阅读说明：本技术 用于骨骼肌内移植的pda-plga细胞支架及其制备方法 (PDA-PLGA cell scaffold for skeletal intramuscular transplantation and preparation method thereof ) 是由 张明 陈立 关越琪 张文友 杨雪晗 于 2021-01-07 设计创作，主要内容包括：本发明属于骨骼肌部位的移植应用技术领域,具体为用于骨骼肌内移植的PDA-PLGA细胞支架及其制备方法,步骤一：配制合适浓度的PLGA纺丝溶液；步骤二：通过静电纺丝法制备连续的无规则纳米纤维,并用金属接收板收集静电纺丝纤维支架,干燥后得到PLGA细胞支架；步骤三：在有氧条件下,将收集好的PLGA细胞支架浸没在含有多巴胺的弱碱性Tris-HCl水溶液中振摇,使其自聚合形成聚多巴胺涂层,干燥后得到PDA-PLGA细胞支架,其结构合理,通过将聚多巴胺能够牢固地粘附在纳米纤维细胞支架上,有效的增强纳米纤维的机械性能和亲水性,对细胞粘附,增殖和生长具有极好的适用性,对组织工程治疗的推进及我国卫生事业的发展有重要的现实意义。(The invention belongs to the technical field of transplantation application of skeletal muscle parts, and particularly relates to a PDA-PLGA cell scaffold for skeletal muscle transplantation and a preparation method thereof, wherein the PDA-PLGA cell scaffold comprises the following steps: preparing PLGA spinning solution with proper concentration; step two: preparing continuous irregular nanofibers by an electrostatic spinning method, collecting an electrostatic spinning fiber scaffold by a metal receiving plate, and drying to obtain a PLGA cytoskeleton; step three: under the aerobic condition, the collected PLGA cytoskeleton is immersed in weak alkaline Tris-HCl aqueous solution containing dopamine to shake, so that the PLGA cytoskeleton is self-polymerized to form a polydopamine coating, and the PDA-PLGA cytoskeleton is obtained after drying.)

1. The preparation method of the PDA-PLGA cytoskeleton for skeletal intramuscular transplantation is characterized by comprising the following steps: the method comprises the following steps:

the method comprises the following steps: preparing PLGA spinning solution with proper concentration;

step two: preparing continuous irregular nanofibers by an electrostatic spinning method, collecting an electrostatic spinning fiber scaffold by a metal receiving plate, and drying to obtain a PLGA cytoskeleton;

step three: and under the aerobic condition, immersing the collected PLGA cytoskeleton in a weak alkaline Tris-HCl aqueous solution containing dopamine, shaking to enable the PLGA cytoskeleton to be self-polymerized to form a polydopamine coating, and drying to obtain the PDA-PLGA cytoskeleton.

2. The method for preparing PDA-PLGA cytoskeleton for intraskeletal muscle transplantation according to claim 1, wherein: the PLGA spinning solution has a mass fraction of 5-25%.

3. The method for preparing PDA-PLGA cytoskeleton for intraskeletal muscle transplantation according to claim 1, wherein: the volume ratio of chloroform to acetone as a solvent in the PLGA spinning solution is 2: 1-5: 1.

4. The method for preparing PDA-PLGA cytoskeleton for intraskeletal muscle transplantation according to claim 1, wherein: when the continuous irregular nanofibers are prepared by the electrostatic spinning method, electrostatic spinning parameters are controlled to be voltage of 5-30 kV, the inner diameter of an electrostatic spinning nozzle is 0.5-2.2 mm, the solution flow rate is 1-10 ml/h, the temperature is 20-30 ℃, and the distance between the electrostatic spinning nozzle and a receiving device is controlled to be 15-30 cm.

5. The method for preparing PDA-PLGA cytoskeleton for intraskeletal muscle transplantation according to claim 1, wherein: the dopamine weak alkaline solution has the dopamine concentration of 1 mg/ml-5 mg/ml.

6. The method for preparing PDA-PLGA cytoskeleton for intraskeletal muscle transplantation according to claim 1, wherein: the pH value of the Tris-HCl aqueous solution is 8.0-8.5.

7. The method for preparing PDA-PLGA cytoskeleton for intraskeletal muscle transplantation according to claim 1, wherein: the PLGA cytoskeleton is immersed in a weak alkaline Tris-HCl aqueous solution containing dopamine and shaken for 12-36 h.

8. The PDA-PLGA cytoskeleton for skeletal muscle transplantation according to the method for preparing PDA-PLGA cytoskeleton for skeletal muscle transplantation of claim 1, which is used for skeletal muscle transplantation after the PDA-PLGA cytoskeleton is obtained.

Technical Field

The invention relates to the technical field of transplantation application of skeletal muscle parts, in particular to a PDA-PLGA cell scaffold for skeletal muscle transplantation and a preparation method thereof.

Background

The tissue engineering mainly utilizes cell scaffold materials to carry out cell culture to form an implant with biological activity for in vivo tissue repair or reconstruction. The cell scaffold for providing three-dimensional space and metabolic environment for cell proliferation becomes an important foundation of tissue engineering, and the preparation method of the cell scaffold mainly comprises the following steps: solvent casting, gas foaming, phase separation, self-assembly, electrostatic spinning, and the like. The nanofiber membrane with a three-dimensional structure can be obtained by an electrostatic spinning method, and is widely used for tissue repair and function reconstruction of bones, nerves, blood vessels, tendons and the like due to the huge bionic potential of an extracellular matrix.

The biological materials currently used as cell scaffolds are mainly natural polymers, natural inorganic substances and synthetic polymers. The polylactic acid-glycolic acid copolymer (PLGA) is a degradable synthetic polymer material which is most widely applied in the biomedical field, the biodegradation speed of the polylactic acid-glycolic acid copolymer is adjustable, the mechanical property and the processing property of the polylactic acid-glycolic acid copolymer are better than those of natural materials, and degradation products are lactic acid and glycolic acid which are nontoxic and harmless to human bodies. PLGA has been approved by the FDA in the united states for clinical use, and is generally used as the main material for sutures, drug delivery devices and tissue engineering scaffolds, but as a synthetic polymer material, its biocompatibility and cell affinity are generally inferior to those of natural polymer materials. The surface of the material is subjected to coating modification, so that the biocompatibility and cell affinity of the material can be effectively improved, and common coatings comprise hyaluronic acid, collagen, hydroxyapatite, polydopamine and the like. Wherein, the poly-dopamine can be firmly adhered on the nano-fiber cell scaffold, effectively enhances the mechanical property and the hydrophilicity of the nano-fiber, and has excellent applicability to cell adhesion, proliferation and growth.

Skeletal muscle, the most abundant tissue in the body, plays an important role in completing various body activities and maintaining normal physiological status. The skeletal muscle is used as an important target organ of glycolipid metabolism, rich blood vessels, nerves, connective tissues and bone tissues are arranged around the skeletal muscle, the skeletal muscle can be used as a potential site for tissue engineering transplantation treatment during muscle repair, bone repair and treatment of glycolipid metabolic diseases, and the cell scaffold can be fixed on the skeletal muscle tissue more easily, so that monitoring and recovery after transplantation are facilitated. In conclusion, the PDA-PLGA cell scaffold which is simple and easy to obtain, has proper mechanical properties, is suitable for skeletal muscle part transplantation, has good biocompatibility and has important practical significance for the promotion of tissue engineering treatment and the development of the national sanitation industry.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

Therefore, it is an object of the present invention to provide a PDA-PLGA cell scaffold for skeletal muscle transplantation and a method for preparing the same, which can achieve effective enhancement of mechanical properties and hydrophilicity of nanofibers and have excellent applicability to cell adhesion, proliferation and growth.

To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions:

the PDA-PLGA cell scaffold for intramuscular transplantation of skeleton and its preparation process include the following steps:

the method comprises the following steps: preparing PLGA spinning solution with proper concentration;

step two: preparing continuous irregular nanofibers by an electrostatic spinning method, collecting an electrostatic spinning fiber scaffold by a metal receiving plate, and drying to obtain a PLGA cytoskeleton;

step three: and under the aerobic condition, immersing the collected PLGA cytoskeleton in a weak alkaline Tris-HCl aqueous solution containing dopamine, shaking to enable the PLGA cytoskeleton to be self-polymerized to form a polydopamine coating, and drying to obtain the PDA-PLGA cytoskeleton.

As a preferred scheme of the PDA-PLGA cell scaffold for skeletal muscle transplantation and the preparation method thereof, the PDA-PLGA cell scaffold comprises the following steps: the PLGA spinning solution has a mass fraction of 5-25%.

As a preferred scheme of the PDA-PLGA cell scaffold for skeletal muscle transplantation and the preparation method thereof, the PDA-PLGA cell scaffold comprises the following steps: the volume ratio of chloroform to acetone as a solvent in the PLGA spinning solution is 2: 1-5: 1.

As a preferred scheme of the PDA-PLGA cell scaffold for skeletal muscle transplantation and the preparation method thereof, the PDA-PLGA cell scaffold comprises the following steps: when the continuous irregular nanofibers are prepared by the electrostatic spinning method, electrostatic spinning parameters are controlled to be voltage of 5-30 kV, the inner diameter of an electrostatic spinning nozzle is 0.5-2.2 mm, the solution flow rate is 1-10 ml/h, the temperature is 20-30 ℃, and the distance between the electrostatic spinning nozzle and a receiving device is controlled to be 15-30 cm.

As a preferred scheme of the PDA-PLGA cell scaffold for skeletal muscle transplantation and the preparation method thereof, the PDA-PLGA cell scaffold comprises the following steps: the dopamine weak alkaline solution has the dopamine concentration of 1 mg/ml-5 mg/ml.

As a preferred scheme of the PDA-PLGA cell scaffold for skeletal muscle transplantation and the preparation method thereof, the PDA-PLGA cell scaffold comprises the following steps: the pH value of the Tris-HCl aqueous solution is 8.0-8.5.

As a preferred scheme of the PDA-PLGA cell scaffold for skeletal muscle transplantation and the preparation method thereof, the PDA-PLGA cell scaffold comprises the following steps: the PLGA cytoskeleton is immersed in a weak alkaline Tris-HCl aqueous solution containing dopamine and shaken for 12-36 h.

As a preferred scheme of the PDA-PLGA cell scaffold for skeletal muscle transplantation and the preparation method thereof, the PDA-PLGA cell scaffold comprises the following steps: after the PDA-PLGA cytoskeleton is obtained by the preparation method of the PDA-PLGA cytoskeleton for skeletal muscle transplantation, the application of skeletal muscle transplantation is carried out.

Compared with the prior art, the invention has the beneficial effects that: through the arrangement of the PDA-PLGA cytoskeleton for skeletal muscle transplantation and the preparation method thereof, the structural design is reasonable, and the poly-dopamine can be firmly adhered to the nanofiber cytoskeleton, so that the mechanical property and the hydrophilicity of the nanofiber are effectively enhanced, the poly-dopamine-polylactic-co-glycolic acid (PDA-PLGA) cytoskeleton has excellent applicability to cell adhesion, proliferation and growth, is beneficial to repair and regeneration of in-situ tissues, and has important practical significance for the promotion of tissue engineering treatment and the development of national sanitation.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail with reference to the accompanying drawings and detailed embodiments, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise. Wherein:

FIG. 1 is a flow chart of the steps of the present invention;

FIG. 2 is a schematic view of an electrospinning process.

FIG. 3 is a scanning electron micrograph of the PDA-PLGA cell scaffold prepared in example 1.

FIG. 4 is a result of hydrophilicity measurement of the PDA-PLGA cell scaffolds prepared in example 1.

FIG. 5 is the measurement result of the mechanical properties of the PDA-PLGA cell scaffolds prepared in example 1.

FIG. 6 shows the results of example 4 on the proliferation of various cells on PDA-PLGA cell scaffolds measured by MTT method.

FIG. 7 shows the adhesion morphology of cells on PDA-PLGA cell scaffolds observed by fluorescence microscopy in example 5.

FIG. 8 shows HE staining of tissue after skeletal muscle transplantation of PDA-PLGA cell scaffolds in example 6.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described herein, and it will be apparent to those of ordinary skill in the art that the present invention may be practiced without departing from the spirit and scope of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Example 1

Preparing PDA-PLGA cell scaffold.

Firstly, 1.0g of high molecular polymer PLGA solid particles serving as main bodies of electrostatic spinning is weighed and dissolved in a mixed solution of 4.2mL of HCL3 and 1.4mLC3H6O (v/v is 3:1), and the mixed solution is stirred for 1H at 25 ℃ until the mixed solution is completely dissolved, so that electrostatic spinning stock solution with the mass fraction of about 12% is prepared. Adding the obtained electrostatic spinning solution into a 5mL syringe, connecting the front end of the syringe with an electrostatic spinning machine through a 17G flat-head metal spinneret, and fixing the syringe on an injection pump, wherein the inner diameter of the metal spinneret is 1.12 mm. The distance between the metal receiving plate and the spinneret was adjusted to determine a receiving distance of 15 cm. The electrospinning process is shown in figure 1, the electrostatic high voltage between a metal spinneret and a receiving plate is controlled to be 15kv during the spinning process, the flow rate of the electrostatic spinning stock solution is controlled to be 1.5mL/h by using an injection pump, the spinning is carried out for 3 hours, and the prepared material is taken out and placed in a fume hood to be dried overnight, so as to obtain the PLGA fiber scaffold.

10mM Tris-HCl buffer solution is prepared, the pH value is adjusted to 8.5 by HCl, 20mg of dopamine hydrochloride is dissolved in 10mL Tris-HCl buffer solution, and 2mg/mL dopamine weak base solution is prepared. Immersing a PLGA fibrous scaffold with a proper size in a dopamine weak alkaline solution, keeping the solution in an aerobic condition, fully shaking to ensure that dopamine is self-polymerized on the surface of the material to form a Polydopamine (PDA) coating, observing that the color of the solution is changed from light brown to black during the self-polymerization of pDA, taking out the scaffold after 24 hours, slightly washing the scaffold with distilled water for 3 times to remove non-attached polydopamine molecules, and drying the prepared material in a fume hood overnight to obtain the PDA-PLGA cell scaffold.

The scanning electron microscope (fig. 2) analysis results show that the surface of the PLGA fiber scaffold with the PDA surface modification has a rougher surface morphology compared to the unmodified PLGA fiber scaffold, and the deposition of the PDA layer and the self-polymerized PDA particles is observed on the surface of the nanofibers, indicating that the PDA coating increases the surface roughness of the PLGA fiber scaffold. The results indicate that the pDA coating changes the surface properties of the PLGA fibrous scaffold.

Example 2

And (3) carrying out hydrophilic detection on the PDA-PLGA cytoskeleton.

Injecting deionized water with the volume of 20 mu L on the surfaces of the prepared PDA-PLGA cytoskeleton and PLGA cytoskeleton materials which are not subjected to coating treatment, placing deionized water drops at six different positions of one sample, measuring a water contact angle for each sample by using a water contact angle measuring instrument (SL200B), and calculating the size of the contact angle by taking an average value.

The water contact angle (fig. 3) measurements show that the water contact angle of the unmodified PLGA cell scaffold is 122 °, the water contact angle of the PDA-PLGA cell scaffold treated with PDA coating is reduced to 103 °, and the surface hydrophilicity is the main factor of the biomaterial affecting the cell activity (e.g. adhesion and migration). Compared with an unmodified PLGA fiber scaffold, the PLGA fiber subjected to PDA surface modification has better hydrophilicity and surface wettability, and is more favorable for cell attachment and proliferation.

Example 3

And (3) detecting the mechanical property of the PDA-PLGA cytoskeleton.

The prepared PDA-PLGA cytoskeleton and PLGA cytoskeleton materials which are not treated by the coating are cut into rectangles with the width of 1cm and the length of 3cm, the thickness is measured by a vernier caliper, four random points on each membrane are measured, and the average value is taken as the thickness (approximately equal to 0.1 mm). The tensile strength and modulus of elasticity of the samples were examined with an electronic universal tester (INSTRON 5948), square clamping positions of 1cmx1cm were left at each end during measurement, the ends of the strip were clamped in hydraulic clamps with an actual working area of 1cmx1cm, after fixation, the material was pulled axially towards both ends at a constant speed of 1mm/min until the material broke, yielding stress-strain curves for both materials, the yield point value of the stress-strain curve was determined as tensile strength, and the modulus of elasticity was calculated from the slope of the linear part of the curve. 4 parallel experiments were performed for each material.

The measurement results of the mechanical properties (figure 4) show that compared with the unmodified PLGA cytoskeleton, the mechanical properties of the PDA-PLGA cytoskeleton subjected to PDA surface modification are obviously improved. The elastic modulus of the unmodified PLGA cytoskeleton is 118.625MPa, the tensile strength is 2.4275MPa, the elastic modulus of the material is improved to 164.75MPa and the tensile strength is improved to 3.365MPa after the material is modified by the PDA coating, and the rigidity and the toughness of the material are obviously improved. In summary, PLGA cell scaffolds can have excellent overall mechanical properties by simply being immersed in a dopamine solution. Covalent bonds and hydrogen bonds can be formed between the PDA coating and the PLGA cytoskeleton nanofibers, so that the mechanical properties of the PLGA cytoskeleton nanofibers can be effectively enhanced, more selection spaces are provided for the transplantation positions and the transplantation modes, and the PLGA cytoskeleton nanofibers have wider application range in the field of tissue engineering.

Example 4

And observing the proliferation of the cells on the PDA-PLGA cell scaffold.

The prepared PDA-PLGA and PLGA cell scaffolds were cut into disks of about 6.5mm in diameter, soaked in 70% ethanol for sterilization for 2h, then rinsed 3 times with sterile phosphate buffered saline (PBS, pH 7.4) and air dried in a clean bench. Preparing a plurality of cells such as mononuclear macrophage (RAW264.7), skeletal muscle cell (C2C12), umbilical vein endothelial cell (HUVEC) and the like into cell suspensions with the density of 1x104/mL, putting the sterilized materials into a 96-well cell culture plate, taking 200 mu L of the cell suspensions, inoculating the cells into the 96-well cell culture plate with the density of 2x103 cells/well, placing the 96-well cell culture plate in a cell constant temperature incubator with the temperature of 37 ℃ and the saturation humidity of 5% CO2 for culturing for 7 days, changing the liquid every 2 days, and detecting the cell viability by using an MTT method at 1, 3, 5 and 7 days after inoculation in order to detect the proliferation of the cells on the stent. 20 μ LMTT (5mg/mL) was added to the 96 well cell culture plates to be tested and incubated for 4 hours. Subsequently, the supernatant was aspirated, and 200. mu.L DSMSO was added to each well, shaken for 10 minutes, and the absorbance value was observed at 570nm by using a microplate reader. The effect of PDA-PLGA and PLGA cell scaffolds on the proliferation of cells was examined.

MTT (FIG. 5) measurements showed a significant increase in cell viability on PDA-PLGA cell scaffolds with PDA surface modification compared to unmodified PLGA cell scaffolds. Because the PDA-PLGA surface forms the PDA coating, the roughness of the surface of the bracket is greatly increased, which is beneficial to the adhesion and adherence of cells, and in addition, the PDA coating generates a large amount of amine and hydroxyl which are beneficial to the adhesion, diffusion and growth of cells. These results indicate that the PDA-PLGA cell scaffolds have good biocompatibility, and the PDA coating can promote the growth and proliferation of cells on the PLGA cell scaffolds.

Example 5

And observing the adhesion morphology of the cells on the PDA-PLGA cell scaffold.

1.0g of polymer PLGA solid particles are weighed and dissolved in a mixed solution of 4.2mLCHCL3 and 1.4mLC3H6O (v/v ═ 3:1), then 280 mu g of rhodamine (≈ 50 mu g/mL) is added, and stirring is carried out at 25 ℃ for 1H until complete dissolution is carried out, so as to prepare the rhodamine-containing electrostatic spinning stock solution. The electrostatic spinning process and the PDA coating treatment process are the same as the example 1, the PDA-PLGA and PLGA cell scaffold containing red fluorescence can be obtained, the prepared PDA-PLGA and PLGA cell scaffold is cut into a wafer with the diameter of about 14mm, the wafer is soaked in 70% ethanol for sterilization for 2h, and then the wafer is washed for 3 times by PBS and dried in a clean bench. Preparing cell suspension with density of 2x104/mL from RAW264.7, C2C12 and HUVEC cells, respectively, putting the sterilized materials into a 24-hole cell culture plate, taking 500 mu L of cell suspension, inoculating the cells into the 24-hole cell culture plate at the density of 1x104 cells/hole, and putting the 24-hole cell culture plate into a cell constant temperature incubator with temperature of 37 ℃, saturation humidity and 5% CO2 for culture. After 24h, the 24-well cell culture plates were removed, the samples were washed 1 time with PBS, fixed with 4% paraformaldehyde for 30 minutes at room temperature, after fixation the samples were washed 3 times with PBS, then the cells were permeabilized with 0.1% Triton X-100 (petunia) for 3 minutes, after permeabilization the samples were washed 3 times with PBS. Then 200. mu.L of Phalloidin-iFluor 488 reagent (1:1000 dilution; abcam) was added to each well, incubated for 40 minutes at room temperature in the dark, and washed 3 times with PBS to remove excess dye. PDA-PLGA and PLGA cytoskeletons are placed on a glass slide, an anti-fluorescence attenuation blocking tablet (Solarbio) containing DAPI is dripped on the cytoskeleton, a cover glass is covered, and the adhesion morphology of the cells after the cells are cultured on the cytoskeleton for 24 hours is observed through an upright fluorescence microscope (Carl Zeiss, Axio imager. Z2).

Cell adhesion (figure 6) the results show that cells can adhere to the cell scaffold and elongated pseudopodia was observed to extend in the 3D cell scaffold nanofiber structure. Cells attach to the surface through elongated filopodia and tend to grow along the polymeric nanofibers at the surface. Compared with PLGA cytoskeleton, the PDA-PLGA cytoskeleton subjected to PDA surface modification shows more cell adhesion and proliferation. Due to its good hydrophilicity, rough surface structure and larger specific surface area, the PDA-PLGA scaffold helps cell adhesion and helps cell infiltration.

Example 6

The PDA-PLGA cell scaffolds prepared in example 1 were subjected to rat animal experiments to evaluate the safety of the PDA-PLGA cell scaffolds.

12 SPF SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.35ml/100g), the skin surface of the hind limb of the rat was incised to expose the muscle tissue, the 12 rats were randomly divided into 2 groups, the hind limb of the rat was incised and sutured only in the sham operation group without implanting material, the material was cut to 1.5X3cm size in the experimental group, rolled into a rod shape, transplanted into the muscle of the rat, and sutured. The animals were routinely fed and observed post-operatively. The post-operation rat animal has good recovery, the incision is well healed, and no infection occurs. The rats fed water normally and moved normally without dyskinesia.

Animal experiment results show that on the 28 th day after operation, the growth activity of the rat is completely normal, the incision is basically healed, only a sutured thin line is seen, and rejection reactions such as obvious congestion, bleeding and the like are not seen. Specimens were cut from the material at a range of 1cm from the muscle graft site on day 28, and tissue sections and HE staining were performed, and the staining results showed that macrophage infiltration at the rat graft site on day 28 was mainly concentrated on the edge of the PDA-PLGA scaffold, inflammatory cell infiltration of the muscle tissue adjacent thereto was substantially disappeared (FIG. 7a), and neovascularization was found in the transplanted PDA-PLGA scaffold (FIG. 7b), which was advantageous for in situ tissue repair and regeneration.

While the invention has been described above with reference to an embodiment, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In particular, the various features of the disclosed embodiments of the invention may be used in any combination, provided that no structural conflict exists, and the combinations are not exhaustively described in this specification merely for the sake of brevity and resource conservation. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.