:

1. the succinyl chitosan is prepared by modifying chitosan, so that the advantages of good biocompatibility, biodegradability, hemostasis, antibiosis and the like of the chitosan are maintained, the characteristic of solubility in water is also endowed, and the application range of the chitosan in the field of biomedicine is expanded; the oxidized hyaluronic acid is obtained by oxidizing hyaluronic acid, so that the advantages of strong water absorption of hyaluronic acid, promotion of wound healing and the like are maintained, and the oxidized hyaluronic acid can be rapidly crosslinked with primary amine groups on succinyl chitosan at room temperature to form dynamic imine bonds.

2. According to the invention, on the basis of oxidizing hyaluronic acid and succinyl chitosan, one or more of calcium ions, polyethylene glycol derivatives, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are introduced, and the self-healing and injectable hydrogel is prepared by adjusting the components and the component content of the hydrogel, so that the hydrogel has the characteristic of adjustable mechanical properties; wherein, the introduction of calcium ions and/or polyethylene glycol derivatives can improve the biocompatibility of the hydrogel; the introduction of calcium ions can also improve the performances of hemostasis, wound healing, regeneration of blood vessels, hair follicles, sebaceous glands and the like of the hydrogel.

3. The injectable hydrogel prepared by the invention is shear thinning hydrogel, and can prevent a gel precursor solution from being injected into a body before gelation in-situ gelation and diluted by body fluid by a mode of firstly gelation and then injection, prolong the gelation time and weaken the treatment effect. The shear thinning self-healing hydrogel is not influenced by the factors, can be injected at any time, is simple and convenient to operate, can quickly exert the treatment effect of the hydrogel, and can reduce the toxicity of ungelled raw materials and cross-linking agents. After the injectable self-healing hydrogel prepared by the invention reaches an affected part through the injection of a common medical injector needle, the injectable self-healing hydrogel is quickly healed into complete hydrogel by utilizing the dynamic Schiff base reaction in the hydrogel, and a bleeding point is covered. The hydrogel integrates the advantages of hyaluronic acid, chitosan, calcium ions, polyethylene glycol derivatives and the like, and has the effects of stopping bleeding and promoting wound healing.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of oxidized hyaluronic acid prepared in example 1;

FIG. 2 is a nuclear magnetic hydrogen spectrum of succinyl chitosan prepared in example 2;

FIG. 3 is a schematic view of the morphology of a hydrogel;

FIG. 4 is a schematic view of self-healing of a hydrogel; in the figure, a is a cut hydrogel, and b is a self-healing hydrogel;

FIG. 5 is a graph of the rheological properties of the NSC-OHA hydrogel prepared in comparative example 1; in the figure, A is a sol-gel transition test chart, and B is a self-healing characteristic test chart;

FIG. 6 is a graph of the rheological profile of the NSC-OHA-EDC2-NHS hydrogel prepared in example 10; in the figure, A is a sol-gel transition test chart, and B is a self-healing characteristic test chart;

FIG. 7 is a statistical plot of cell viability of L929 cells in hydrogel leachate; the ordinate in the figure is the cell viability (%);

FIG. 8 is a test chart of hemostatic properties of hydrogels; the ordinate in the figure is the amount of bleeding (g);

FIG. 9 is an image of the skin of a mouse at day 0, 3, 5, 10 after full-thickness defect of the back skin;

FIG. 10 is a bar graph of wound healing rates of skin on the back of mice; the ordinate in the figure is the wound healing rate (%);

FIG. 11 is a photograph of the endothelial growth factor stain on day 10 after hydrogel application to the skin wound on the back of the mouse; the scale bar in the figure is 100 μm;

FIG. 12 is a three color masson stain image of mice on day 10 after application of hydrogel to dorsal skin wounds; the magnification in the figure is 200 ×.

Detailed Description

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some embodiments of the invention are shown. The invention has not been described in detail, but is in accordance with conventional techniques in the art.