Cyclic testing machine for biological structures under cell culture conditions

Abstract

Cyclic testing machine (100) for a specimen (11) of biological structures under cell culture conditions, the machine (100) comprising: - a base (1), - a crosspiece (2) hinged at one of its first end (2') by means of a cylindrical hinge (6), in turn integral with a first end (1') of the base (1), so as to operate a cyclic rotation motion around the cylindrical hinge (6), - a weight-holding basket (8) removably fixed on the crosspiece (2), - an anchoring cylinder (14) fixed to a second end (2') of the crosspiece (2), - a stepper motor (10) supported by a support (3), in turn integral with a second end (1'') of the base (1), - a pulley (15) keyed to one end of the stepper motor (10), said machine (100) being characterised in that - an inextensible cable (17) fixed to the anchoring cylinder (14) is wound in a groove (18) of the pulley (15), which rotates by defined angles by means of the stepper motor (10) and allows the movement of the crosspiece (2) itself by means of the hinge (6); - a pressing plate (12) integral with the crosspiece (2) configured to apply a compressive load to the specimen (11).

Description

[0001] CYCLIC TESTING MACHINE FOR BIOLOGICAL STRUCTURES

[0002] UNDER CELL CULTURE CONDITIONS

[0003] DESCRIPTION

[0004] Technical field of the invention

[0005] The present invention relates to a testing machine capable of applying, under load control , a cyclic compressive load on biological structures under cell culture conditions .

[0006] Known Art

[0007] As is known, cyclic testing machines for biological structures under cell culture conditions are available inside bioreactors for tissue engineering .

[0008] A bioreactor is defined as any device capable of providing a suitable environment for the growth of biological organisms . In particular, bioreactors intended for the growth of cells or tissues for therapeutic or experimental purposes , have a speci fic structure . Many cells and tissues , especially mammalian ones , require a suitable support upon which to grow, and continuously agitated environments can be detrimental to them. Also nutritional requirements and factors necessary for growth are significantly higher .

[0009] Bioreactors are expensive, often costing several thousand euros. Currently, on the market bioreactors for tissue engineering are available that use more or less sophisticated cyclic loading systems, characterized by high costs which can range from several thousands to tens of thousands of euros. These are testing machines being an integral part of the bioreactor that have been designed and optimized with the bioreactor itself. Different types of bioreactors are known in the state of the art: some to prompt / stimulate the vascular tissue, others the cartilage tissue, others specific bone segments, etc.

[0010] Furthermore, commercially available bioreactors have rather limited load variability ranges.

[0011] Also known are static loading systems based on the use of the leverage and specifically designed and engineered for the civil engineering field, and therefore they are unsuitable for limited and controlled environments such as those of an incubator . Alternatively, static loading systems are based on the leverage and on the use of a gear wheel , in which the loading action of the test specimen occurs through the rotation action of a gear wheel that meshes with a rack and pinion and is operated by a crank . Also these solutions cannot be suitable for loading biological structures under cell culture conditions inside an incubator . Further static loading systems , based on the leverage , involve , for example , the use of a counterweight with suspended weights that are designed to measure the friction force at the piston-cylinder interface . Vibrations which would result from the counterweight system would af fect the correct application of the load on the test specimen and are therefore unusable for the purposes of the present invention .

[0012] A last example of a known technology is represented by a loading system based on the use of a second-class leverage and of a counterweight system .

[0013] However a cyclic loading is not described, and the system cannot be used inside an incubator for two reasons : ( i ) the resulting vibrations would be great and signi ficant ; ( ii ) a large amount of space is required, which is not available inside the incubator . The loading system includes a hinged support for loading the specimen but does not include a system for adj usting the relative distance between the pressing plate and the upper surface of the specimen .

[0014] None of them are designed to cyclically load the biomaterial specimen and to be contained inside an incubator . Such loading systems allow for solving speci fic problems inherent in experimental testing, but none of them are designed to conduct cyclic loading tests in a limited and controlled environment such as that inside a cell culture incubator .

[0015] There is therefore a need for implementing a small , cost-ef fective cyclic testing machine capable of loading biological structures under cell culture conditions inside an incubator .

[0016] Summary of the Invention The cyclic testing machine which is the subj ect of the present invention is a novel device that can be easily installed inside a cel l culture incubator without requiring any modi fications to the incubator itsel f .

[0017] Advantageously, the machine is small enough to fit easily inside a cell culture incubator and has a low manufacturing cost , estimated at j ust a few hundred euros . The possibility of installing the testing machine inside an incubator represents an excellent alternative to the costly bioreactor solution . Incubator manufacturers may be interested in manufacturing the machine which is the subj ect of the present invention, and in reselling it as an integrated device to complement the incubator for mechanobiological analyses aimed at investigating the ef fects of mechanical loading on the regrowth or remodeling process of biological tissue .

[0018] Advantageously, the subj ect machine is capable of cyclically prompting / stimulating biological structures without the use of a bioreactor, but rather by means of a simple cell culture incubator, inside which the machine can be positioned.

[0019] Furthermore, unlike commercially available bioreactors, which have specific functions and therefore have a rather limited load variability ranges, the subject machine, due to its leverage system and therefore to the possibility of continuously varying the distance of the weightholding basket from the cylindrical hinge and the mass of the weights allocated in the basket itself, allows for a wide variability of the applied compression load. This load can vary from a few tens of grams to a few kilograms.

[0020] It is worth noting that, due to the leverage system used by the machine, it is possible to monitor the applied load without the need for a load cell.

[0021] Therefore, according to the present invention, an innovative cyclic testing machine for biological structures is defined, as specified in the appended independent claim.

[0022] The dependent claims outline specific and further advantageous aspects of the invention .

[0023] Brief description of the drawings

[0024] The invention will be further described by means of the following drawing tables , which are completely exemplary and not limiting :

[0025] - Figure 1 is an overall perspective view of the device according to the present invention ;

[0026] Figure 2 is front perspective view of the device of Fig . 1 ;

[0027] - Figure 3 is a bottom perspective view of the device of Fig . 1 ;

[0028] - Figure 4 is a detailed perspective view of the pressing plate of the device of Fig . 1 ;

[0029] - Figure 5 is a sectional view of the device of Fig . 1 for the calculation of the masses to be used in the weight-holding basket ;

[0030] - Figure 6 shows a scheme for the calculation of the masses to be placed in the weight-holding basket , in order to apply to the specimen a compression load of 6 kg = 58 . 86 N in the loading phase ; Figure 7 shows a scheme for the calculation of the force applied by the stepper motor, in order to lift the masses placed in the weightholding bascket in the unloading phase ;

[0031] - Figure 8 shows the scheme for the analysis of the movements occurring at the stepper motor and the specimen to be tested according to the invention .

[0032] Detailed description

[0033] According to the present invention and to the annexed Figures 1- 8 , a cyclic testing machine for biological structures under cell culture conditions is shown .

[0034] As is shown in Figure 1 , the cyclic testing machine 100 according to an embodiment of the present invention comprises : a base 1 , a crosspiece 2 , a stepper motor 10 , a support 3 for the motor, a pair of brackets 4 for the application of the load, wherein each pair of brackets 4 is provided with a regulating slot 5 , a cylindrical hinge 6 , a support 7 for the cylindrical hinge , a weight-holding basket 8 , a wing screw 9 for blocking the weicht-holding basket 8 , a pressing plate 12 , an anchoring cylinder 14 , a pulley 15 keyed to the stepper motor, as is shown in Figure 2 .

[0035] The base 1 is appropriately configured with the side walls 13 slightly raised ( Figure 2 ) on its upper portion 21 in order to ensure the necessary rigidity so that the loading system does not excessively warp and deform during the compression tests .

[0036] The crosspiece 2 is hinged at its first end 2 ' by means of the cylindrical hinge 6 , to a first end 1 ' of the base 1 . It has a rectangular section at its first end 2 ' at the cylindrical hinge 6 , and a double " T" section moving towards its second end 2" , in order to ensure the sliding of the weight-holding trolley 8 on the crosspiece 2 itsel f . Due to the presence of the guide , the weight-holding basket 8 exerts its force on the crosspiece 2 ; it is therefore not suspended but rests upon the same .

[0037] Advantageously, the crosspiece 2 is configured in a sigmoid shape to minimi ze the overall dimensions along the vertical direction and thus to allow the loading system to be easily inserted between the support shelves inside the incubator .

[0038] The crosspiece 2 acts as a sliding guide on which the weight-holding basket 8 moves . The latter can be positioned at a variable distance along the crosspiece 2 , with respect to the cylindrical hinge 6 , and is removably fixed to the crosspiece 2 in a predefined position by means of the wing screw 9. In particular, the weight-holding basket 8 includes a cavity in which the weights to be applied to the specimen 11 are located and engages with the portion of the crosspiece 2 ( Figure 1 ) having a double " T" section acting as a guide . Due to the sliding of the weight-holding basket 8 , it is possible to continuously adj ust the value of the load acting on the specimen to be tested .

[0039] The cylindrical hinge 6 is connected to a support 7 secured to the first end 1 ' of the base 1 , which represents the fulcrum for the crosspiece 2 .

[0040] The stepper motor 10 is supported by the support 3 , which is fixed by pins to a second end 1" of the base 1 . The support 3 holds the stepper motor 10 so that it is parallel to the anchoring cylinder 14 .

[0041] The anchoring cylinder 14 is fixed to a second end 2" of the crosspiece 2 ( Figure 2 ) . An inextensible cable 17 , knotted to the anchoring cylinder 14 , is wound into a groove 18 of the pulley 15 keyed to one end of the stepper motor 10 , which, by rotating through appropriate angles , allows the crosspiece 2 to be li fted by def ined displacements . The keying of the pulley 15 occurs by engaging a flat cylindrical surface made on the hub of the pulley with a corresponding flat cylindrical surface belonging to the rotor of the stepper motor 10 .

[0042] Advantageously, the leverage system equipped with the stepper motor 10 cyclically li fts and releases the crosspiece 2 , hinged at its end 2 ' , at a frequency that can be managed and controlled by the user .

[0043] Advantageously, the crosspiece 2 , by means of the pressing plate 12 , exerts a pure compressive load on the biological structure to be tested and positioned beneath the plate 12 itsel f .

[0044] Advantageously, the basket 8 , within which weights of various materials and si zes can be placed, moves continuously on the crosspiece 2 , configured as a sliding guide . By appropriately varying the weights and distances of the basket 8 with respect to the cylindrical hinge 6 located at the end 2 ' of the crosspiece , it is possible to continuously vary the compressive force applied to the specimen to be tested, with a wide variability range .

[0045] In the vicinity of the cylindrical hinge 6 , the pair of brackets 4 for applying the load ( Figure 1 ) are symmetrically fixed with respect to a plane X passing through a median plane in the longitudinal direction of the crosspiece 2 . The brackets 4 are connected to the crosspiece 2 with removable fastening means of a known type , for example bolts , and are provided with an adj ustment slot 5 through which it is possible to continuously vary the distance between the base 1 and the pressing plate 12 . The pressing plate 12 , illustrated in detail in Figure 4 , is connected to the lower end of each bracket 4 of the pair of brackets , also with removable fastening means of a known type , for example bolts . Therefore , by means of the adj ustment slots 5 it is also possible to continuously vary the relative height between the pressing plate 12 and the upper surface of the specimen 11 to be tested ( Figure 1 ) . The slots 5 are si zed so as to leave space for the insertion, beneath the pressure plate , of the specimen 11 to be tested and of any container, such as a Petri-dish or a Petri capsule , within which the specimen is cultured .

[0046] By appropriately lubricating the seat of the bolt connecting the pressing plate 12 to the pair of brackets 4 , it is possible to make the pressing piston 12 float with respect to the connecting bolt , which constitutes its axis of rotation . This ensures the regular rotation of the pressing plate 12 around the bolt as the crosspiece 2 rotates around the cylindrical hinge 6 . In this way, the parallelism is maintained between the lower base 19 of the pressing plate 12 and the surface of the base 1 ( Figure 4 ) , or with the upper surface of the specimen 11 . This ensures that a pure compression load is applied to the specimen 11 to be tested, without bending ( resulting from of f-axis loading) . The pair of adj ustment brackets 4 ensure that the distance between the lower base 19 of the pressing plate 12 and the upper surface of the specimen 11 to be tested can be adj usted according to the height of the specimen 11 itsel f .

[0047] Due to the adj ustment slots 5 , it is possible to test specimens of varying dimensions . The space in the base 1 beneath the pressing plate provides suf ficient space to accommodate the Petri-dishes or the cell culture devices within which the specimen to be tested can be cultured .

[0048] As soon as the stepper motor 10 is activated, the inextensible wire 17 li fts the crosspiece 2 with the weight-holding basket 8 until it reaches a determined height . When the motor changes its direction of rotation, the crosspiece 2 lowers until the lower base 19 of the pressing plate 12 does not adhere to the upper surface of the specimen 11 . At that point , the inextensible wire 17 is no longer tensioned, it becomes slack, and the entire load of the weightholding basket 8 acts on the specimen .

[0049] The base 1 in its lower portion 20 , illustrated in Figure 3 , includes cavities 16 appropriately shaped to prevent deformation and to minimi ze the masses , making the testing machine easier to handle for use in a cell culture incubator .

[0050] Figure 5 illustrates the calculation of the masses to be allocated in the weight-holding basket 8 in order to apply a load of known magnitude on a specimen . In particular, Figure 5 shows the two extreme positions Al and A2 that the weight-holding basket 8 can assume and their distance from the cylindrical hinge 6 . The dimensions of the testing machine 100 are such that the machine itsel f can be allocated inside a common cell culture incubator . For example , assuming, as is shown in the graph in Figure 6, that a load P = 6 kg = 58.86 N is applied to the specimen to be tested, for the equilibrium of the moments it turns out that the mass W to be allocated in the weight-holding basket is equal to : W = 750 g in the case of b = bmax = 200 mm (W = Pa / bmax, a = 25 mm) , and W = 1250 g in the case of b = bmin = 120 mm (W = Pa / bmin, a = 25 mm) . The scheme in Figure 6 obviously refers to the loading phase, that is, the phase in which the crosspiece 2 lowers, loading the specimen. It is certainly interesting to investigate what happens during the unloading phase, that is, the phase in which the crosspiece is lifted by the stepper motor (Figure 7) .

[0051] For the equilibrium of moments, the lifting force S exerted by the stepper motor to lift a weight of W = 750 g is equal to : S = 484 g. Similarly, the force exerted by the motor to lift a weight of W = 1250 g is equal to : S = 806 g.

[0052] An analysis of the displacements undergone by the different points of the crosspiece 2 was also carried out (Figure 8) . If 0 is the angle of rotation undergone by the crosspiece 2 and if this angle is sufficiently small (which is what happens and can be legitimately hypothesized) , then the inextensible wire will have to wind / unwind inside the groove 18 of the pulley 15 by an amount equal to A « c • 0 (where 0 must be expressed in radians) . The corresponding displacement undergone by the surface of the pressing plate 12 (acting on the specimen) will instead be equal to 5 = a • 0. From the previous relations it follows: 5 = a • A / c. Assuming c = 310 mm (Figure 5) and a = 25 mm (Figure 5) , we have 5 = 0.081 • A, meaning that the displacement experienced by the surface of the pressing plate 12 is less than 10% of the winding / unwinding displacement of the inextensible wire 17, wound on the pulley 15.

[0053] Based on the above, it can be concluded that, due to the use of the leverage system, the masses required in the weight-holding basket 8 are significantly smaller than those that would be required if the leverage system were not used (Figure

[0054] 6) . In general, smaller masses imply smaller inertia, and therefore smaller vibrations .

[0055] Advantageously, the force exerted by the stepper motor 10 to li ft the crosspiece 2 is signi ficantly smaller than that which would be required i f the leverage system were not used ( Figure 7 ) . In other words , this allows the use of a motor with a lower static torque ( "holding torque" ) and therefore a smaller motor, easier to manage within a cell culture incubator .

[0056] Advantageously, the leverage system functions as a motor reduction gear ( Figure 8 ) . The li f ting / lowering movement of the surface of the pressing plate 12 acting on the specimen is less than 10% of the winding / unwinding motion of the wire 17 in the pulley 15 keyed to the stepper motor 10 .

[0057] The calculations and tests were performed on a testing machine with dimensions compatible with those of a traditional cell culture incubator with a useful volume of 470 x 605 x 530 mm . Speci fic considerations must be made i f incubators with larger or smaller dimensions than those considered are used . Advantageously, by appropriately setting the driver for the control of the stepper motor 10, it is possible to set variable frequency values with which the motor lifts and releases the crosspiece 2. In other words, the system allows for the continuous variation of the frequency with which the cyclic compressive load is applied to the specimen to be tested. A limitation is that excessively high frequency values (i.e. > 10 Hz) cannot be set, especially if the applied compressive loads exceed several tens of Newtons (N) . When the frequencies are excessively high and, simultaneously, the masses deposited in the basket are large (i.e. over 20 N) , significant vibrations are triggered on the crosspiece, which are greater the lower the "Young's modulus" of the material from which it is made. Preliminary analyses have shown that such oscillations are large in the case of a crosspiece made of acrylonitrile butadiene styrene (ABS) , but they are expected to be significantly reduced if the crosspiece is made of metal, for example titanium, using rapid prototyping techniques such as selective laser sintering ( SLS ) or selective laser melting ( SLM) , both 3D printing technologies . It is worth noting, however, that such high frequencies are not required for mechanobiological tests . Typically, the frequencies used in previous experiments conducted on biological structures subj ected to compression never exceed 1 Hz . The reason for this is linked to the fact that these tests are often aimed at simulating the regrowth or di f ferentiation process of mesenchymal tissue in patients who , for example , have suf fered a bone fracture . It is reasonable to assume that in such patients , movements occur gradually and " slowly" , which makes testing with a cyclic compressive load with a frequency greater than 1 Hz uninteresting .

[0058] Advantageously, due to the need to ensure the sterility of the pressing plate ( its surface , in fact , comes into contact with the upper surface of the specimen) during testing, and therefore to the need to heat it up to high temperatures incompatible with ABS , the plate itsel f was machined from aluminum .

[0059] While at least one exemplary embodiment has been presented in the summary and detailed descriptions , it should be understood that there are a large number of variations that fall within the scope of the invention . Furthermore , it should be understood that the embodiment ( s ) presented are merely examples that are not intended to limit in any way the protection scope of the invention or its application or configurations . Rather, the summary and detailed descriptions provide the person skilled in the art with a convenient guide for implementing at least one exemplary embodiment , it being clear that numerous variations can be made to the function and assembly of the elements described therein, without departing from the scope of the invention as established by the appended claims and their technical-legal equivalents .

Claims

CLAIMS1. A cyclic testing machine (100) for a specimen (11) of biological structures under cell culture conditions, the machine (100) comprising:- a base ( 1 ) ,- a crosspiece (2) hinged at one of its first ends (2' ) by means of a cylindrical hinge (6) , in turn integral with a first end (1' ) of the base (1) , so as to operate a cyclic rotation motion around the cylindrical hinge (6) ,- a weight-holding basket (8) removably fixed on the crosspiece ( 2 ) ,- an anchoring cylinder (14) fixed to a second end (2") of the crosspiece (2) ,- a stepper motor (10) supported by a support (3) , in turn integral with a second end (1") of the base (1) ,- a pulley (15) keyed to one end of the stepper motor (10) , said machine (100) characterized in that- an inextensible cable (17) fixed to the anchoring cylinder (14) is wound in a groove (18) of the pulley (15) , which rotates by defined angles by means of the stepper motor (10) and allows the movement of the crosspiece (2) itself by means of the hinge (6) ;- a pressing plate (12) integral with the crosspiece (2) configured to apply a compression load to the specimen (11) .

2. Machine (100) according to claim 1, in which the cylindrical hinge (6) is configured as the fulcrum of the crosspiece (2) and is connected to a support (7) in turn integral with a first end (1' ) of the base (1) •3. Machine (100) according to claim 1 or 2, comprising a pair of brackets (4) to the lower end (4' ) of each bracket of which the pressing plate (12) is removably connected.

4. Machine (100) according to claim 3, wherein each bracket of the pair of brackets (4) is provided with an adjustment slot (5) of the distance between the pressing plate (12) and the base (1) .

5. Machine (100) according to claim 4, wherein the pressing plate (12) is configured to be floating with respect to its rotation axis so that, as the position of the crosspiece (2) varies due to its cyclic rotation motion, the lower base (19) of the pressing plate (12) remains parallel to the base (1) .

6. Machine (100) according to one of the preceding claims, wherein the base (1) comprises side walls(13) on its upper portion (21) and cavities (16) in its lower portion (20) .

7. Machine (100) according to one of the preceding claims, wherein the crosspiece (2) has a rectangular section at its first end (2' ) and a double "T" in the remaining portion up to its second end (2") .

8. Machine (100) according to claim 4, wherein the crosspiece (2) is configured in a sigmoid shape to minimize the overall dimensions along the vertical direction .

9. Machine (100) according to one of the preceding claims, wherein the weight-holding basket (8) comprises a cavity in which the weights are housed and engages with the portion of the crosspiece (2) having a double "T" section by means of a wing screw(9) .

Images