Abstract:
A mechanical testing device has a rigid frame and a piezo translator connected to the frame. A Wheatstone bridge is connected to the translator to produce an electrical signal related to the compression of the translator, wherein a sample positioned between the piezo translator and the frame is subjected to loads by the movement of the translator. A sensor detects the force applied to the sample by the piezo translator, and produces a signal indicative of the force. A computer receives the Wheatstone bridge electrical signal and the signal indicative of the force applied to the sample. The computer controls the advancement of the translator to allow the application of precise amounts of compression to the sample.

Description:
CROSS REFERENCES TO RELATED APPLICATIONS  
       [1]    1. This application is a divisional of U.S. application Ser. No. 09/110,503, filed Jul. 6, 1998, which claimed priority based on U.S. provisional application No. 60/052,587, filed Jul. 15, 1997, the disclosures of both said applications being incorporated by reference herein.  
     
    
     
       STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT  
         [2]    2. Not applicable.  
         BACKGROUND OF THE INVENTION  
         [3]    3. The present invention is in the field of biotechnology and specifically relates to the study of the physiological and physiochemical processes which govern and underlie the formation, growth and resorption of human and animal bone. In particular the invention provides novel means for the study of responses of the mammalian musculoskeletal system to stress and potentially may lead to the discovery of novel substances produced by bone during these responses. The instant system may lead to a better understanding of diseases such as osteoporosis and the perfusion chamber means provides means for the study of the effects of drugs and other substances added to the perfused medium.  
           [4]    4. It has been known for over 150 years that bone responds to mechanical loading. Although the effects of exercise and mechanical loading on the musculoskeletal systems have been well documented, the actual mechanisms by which mechanical loading acts at the cellular level in the maintenance of skeletal integrity are not completely understood. Although greater attention is being given to exercise and nutrition as a means of preventing and/or treating osteoporosis, the regulatory mechanisms that control skeletal response to mechanical loading, growth factors and nutrition are not yet delineated.  
           [5]    5. There is speculation about the biophysical structure and properties of the sensory and biochemical and molecular biological mechanism of mechano-transduction. When controlled loads of a given magnitude and frequency are applied, in vivo, either in an isolated wing preparation or a rat tibia, bone mineral density is known to increase to an extent which is approximately proportional to the load applied. However, according to the prior art, it is not possible to assess quantitatively the bone-specific regulatory control product and their mechanisms nor to monitor the bone production of local growth factors and cytokines, in these in vivo preparations.  
           [6]    6. Whilst cell culture preparations do permit an investigator to quantify second messengers, cytokines and local growth factors, they do not permit one to monitor the responses of bone cells to mechanical deformation of the bone matrix which are so important in maintaining and/or remodeling of the skeletal system.  
           [7]    7. Although growth factors have been shown to enhance the development of new bone, clearly and without the presence of mechanical loading, under these circumstances, the new matrix is not formed along lines of strain and it is that feature, in life, which induces maximum integrity of the new bone so formed. The present authors have been associated with previous work in which the viability of osteoblasts from 2 to 4 week old pigs was successfully maintained, in culture, for 68 days. Careful consideration of these findings led to the hypothesis that, in a suitable novel system, which would permit continuous perfusion and mechanical loading of suitable explanted samples of trabecular bone from mature pigs, viability might be maintained for 10 to 12 days or longer. If this were to be achieved, such a time frame would permit measurements of the rate of bone formation and resorption of the trabecular bone, not available using the systems, apparatus and methods of the prior art. Further, such a novel system would be applicable to the study of human bone.  
           [8]    8. Up to now, prior art apparatus and systems for investigating bone have either comprised cell culture apparatus of a variety of well-known types or mechanical means for applying three point and four point bending forces to a biological test subject. An example of the three point type is disclosed in U.S. Pat. No. 5,406,853 to Lintilhac and Vesecky and an example of the four point type is disclosed in U.S. Pat. No. 5,383,474 to Recker and Akhter.  
           [9]    9. The present authors are not aware of any prior art system or apparatus which provides means for simultaneous, contemporaneous and continuous study of axially loaded viable mammalian bone undergoing concurrent continuous perfusion and the effluent medium therefrom.  
         References  
         [10]    10. 1. Baron R., Vignery A., Neff L., Silverglate A., Santa Maria A. (1983). Processing of undecalcified bone specimens for bone histomorphometry. In: ed, Recker R. R., Bone Histomorphometry: Techniques and Interpretation CRC, Boca Raton, Fla., 13-35.  
           [11]    11. 2. Brighton C. T., Sennett B. J., Farmer J. C., lanotti J. P., Hansen C. A., Williams J. L., Williamson J. The inositol phosphate pathway as a mediator in the proliferative response of rat calvarial bone cells to cyclical biaxial mechanical strain. J. Orthop. Res. 10:385-393; 1992.  
           [12]    12. 3. Carvalho R. S., Scott J. E., Suga D. M., Yen E. H. K. Stimulation of signal transduction pathways in osteoblasts by mechanical strain potentiated by parathyroid hormone. J. Bone Min. Res. 9::999-1011; 1994.  
           [13]    13. 4. Crenshaw T. D., Thomson B. M., Noble B. S., Milne J. S. and Loveridge N. Prostaglandin E2 inhibits proliferation of porcine progenitor osteoblast cells. J. Bone Min. Res. 8(1):S362, 1993.  
           [14]    14. 5. Currey J. The Mechanical Adaptation of bones. Princeton N.J.: Princeton University Press, 1984.  
           [15]    15. 6. Dalsky G. P., Stocke K. S., Ehsani A. A., Slatopolsky E., Lee W. C. and Birge S. J. Weight-bearing exercise training and lumbar bone mineral content in post menopausal women. Ann. Intern. Med. 108:824-828, 1988.  
           [16]    16. 7. E1 Haj A. J., Minter S. L., Rawlinson S. C. F., Suswillo R. and Lanyon L. E. Cellular responses to mechanical loading into vitro. J. Bone Min. Res. 5:923-932, 1990.  
           [17]    17. 8. Frost H. M. (1983). Bone histomorphometry: analysis of trabecular bone dynamics. In: ed, Recker R. R. Bone Histomorphometry: Techniques and Interpretation CRC Press, Boca Raton, Fla., 109-142.  
           [18]    18. 9. Gleeson P. B., Protas E. J., Le Blanc A. D., Schneider V. S. and Evans H. J. Effects of weight lifting on bone mineral density in premenopausal women. J. Bone, Min. Res. 5:153-157, 1990.  
           [19]    19. 10. Hock J. M., Centrella M. and Canalis E. Insulin-like growth factor I (aIGF-1) has independent effects on bone matrix formation and cell replication. Endocrin. 122:254-260; 1988.  
           [20]    20. 11. Hsieh H-J., Li N. Q., Frangos J. A. Shear stress increases endothelial platelet-derived growth factor mRNA levels. Am. Physiol J. 260:H642-646; 1991.  
           [21]    21. 12. Jones D. B., Nolte H., Scholubbers J. G., Turner E., Veltel D. Biochemical signal transduction of mechanical strain in osteoblast-like cells. Biomaterials. 12:101-110; 1991.  
           [22]    22. 13. Lanyon L. E. Control of bone architecture by functional load bearing. J. Bone Min. Res. 7:S369-S375, 1992.  
           [23]    23. 14. Murray D. W. and Rushton N. The effect of strain on bone cell prostaglandin E2 release: a new experimental method. Calcif. Tissue Int. 47:35-39, 1990.  
           [24]    24. 15. Mundy G. R. Bone resorbing cells. In: Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. Favus M. J. (ed), Kelseyville, Calif. American Society for Bone and Mineral Research (PP.18-22) 1990.  
           [25]    25. 16. Notelovitz M., Martin D., Tesar R., Khan F. Y., Probart C., Fields C. and McKenzie L. Estrogen therapy and variable resistance weight training increase bone mineral in surgically menopausal women. J. Bone Min. Res. 6:583-590, 1991.  
           [26]    26. 17. Parfitt A. M., Drezner M. K., Glorieux F. H., Kanis J. A., Malluche H., Meunier P. J., Ott S. M., Recker R. R. (1987). Bone histomorphometry: standardization of nomenclature, symbols and units. J. Bone Min. Res. 2:595-609.  
           [27]    27. 18. Parfitt A. M., Mathews C. H. E., Villanueva A. R., Kleerekoper M., Frame B., Rao D. S. ( 1983 ). Relationships between surface, volume and thickness of iliac trabecular bone in aging and in osteoporosis. J Clin Inv 72:1396-1409.  
           [28]    28. 19. Pead M. J., Suswillo R., Skerry, Vedi S., Lanyon L. E. Increased [3H]uridine levels in osteocytes following a single short period of dynamic bone loading in vivo. Calcif. Tissue Int. 43:92-96; 1988.  
           [29]    29. 20. Parfitt A. M. The physiologic and clinical significance of bone histomorphometric data. In: Bone Histomorphometry: Techniques and interpretation. Recker R. R. (ed) (PP.143-223) 1983.  
           [30]    30. 21. Raab D. M., Crenshaw T. D., Kimmel D. B., Smith E. L. A histomorphometric study of cortical bone activity during increased weight-bearing exercise. J. Bone Min. Res. 6:741-749, 1991.  
           [31]    31. 22. Raab-Cullen D. M., Akhter M. P., Dimmel D. B. and Recker R. R. Periosteal bone formation stimulated by externally induced bending strains. J. Bone Min. Res. 9:1143-1152; 1994.  
           [32]    32. 23. Raab-Cullen D. M., Thiede M. A., Petersen D. N., Kimmel D. B., Recker R. R. Mechanical loading stimulates rapid changes in periosteal gene expression. Calcif. Tissue Int. 5:473-478; 1994.  
           [33]    33. 24. Rawlinson S. C. F., Mohan S., Baylink D. J., Lanyon L. E. Exogenous prostacyclin, but not prostaglandin E2, produces similar responses in both G6PD activity and RNA production as mechanical loading and increases IGF-II release in adult cancellous bone in culture. Calcif. Tissue Int. 53:324-329; 1993.  
           [34]    34. 25. Rubin C. T. and Lanyon L. E. Regulation of bone mass by mechanical strain magnitude. Calcif. Tissue Int. 37:411-417, 1985.  
           [35]    35. 26. Smith E. L. and Gilligan C. Dose-response relationship between physical loading and mechanical competence of bone. Bone 18:45S-50S; 1996.  
           [36]    36. 27. Smith E. L., Gilligan C., McAdam M., Ensign C. P. and Smith P. E. Deterring bone loss by exercise intervention in premenopausal and postmenopausal women. Calcif. Tissue Int. 44:312-321, 1989.  
           [37]    37. 28. Tommerup L. J., Raab D. M., Crenshaw T. D. and Smith E. L. Does weight-bearing exercise affect non-weight-bearing bone? J. Bone Min. Res. 8(9): 1053-1058, 1993.  
           [38]    38. 29. Turner C. H., Woltman T. A. and Belongia D. A. Structural changes in rat bone subjected to long-term in vivo mechanical loading. Bone 13:417-422, 1992.  
           [39]    39. 30. Turner C. H. Mechanical loading thresholds for lamellar and woven bone formation. J. Bone Min. Res. 9:87-97, 1994.  
         OUTLINE OF THE PRESENT INVENTION  
         [40]    40. In the instant system, apparatus means is provided for the perfusion and axial mechanical loading of an explanted sample of mammalian trabecular bone which has been prepared in an appropriate manner. During use, a prepared trabecular bone biopsy core is placed within the apparatus and is then loaded mechanically to induce tension and/or compression to the bone matrix. The bone explant perfusion and loading apparatus of the instant system is provided with means for maintaining an environment with stable oxygen, carbon dioxide, nutrients and systemic hormones.  
           [41]    41. Prepared bone biopsies are in the form of trabecular bone cores, 10-12 mm in diameter and 3 to 5 mm thick. These are surgically extracted, under sterile conditions, from suitable long bones of the subject. This procedure is carried out with care and precision, using suitable cutting means and cooling means, to ensure that the resultant bone disk samples are not subjected to temperature rises during cutting and that extreme dimensional accuracy and disk flatness are achieved.  
           [42]    42. Cutting means are in the form of a surgical hand saw used to cut gross samples, a diamond tipped keyhole saw to remove bone cores from the gross samples and an ultra high precision band saw with a diamond tipped bladed, operated in conjunction with jig means, to cut bone disk. Trabecular bone sample disks produced are flat (±100 nm) and have parallel end surfaces (±2-5 μm). Cooling means comprise suitable phosphate buffered saline (PBS) at 6° C. which is used to flood the work piece during cutting. Each trabecular bone sample disk, so prepared, is intended to supply about 3,500,000-11,000,000 cells (based on an estimate of 10,000-20,000 cells per cubic mm of bone (Mundy 1990; Parfitt 1983). Extracted bone disk samples are perfused and maintained with suitable circulating medium, Hepes and fetal calf serum.  
           [43]    43. The apparatus of the instant invention provides means for concurrent mechanical loading of the prepared bone explant sample disk, located within a novel perfusion chamber, in a controlled manner. The maximum compressive strain applied to each sample is 0.5% (5,000 μE) generally at 1 Hz, with the capability of using steeper rise times, if desired. These figures translate to a maximum compression, in each sample, of 20 μm, at a rate of 50,000 μE sec −1 . Further, the apparatus applies to the bone sample disks, controlled deformations of 200 nm. The apparatus applies forces of up to 800N, at frequencies in the physiological range, of up to 15 Hz and maximum strain rates of between 10,000 μE sec −1  and 50,000 μE sec −1 . These data are appropriate to samples of spongy mammalian bone in which Young&#39;s modulus varies between 400 MPa and 1200 Mpa.  
           [44]    44. The apparatus of the instant system also provides an environment in which many factors can be investigated. Because whole tissue is used, bone cells can be studied in a near-natural environment of bone matrix and bone marrow. The apparatus provides means for the user to monitor cellular response but additionally and in a novel manner, to monitor the architecture, strain characteristics and strength of the bone disk and changes therein.  
           [45]    45. The bone explant perfusion and mechanical loading apparatus of the present invention preserves the hard matrix of the bone sample and permits the collection of second messengers and growth factors in the perfusion medium. The instant system thus has many of the advantages of cell culture, whilst retaining the bone matrix encountered in vivo.  
           [46]    46. Means provided within the instant system permit recording of changes in the explanted trabecular bone core sample and further permit the calculation of strain, load and Young&#39;s modulus for each such sample. Thus, the instant system permits not only the monitoring of second messengers, cytokines and growth factors but further permits study of how these factors, in conjunction with mechanical loading, will maximize skeletal response to varied stimuli both alone and in combination.  
           [47]    47. In the instant system there are provided perfusion loading apparatus means, power means, control means, computer hardware means, software means and sampling and analysis methods.  
           [48]    48. The perfusion loading apparatus comprises frame means, adjustable biasing pre-loading means, translator loading means, force sensor means and perfusion chamber means. Most components are substantially cylindrical and are accurately machined in corrosion resistant metal, conveniently stainless steel.  
           [49]    49. Frame mounting means are in the form of a relatively massive cylindrical frame housing, comprising a base, a lower frame section, an upper frame section and a cap, each adapted to fit together. These components are secured together with a series, conveniently of 6, partially male-threaded hardened steel bolts which pass through the frame components and are each tightened down with a female-threaded nut. The frame is about 150 mm high and about 80 mm in diameter. The lower part of the frame is substantially solid and has an axial cylindrical hole to accept a ceramic stacked piezo translator which is secured in place by virtue of a close fit in the lower frame and also by screw means through the base.  
           [50]    50. The top part of the frame provides mounting means for adjustable biasing pre-loading means provided by adjustable screw means located axially in and through and the frame cap and secured thereto by threaded means. Within the adjustable biasing pre-loading means there is provided locating and bearing means for force sensor means in the form of an annular quartz crystal force sensor in a precision welded housing.  
           [51]    51. The perfusion chamber assembly is located axially and centrally in the upper section of the frame and comprises a stainless steel bottom bearing cap which provides mounting means for a perfusion chamber body made in durable biologically inert, non-leaching plastics, preferably polycarbonate. A piston, conveniently made in stainless steel, is provided with sealing means in the form of an ‘O’ ring, made from resilient and biologically inert material, preferably neoprene, engages with the upper part of the perfusion chamber body and under the influence of the pre-loading and loading entities, bears down upon a cylindrical explanted trabecular bone sample placed therein. Fluid pathways formed in the perfusion chamber body are disposed so as to ensure that perfusing fluid reaches all parts of the bone sample. Spigots provide connecting means for suitable tube means for delivering perfusing fluid to the assembled perfusion chamber and for collecting effluent from it.  
           [52]    52. The upper and lower components of the perfusion chamber are provided with locating and compression centering means and the assembly is located axially above and upon the translator loading means and directly beneath and in contact with the adjustable pre-load means which drive through push rod and ball bearing coupling means.  
           [53]    53. The piezo translator is provided, via cable connecting means, with a suitable control interface having a microprocessor controlled digital to analogue converter, low voltage driver, controller and power supply, a high voltage amplifier and display unit, all having performance and operating characteristics appropriate to the functional applications of the instant system.  
           [54]    54. The force sensor is provided, via cable connecting means, with a suitable force amplifier having an appropriate power supply and display unit, all having performance and operating characteristics appropriate to the functional applications of the instant system.  
           [55]    55. It will now be apparent that frame means, in co-operation with adjustable biasing pre-load means having force sensor means, translator loading means and perfusion chamber means, as hereinbefore described, constitute perfusion means and instrumented axial press means for the perfusion and mechanical loading of an explanted human or animal bone sample.  
           [56]    56. An explanted trabecular bone sample, prepared as hereinbefore described, is placed within the perfusion chamber, which is then assembled to the frame and loading apparatus. With connections established, power on, and perfusing fluid flowing, the adjustable biasing pre-loading sub-system is adjusted to remove lost motion from and to apply a biasing force to the load train. The biasing force is applied using a large load adjustment knob situated above the frame which drives the adjustable biasing pre-loading means via fine-threaded screw means. A suitable biasing pre-load may also be established using electro-mechanical means via regulator loop means provided in the translator controller. Establishment of a biasing force allows system integrity to be checked. The desired working load or linear translation for the experiment in hand may then be effected using the translator and translator controller.  
           [57]    57. Serial samples of effluent may be collected and assayed for one or more selected factors. Voltage outputs from the translator and charge output from the force sensor are processed and displayed visually. These are used for input to a suitable standard personal computer employing a standard operating system and running a bespoke software program for manipulating data. The program provides software means which produce outputs, via a standard interface, to the system for set-up, configuration, calibration and control of hardware as well as for calculation of relaxation and Young&#39;s modulus. Numerical and graphical results may be output to a suitable monitor and printing device connected to the computer.  
           [58]    58. The instant system allows assessment of bone cellular response to specific stimuli, under controlled conditions. An understanding of these mechanisms will allow their manipulation which may possibly lead to the alleviation or control of osteoporosis and other deleterious skeletal changes. The instant system advances the state art in permitting investigators to study physiological responses of bone tissue under specified conditions. The instant system also advances the state of the art in permitting study of human bone biopsies in a controlled environment. It provides means for identifying morphologic changes occurring in different bone diseases and potentially, for the determination of the physiologic and genetic determinants in such diseases.  
           [59]    59. It is thus a first and most important object of the present invention to provide a novel system for continuous perfusion in conjunction with mechanical loading and for collecting and monitoring second messengers, cytokines and growth factors produced by a viable explanted bone sample in order to study skeletal response to varied stimuli both alone and in combination.  
           [60]    60. It is a second important object of the present invention to provide novel means within the instant system for recording changes in thickness of an explanted bone sample during mechanical loading and further for the calculation of strain, load and Young&#39;s modulus for each such sample.  
           [61]    61. It is a third important object of the present invention to provide novel apparatus means for concurrent perfusion and axial mechanical loading of an explanted sample of mammalian bone, prepared in an appropriate manner, for an extended period during which the bone is to be kept viable.  
           [62]    62. It is a fourth object of the present invention to provide suitable control and recording means for novel apparatus means for concurrent perfusion and axial mechanical loading of an explanted sample of mammalian bone.  
           [63]    63. The instant system will now be described in more detail in conjunction with the following drawings.  
       
    
    
     DESCRIPTION OF THE DRAWINGS  
       [64]    64. In order that the present invention may be more readily understood, reference will now be made to the following drawings in which:  
         [65]    65.FIG. 1, is a diagrammatic front view of the assembled mechanical and electro-mechanical components of a bone explant perfusion and mechanical loading system, according to the present invention.  
         [66]    66.FIG. 2, is a diagrammatic exploded upper perspective axial view of the mechanical and electro-mechanical components of FIG. 1.  
         [67]    67.FIG. 3, is a diagrammatic exploded inverted perspective axial view of the mechanical and electro-mechanical components of FIG. 1.  
         [68]    68.FIG. 4, is a diagrammatic exploded section of the components of the perfusion chamber assembly and a prepared bone sample.  
         [69]    69.FIG. 5, is an underplan view of the perfusion chamber body of the present invention.  
         [70]    70.FIG. 6, is a side section of the assembled components of the perfusion chamber assembly with a prepared trabecular bone sample located therein.  
         [71]    71.FIG. 7, is a schematic diagram of the instant system particularly illustrating electronic control equipment used in conjunction with the electro-mechanical equipment of the present invention.  
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [72]    72. With general reference to FIGS. 1-7, there is described a preferred embodiment of a novel, combined perfusion and mechanical loading system for explanted bone, generally designated by the numeral  10 .  
         [73]    73. Referring to the FIGS. 1-7, there are shown the principal assemblies of a perfusion and mechanical loading system  10 , comprising metal frame housing means  100 , adjustable biasing pre-loading means  400 , translator loading means  600 , force sensor means  700 , perfusion chamber means  800 , and electronic control means  900 .  
         [74]    74. As may best be seen with reference to FIGS. 2 and 3, metal frame housing means  100 , are in the form of a substantially cylindrical and relatively massive metal frame, preferably made from solid stainless steel and comprising several accurately machined parts. In this embodiment, a frame base  102 , is substantially circular, conveniently having a diameter and general thickness of 78 mm and 15 mm, respectively. Additionally, frame base  102 , has a substantially circumferential rim  104 , conveniently 4 mm wide and extending somewhat less than 5 mm above general upper surface  106 , of  102 . A central circular through hole  108 , in frame base  102 , conveniently 4 mm in radius, has a countersunk recess  110 , on the under surface  112 , of  102 . A central circular recess  114 , provided in upper surface  106 , of frame base  102 , is co-axial with central hole  108 . Circular recess  114 , has a radius and depth conveniently of 12.5 mm and 5 mm, respectively. A parallel sided recess  116 , conveniently 9 mm wide, extends from recess  114 , to the edge  118 , of frame base  102 , interrupting rim  104 , thereof. The depth of parallel sided recess  116 , is substantially similar to that of circular recess  114 . A plurality of clearance fixing holes,  120 - 130 , have centers disposed at equal angles around a pitch circle  132 , shown as a center line, conveniently of radius 25 mm and concentric with  108 . Fixing holes,  120 - 130 , are each adapted by the provision of a counter bore  134 - 144 , on under surface  112 , of frame base  102 . Frame base  102 , is provided with a counter bore  146 , disposed in its upper surface  106 , having a diameter and depth, conveniently, of 6 mm and 7 mm, respectively. Counter bore  146 , is centered on a line bisecting the angle between holes  124 ;  126 , substantially midway between pitch circle  132 , and rim  104  of  102 .  
         [75]    75. A lower frame section  148 , is substantially of similar radius to frame base  102 . Lower frame section  148 , conveniently has a height of 61 mm and is provided with substantially circumferential right angled rebates of 5 mm, top and bottom, indicated at  150  and  151 , respectively. A central circular through-hole  152  and a series of fixing holes  154 - 164 , are provided which have substantially similar diameters to and relative spatial dispositions corresponding with  114  and  120 - 130 , respectively, of frame base  102 . Similarly, a parallel sided recess  166  and a counter bore  168 , are provided in the underside  170 , of lower frame section  148 , having dimensions and positions which correspond with  116  and  146  of  102 . Counter bore  168 , is adapted by the provision of female thread means  172 . A locating stud  174 , adapted by the provision of partial male thread means  176 , threadedly engages with the female thread means  174 , of counter bore  168 . Lower rebate  151  and locating stud  174 , of lower frame section  148 , together with rim  104  and counter bore  146 , of frame base  102 , constitute adaptations for mutual location and secure positioning means.  
         [76]    76. An upper frame section  178 , has a height, conveniently, of 65 mm and is of similar radius to  102  and  148 . Additionally and also similarly, upper frame section  178 , is provided with a series of fixing holes  180 - 190 , which have substantially similar diameters to and relative spatial dispositions corresponding to  154 - 164 , of lower frame section  148  and  120 - 130 , of frame base  102 . A central circular hole  192 , has a diameter, conveniently, of 37 mm. Observation means are in the form of a parallel sided fenestration slot  194 , conveniently 30 mm wide, extending from  192 , through wall  196 , of upper frame section  178 , from its upper surface  198 , to a depth, conveniently, of 50 mm. Fenestration slot  194 , is symmetrically disposed about fixing hole  184  and has a deep chamfer  200 , extending out to a width, conveniently, of 53 mm at the outer surface  202 , of  178 . Lower surface  204 , of upper frame section  178 , is adapted by the provision of a machined recess  206 , to form a rim  208 , so dimensioned as to constitute engagement location means for positioning upon upper rebate  150 , of lower frame section  148 . A further parallel sided slot  210 , has substantially the same width and is in a corresponding position to recesses  116 ;  166 , of frame base  102  and lower frame section  148 , respectively. Slot  210 , extends from central hole  192 , through wall  196 , to outer surface  202  and extends to a depth, conveniently of 25 mm, from upper surface  198 , of the  178 . A counter bore  212 , in upper surface  198 , of  178 , is provided with female thread means  214 . A locator stud  216 , is provided with partial male thread means  218 , for threaded engagement with female thread means  214 , of counter bore  212 . Counter bore  212  and locator stud  216 , are of substantially similar dimensions and are similarly spatially disposed to  168  and  174 , of lower frame section  148 . Upper surface  198 , of upper frame section  178 , has a circumferential rebate  220 , substantially similar to  150 , of lower frame section  148 .  
         [77]    77. A frame cap  222 , has a similar diameter and thickness to frame base  102 . Frame cap  222 , is provided with fixing holes  224 - 234 , substantially similar in diameter and disposition to, those of frame base  102 . Fixing holes,  224 - 234 , are each adapted by the provision of a counter bore  236 - 246 , in upper surface  248 , of  222 , substantially similar to  134 - 144 , in  112 , of frame base  102 . Additionally, frame cap  222 , has a rim  250 , disposed circumferentially about its lower surface  252 , substantially similar to rim  104 , of frame base  102 . A counter bore  254 , is adapted and positioned to receive locator stud  216 , of upper frame section  178 . Rim  250  and counter bore  254 , of frame cap  222 , together with rebate  220  and locating stud  216 , of upper frame section  178 , constitute adaptations for mutual location and secure positioning means. A central circular through-hole  256 , adapted by the provision of female thread means  258 , has a diameter, conveniently, of 30 mm. A central circular recess  260 , in upper surface  248 , of frame cap  222 , is conveniently 40 mm in diameter with a filleted circumference to its full depth, conveniently of 4 mm. Rim  250 , is partially interrupted by a shallow cut out  262 , so sized and positioned as to correspond with fenestration slot  194 , of  178 , in order to extend observation means in metal frame housing  100 .  
         [78]    78. Metal frame housing  100 , is assembled by mutually locating and positioning  102 ,  148 ,  178  and  222  and securing them together with mutual threaded securing means. These means are in the form of a series of bolts, preferable made in hardened steel and indicated in FIGS. 2 and 3, by example bolt  264 . Each bolt has a male-threaded portion, shown on  264 , at  266  and extending through corresponding fixing holes  120 - 130 , in  102 ;  154 - 164 , in  148 ;  180 - 190 , in  178  and  224 - 234 , in  222 . Bolt heads, indicated on  262 , by example bolt head  268 , locate in corresponding counter bores  236 - 246  of  222 . Bolts, exemplified by bolt  264 , are each secured by threaded nut means, in the form of nuts, preferably made of hardened steel and indicated in FIGS. 2 and 3, by example nut  270 , such that each is located in corresponding counter bores  236 - 246  of frame cap  222 . Nuts and bolts are evenly tightened using a torque wrench.  
         [79]    79. Adjustable biasing pre-loading means  400 , are in the form of a plurality of substantially cylindrical, accurately machined components preferably made of stainless steel. A housing  402 , is conveniently 37 mm in maximum diameter and of 65 mm height. A lower portion  404 , is conveniently, 15 mm in height with a diameter of 30 mm and is adapted by the provision of male-threaded means  406 , to threadedly engage from above with female thread means  258 , of axial circular hole  256 , of frame cap  222 . An axial lower counter bore  408 , having a diameter conveniently of 20 mm, extends upwards from lower surface  410 , of  402 , conveniently for 30 mm. A through hole  412 , conveniently of 4 mm diameter, extends radially through the wall  414 , of lower male-threaded portion  404 , of  402 , into lower counter bore  408 , thereof and is adapted by the provision of female-threaded means  416 , throughout its length. An axial upper counter bore  418 , having substantially the same diameter as  408 , extends downwards from upper surface  420 , of  402 , for 15 mm. A further and larger axial upper counter bore  422 , in  420 , conveniently having a diameter of 27 mm and a depth of 10 mm, is adapted by the provision of female thread means  424 . Upper counter bores  418 ;  422  and lower counter bore  408 , are united by an axial circular through hole  426 , conveniently 10 mm in diameter.  
         [80]    80. An adjusting axial screw  428 , conveniently has a length of 60 mm. A lower portion  430 , has a diameter conveniently of 10 mm and a length of 25 mm and is adapted by the provision of male thread means  432 , conveniently extending 22 mm, the remaining 3 mm, indicated at  434 , being plain. An upper plain portion  436 , of  428 , conveniently has a diameter of 10 mm and a length of 30 mm. Upper, plain portion  436 , is provided with radial plain blind hole  438 , conveniently situated half way along its length and having a diameter of 4 mm and extending 2 mm in depth. A central plain portion  440 , of  428 , conveniently has a diameter of 20 mm and a length of 5 mm.  
         [81]    81. A locking collar  442 , is conveniently 27 mm in diameter and 10 mm in depth and is adapted on its outer surface  444 , by the provision of male thread means  446 , to provide threaded engagement means for receival by the female-threaded means  424 , of counter bore  422 , of pre-load adjuster housing  402 . Locking collar  442 , is further adapted by the provision of an axial through hole  448 , having a diameter sufficient to provide sliding engagement means for the upper plain portion  436 , of pre-load adjusting axial screw  428 . Upper surface  450 , is provided with a pair of counter bores  452 ;  454 , disposed on the same diameter of  442 , either side of and equidistant from axis  456 . Counter bores  452 ;  454 , each conveniently have a diameter of 3 mm and extend to a depth of 5 mm and constitute tightening drive means for  442 .  
         [82]    82. A knob  458 , conveniently has a diameter of 60 mm and a maximum depth of 20 mm. An upper portion  460 , having the full diameter of  458 , is conveniently 10 mm deep and has a knurled outer surface  462 . A lower portion  464 , conveniently has a diameter of 30 mm. Knob  458 , is adapted by the provision of an axial through hole  466 , having a diameter such as to provide, in co-operation with upper plain portion  436 , of load adjusting axial screw  428 , easy push-fit means. Knob  458 , is further adapted by the provision of a slot  468 , conveniently 12 mm wide and extending through the full depth of upper portion  460 . Slot  468 , has a semi-circular inner margin  470 , adapted by the provision of a small central hole  472 , extending radially into central through hole  466  and is further adapted by the provision of female thread means  474 , for the receival of a grub screw  476 . Grub screw  476 , constitutes engagement locking means between pre-load adjuster knob  458  and pre-load adjusting axial screw  428 .  
         [83]    83. An actuator  478 , has external dimensions such that, in cooperation with lower counter bore  408 , of pre-load adjuster housing  402 , these elements provide fully engageable sliding push-fit means. The upper surface  480 , of  478 , is provided with an axial counter bore  482 , conveniently having a depth of 23 mm, adapted by the provision of internal female thread means  484 , for threaded engagement with lower male-threaded portion  430 , of pre-load adjusting axial screw  428 . Counter bore  482 , is further adapted, at its lower end  486 , by the provision of countersink means, the position of which is indicated by arrow  488 , in FIG. 2. Countersink means  488 , are for the receival of the head  490 , of countersunk screw means  492 , conveniently of 4 mm diameter. Lower surface  494 , of  478 , is provided with a central counter bore  496 , conveniently having a diameter of 6 mm and which communicates with counter bore  482 . External surface  498 , of  478 , is provided with vertical, parallel sided groove  500 , of such a width and depth as to co-operate, in the assembled condition, with a blunt-nosed grub screw  502 , provided with partial male thread means  504 , received threadedly in and through hole  412 , of housing  402 , to provide engagement guiding and rotation restraining means. It will now be understood that with  502 , engaged in  500 , the latter will be prevented from rotating when knob  458 , is used to turn adjusting axial screw  428  and that instead, actuator  478 , will be driven up or down, within lower counter bore  408 , of housing  402 , according to the direction in which knob  458 , is turned. It will be further understood that this arrangement together with the threaded engagement between internal female thread means  484 , of counter bore  482 , of  478 , with male-threaded portion  430 , of pre-load adjusting axial screw  428 , constitute drive means for setting or altering biasing pre-load.  
         [84]    84. A push rod  506 , has an upper portion  508 , so sized as to co-operate, slidingly, both with counter bore  496 , in lower surface  494 , of actuator  478  and also with force sensor  700 , hereinafter described. Upper portion  508 , of  506 , is adapted by the provision of a central counter bore  510 , provided with female thread means  512 , for the threaded receival of countersunk screw means  492 , which is introduced down upper counter bore  482 , actuator  478 . Push rod  506  also has a lower portion  514 , having a greater diameter than  508 . Lower surface  516 , of  514 , is provided with a small peripheral chamfer  518  and is adapted by the provision of a central, substantially hemispherical, recess  520 , providing receival means and compression, locating means for a ball bearing  522 , conveniently having a diameter of 6 mm. Ball bearing  522 , provides part of means for transmitting and centering loads applied to perfusion chamber means  800 , hereinafter described. Upper surface  524 , of lower portion  514 , provides shoulder bearing means for push rod  506 , against force sensor  700 .  
         [85]    85. Translator loading means are in the form of a ceramic stacked piezo translator  600 , incorporating multiple strain gauge means and having a maximum translational range of 40 μm. Piezo translator  600 , may conveniently be a commercial product such as a P-239.30, incorporating an optional module P-177.10, having four strain gauges (Physik Instrumente GmbH, Germany). Piezo translator  600 , is substantially cylindrical in form and has a base portion  602 , for which circular recess  114 , in upper surface  106 , of frame base  102 , provides gentle push-fit location means. Translator base portion  602 , has a central counter bore  604 , which is provided with female thread means  606 . Securing means between  600  and  102 , comprise countersunk male-threaded screw means  608 , having a male-threaded shank  610 . Shank  610 , passes through central hole  108 , in frame base  102  and engages, threadedly, with  606 . Countersunk head  612 , of  608 , is tightened against countersunk recess of  110 , of under surface  112 , of  102 .  
         [86]    86. Piezo translator  600 , has a main body portion  614 , having such a diameter that it engages central hole  152 , of lower frame section  148 , with a sliding push-fit. Main body portion  614 , is of such a height that, when fully engaged in assembled metal frame housing  100 , its upper surface  616 , is substantially level with the bottom of fenestration slot  194 , of upper frame section  178 . Upper surface  616 , of piezo translator  600 , is adapted by the provision of an axial counter bore  618 , adapted by the provision of female thread means  620 , for the threaded receival of a small, substantially cylindrical drive pin  622 , having a shank  624 , provided with male thread means  626 . Drive pin  622 , may conveniently be a commercial product such as a P-239.95 (Physik Instrumente GmbH, Germany) described by the manufacturer as a ‘top piece’. Body portion  628 , of drive pin  622 , constitutes boss mounting means for an upper portion  630 , which is substantially hemispherical and has a diameter conveniently the same as ball bearing  522 , of push rod  506 . It is to be understood that  630 , provides the remainder of means, hereinbefore described with reference to ball bearing  522 , means for transmitting and centering loads applied to perfusion chamber means  800 , hereinafter described.  
         [87]    87. When assembled to metal frame housing  100 , connecting means for piezo translator  600 , in the form of cable means are so disposed as to lie in parallel sided recesses  116 , in upper surface  106 , of frame base  102  and  166 , in lower surface  170 , of  148 , providing aperture means constituting access means for cable means to connectors  632  and  634 . Piezo translator  600 , is provided, via connectors  632  and  634 , with electronic control means  900 , all having performance and operating characteristics appropriate to the functional applications of the instant system best seen and described hereinafter, with reference to FIG. 6.  
         [88]    88. Force sensor means are in the form of a quartz crystal force sensor  700 , housed in an extremely rigid, precision welded, substantially cylindrical housing  702 , having dimensions, conveniently, of outside diameter 14.5 mm, inner diameter 6.5 mm and height 8 mm. Force sensor  700 , may conveniently be a commercial product such as a model 9011A device (Kistler AG, Winterthur, Switzerland). Force sensor  700 , has an axial through hole  704 , for smooth sliding engagement with upper portion  508 , of push rod  506 . In the assembled condition, lower surface  706 , of housing  702 , located on  508 , bears directly upon upper surface  524 , of lower portion  514 , which provides shoulder bearing means for push rod  506 . Upper surface  708 , of  702 , is borne upon by lower surface  494 , of actuator  478 . During use of the instant system, compression of  702 , between  506  and  478 , provides reactive force means for operation of force sensor  700 . Force sensor  700 , is provided with connecting means in the form of cable means which, in the assembled condition, pass through parallel sided slot  210 , of upper frame section  178 . Parallel sided slot  210 , constitutes aperture means in  178 , for access means for cable means to cable connector  710 . Cable connecting means extend from cable connector  710 , to electronic control means  900 , best seen in and hereinafter described with reference to, FIG. 6.  
         [89]    89. Perfusion chamber means  800 , best seen in FIGS. 4, 5 and  6 , comprise three principal, substantially cylindrical components, a bottom bearing cap  802  and a piston  804 , both machined in suitable grades of stainless steel and a perfusion  806 , preferably machined from a block of suitable biologically inert, non-leaching plastics, preferably polycarbonate and provided with connection means for perfusion fluid. The choice of plastics is very important since many materials leach substances which are toxic or lethal to cells. Minimally different embodiments may be made in which  806 , may be made from suitably biologically inert stainless steels. The preferred embodiment confers the advantage, by virtue of fenestration slot  194 , in upper frame section  178 , of observability of perfusion during use of system  10 .  
         [90]    90. Bottom bearing cap  802 , has a lower portion  808 , conveniently having a radius of 25 mm and a depth of 5 mm. Lower surface  810 , of  808 , is adapted by the provision of an axial, substantially hemispherical, recess  812 , providing receival means and compression, locating and centering means for upper hemispherical portion  622 , of drive pin  618 , of piezo translator  600 . Upper portion  814 , of  802 , conveniently has a diameter of 15 mm and a depth of 5 mm. The upper surfaces  816 ;  818 , of  808  and  814 , respectively, are precision ground to flatness and finished by polishing. Upper portion  814 , of  802 , is provided with male thread means  820 .  
         [91]    91. Piston  804 , conveniently has a diameter of 12 mm and a height of 8 mm. Upper surface  822 , of  804 , is adapted by the provision of an axial, substantially hemispherical, recess  824 , providing receival means and compression, locating and centering means for ball bearing  522 , of push rod  506 . Lower surface  826 , of piston  804 , is precision ground to flatness and finished by polishing. Circumferential wall  828 , of  804 , is adapted by the provision of an upper and a lower annular groove, indicated at  830  and  832 , respectively and mutually disposed apart in a parallel manner to upper and lower surfaces  822  and  826 , respectively. Circumferential wall  828 , is finished by micro-fine machining and polishing. Lower annular groove  832 , constitutes an adaptation for the receival of a sealing means in the form of an ‘O’ ring  834 , made from inert resilient sealing material, preferably neoprene.  
         [92]    92. Perfusion chamber body  806 , conveniently has an outer diameter of 25 mm and a height of 15 mm. Lower surface  836 , of  806 , is provided with an axial counter bore  838 , of such a depth and diameter as to provide, in conjunction with suitable female thread means  840 , receival and sealing means for upper portion  814 , of bottom bearing cap  802 . Lower surface  836 , of  806 , is precision ground to flatness and is adapted to cooperate with upper surface  816 , of lower portion  808 , of bottom bearing cap  802  and suitable biologically inert non-leaching adhesive means, to provide additional sealing means between the two components. Lower surface  836 , of  806 , is further adapted by the provision of a machined annular channel  842 , conveniently having a semi-circular cross-section of 2.5 mm diameter and lying on a pitch circle, conveniently 19 mm in diameter and indicated with center line at  844 , in FIG. 5. Two small counter bores  846  and  848 , having the same diameter as  842 , are each centered on the intersection of a diameter of  842  and center line  844 , one either side of central axis  850 . Small counter bore  846 , extends to a depth somewhat less than that of the main lower axial counter bore  838 . A radial hole  852 , having the same diameter as counter bore  846 , extends through wall  854 , of  806 , so as to meet  846 , at right angles, forming substantially continuous lumen means. An upper axial counter bore  856 , is so sized and adapted that it may receive an explanted trabecular bone sample  858 , prepared as hereinafter described, as a sliding fit and also may engage the greater part of piston  804 , as an easy push fit. Lower annular groove  832 , of piston  804  and ‘O’ ring  834 , located therein, are particularly included in the engagement between piston  804  and perfusion chamber body  806 . Inner surface  860 , of upper axial counter bore  856 , is adapted by the provision of a parallel sided, annular channel,  862 , conveniently 4 mm wide and about 2 mm deep. The position of  862 , is such that it substantially surrounds the outer margin or wall  864 , of explanted trabecular bone sample  858 , when this is inserted in  856 , of  806 . Second small counter bore  850 , extends upwards into  806 , to a depth somewhat greater than the depth of main lower axial counter bore  838 , such that it terminates at a point substantially level with the mid point of the height of inserted explanted bone sample  858 . A diameter hole  866 , having the same diameter as small counter bores  846 ;  848 , extends through wall  854 , of  806 , following the line of radial hole  852 , hereinbefore described, intersecting annular channel  862  and also intersecting second small counter bore  848 , at right angles, at the limit of its depth, forming further substantially continuous lumen means. Spigots  868  and  870 , conveniently fabricated in stainless steel, are adapted to engage, respectively, with radial hole  852  and that portion  872 , of diameter hole  866 , which lies on the same side of  806 , as  852 , with a forced, sealing, press-fit. A small cylindrical plug  874 , of the same material as  806 , is adapted to engage with diameter hole  866 , on the opposite side to radial hole  852 , with a press-fit in conjunction with suitable biologically inert, non-leaching, adhesive means to provide sealing means between the two components. Plug  874 , is of such a length that it extends up to but does not substantially encroach into, second small counter bore  848 .  
         [93]    93. Annular channel  842 , of lower surface  836 , of perfusion chamber body  806 , in cooperation with the upper surface  816 , of lower portion  808 , of bottom bearing cap  802  and adhesive sealing means; small counter bores  846  and  848 , annular groove  862 , of upper counter bore  856 , radial hole  852  and plugged portion  876 , of diameter hole  866 , constitute substantially continuous fluid pathway means for perfusing fluid. Spigots  868  and  870 , constitute connecting means for suitable tube means in the form of tubes conveniently made of silicone rubber and indicated at  878  and  880 , for delivering perfusing fluid to the assembled perfusion chamber and for collecting effluent from it for monitoring and analysis.  
         [94]    94. It will now be understood that the substantially cylindrical elements of frame means, in co-operation with adjustable biasing pre-loading means having force sensor means, translator loading means having electronic control means and connection means and perfusion chamber means having connecting means for perfusing fluid, as hereinbefore described, constitute instrumented perfusion and axial press means for the perfusion and mechanical loading of an explanted trabecular bone sample.  
       Performance and Function of Piezo Translator. Force Sensor and Electronic Control Means  
       [95]    95. With particular reference to FIG. 6, as well as continuing reference to FIGS. 2 and 3, electronics control means  900 , comprises a rack  902 , in which are mounted several major components. A 220 V AC power supply  904 , also houses a display module  906 , which gives readings of high voltage or compression values. A high voltage amplifier  908 , provides the high operating voltage (−1000 V) to drive piezo translator  600 . A controller module  910 , includes a compression signal amplifier (not seen) and regulator loop (not seen), to force piezo translator  600 , to a required position, within its translational range of 40 μm, corresponding to a given value of high voltage or compression. This range is satisfactory for applications involving explanted bone samples in the instant invention. Controller module  910 , may conveniently be a commercial product such as E-255 PZT Interface and Controller (Physik Instrumente GmbH, Germany) which incorporates a digital-analogue converter (DAC). Controller module  910 , is linked by cable means (not seen) to low voltage driver and controller  912 , which may conveniently be a commercial product such as LVPZ Driver and Controller E-809 (Physik Instrumente GmbH, Germany). Controller module  910 , is also linked by cable means (not seen) to a force signal amplifier  914 , which is a charge amplifier for amplifying output from force sensor  700 .  
         [96]    96. A personal computer  916 , is equipped with a microprocessor of at least 386 rating and is provided, internally, with an additional plug-in card (not seen) which provides a control interface between an analogue-digital converter (ADC) and DAC of  910 . Cable  918 , connects the additional plug in card of  916 , to a compression signal amplifier output provided on low voltage driver and controller  912 . Cable  920 , connects the additional plug in card of  916 , to force signal amplifier  914 . Cable  922 , connects between a communications port COM- 1  (not seen) of  916 , to the digital-analogue converter of  910 . Cable  924 , connects between a communications port COM- 2 , (not seen) of personal computer  916  and a mouse  926 . Personal computer  916 , is also provided with a local printer terminal port (not shown) for the connection of a suitable printer (not shown). Personal computer  916 , is also equipped with a graphics monitor  928 , functioning to EGA, VGA or higher standard to which it is connected by a monitor cable  930 . A suitable operating system, such as DOS™ 3.2 or higher or Windows 3.1™ or Windows 95™, is installed on personal computer  916 , together with custom software which provides means for coordinating and calibrating the electro-mechanical elements of the system as well as for collecting, collating and displaying data and making calculations thereon and displaying the results thereof.  
         [97]    97. In FIG. 6, frame means  100  and adjustable biasing preloading means  400 , are shown in side view to reveal connecting means for cable means. Cables  932  and  934 , connect high voltage amplifier  908 , to piezo translator  600 , at connectors  632  and  634 , respectively. Cable  936 , connects force sensor  700 , to controller module  910 , at connector  710 .  
         [98]    98. Piezo translator  600 , incorporates four strain gauges (not seen) attached internally to the ceramic stack and arranged in a full Wheatstone bridge circuit. The multiple strain gauge arrangement may conveniently be in the form of an optional commercial module P-177.10 (Physik Instrumente GmbH, Germany). In conjunction with controller module  910 , the bridge arrangement allows a positioning accuracy of 0.2% of the nominal expansion of piezo translator  600 , to be achieved.  
         [99]    99. Force sensor  700 , is a quartz crystal force sensor for measuring dynamic and quasi-static forces, having a range of 15 kN, a very high resolution of 0.01N under any pre-load, sensitivity of z≈ — −4.3pC/N, modulus of 3.6 GPa and very high rigidity of ≈ — −1.8 kN/μm. These characteristics are satisfactory for applications involving explanted bone samples in the instant invention.  
       Experiment 1  
     Calibration And Validation of Loading Elements Of The System  
       [100]    100. The instant system was validated and characterized by the following methods:  
         [101]    101. a. determination of any errors in the system  
         [102]    102. b. identifying deformation accuracy, force application, frequency of loading and calculation of E (Young&#39;s modulus) on known materials and determining the physical compliance in the system.  
         [103]    103. Calibration and validation was accomplished by comparing nondestructive test results of the instant mechanical loading, translator and force sensor elements of the instant system to identical tests run on an MTS (Bionix) servohydraulic test machine. Homogeneous materials, with moduli that span the expected range of cancellous bone, were used (e.g. nylon, aluminum, teflon). These materials had strain gauges applied to the vertical surfaces. Strain was monitored on the same materials in both systems and the results were compared. In addition, a precision extensometer was placed between the platens on the MTS machine to provide specimen deformation, as well as load and thus compute the strain. The current required to achieve similar deformations, strains and loads was recorded. The systems were compared with ramp and sinusoidal wave forms. Hysteresis was noted together with time dependent responses in the materials and test system. The system was the materials and test system. The system was tested quasistatically and at increasing frequencies up to 10 Hz (a functional limit for the MTS system). The system was also tested throughout the range of functional deformation rates available with the piezo crystal translator. Similar specimens were taken to failure and the total material behavior curves of the MTS system and the instant system using the piezo crystal translator, were compared.  
         [104]    104. Correlation of a very high order was established, validating the prospective deployment of the novel mechanical loading system, in conjunction with the novel perfusion means of the instant invention in explanted trabecular bone samples.  
         [105]    105. It was determined that the mechanical and electro-mechanical elements of the instant system are capable of applying controlled deformations, accurate to 200 nm, and applying forces of up to 800 N, at frequencies in the physiological range of up to 15 Hz and maximum strain rates of between 10,000 μE sec-1 and 50,000 μE sec-1. Young&#39;s modulus for trabecular bone varies from E=400 MPa to, typically, E=1200 MPa in the adult pig.  
       Experiment 2  
     Perfusion  
       [106]    106. Preparation of Explanted Bone Samples  
         [107]    107. Features considered when determining the optimal size of the bone sample for the instant system were:  
         [108]    108. 1. The practicality of using cow and pig bone samples in the first instance and the feasibility of using human bone samples, subsequently, having the same dimensions.  
         [109]    109. 2. The volume of bone and means for achieving adequate perfusion through it.  
         [110]    110. 3. The amount of tissue which would be necessary to produce the desired biochemical markers, in quantities sufficient to make the required measurements.  
         [111]    111. The selection of pig and cow trabecular bone was based on earlier studies by the present authors and other workers. In particular the studies of an associate, Dr. Kit Mui Chiu whose observations were recorded in a doctoral thesis at the University of Wisconsin, presented in 1996 and entitled “The effect of camitin dehydroepiandrosterone sulfate on young senescent osteoblast-like cells”, were important. In these studies pig osteoblasts were kept viable, in culture, for 68 days. Careful consideration of these findings and other prior art, led to the conclusion that, in a suitable novel system, providing continuous perfusion means and suitable loading means, viability might be maintained for a worthwhile period of study which could be up to 14 days or more.  
         [112]    112. The bone cores for our experiments were obtained from the trabecular bone of distal ulnae or femurs of 2 to 3 year old cows or femora or humeri of 2 to 3 year old pigs. Under sterile conditions throughout, the limb is first excised and then a 2.5 cm×2.5 cm×4.5 cm (proximo-distal dimension) sample of trabecular bone is cut from the central region of the proximal or distal metaphysis of the bone with a surgical hand saw and the proximal end is marked. The specimen is visually inspected under a dissecting microscope at 10X to assure that no growth plate scars are present.  
         [113]    113. Following isolation of the gross sample, 6×5 mm thick subspecimens are cut from it, under running sterile PBS at room temperature, using a band saw having a diamond tipped blade (Exact, Germany). Six bone core disks are then drilled in the proximo-distal direction, under sterile PBS, from each of the sub-specimens, using a 10 mm or 12 mm diamond tipped keyhole drill (Exact, Germany). The 6 bone cores from each 5 mm sub-specimen are randomized.  
         [114]    114. Each bone core disk is immediately marked on the proximal surface and placed in serum free medium for 20 minutes prior to placing it in the perfusion chamber apparatus of the present invention. Each sample is placed in the perfusion chamber such that it will be loaded from proximal to distal. The sample is then allowed 48 hours in the perfusion chamber in order to adapt, prior to any intervention. Thus, all experiments conducted using this protocol extend over 16 days, comprising 2 days for core adaptation and 14 days of intervention.  
         [115]    115. Using this method, bone disks may be cut with the necessary extreme precision to a flatness of ±0.2 microns and a parallelism of ±0.1 microns. The dimensions were selected in order to produce samples of a practical size for perfusion and in order to supply between 3,500,000-11,000,000 cells, based on an estimate of 10,000-20,000 cells per millimeter cube of bone (Mundy 1990; Parfitt 1983), which was considered sufficient to provide an adequate yield of markers for study.  
         [116]    116. Disk samples of trabecular bone, prepared according to the method immediately hereinbefore described, were perfused and maintained with circulating medium. The medium used was Ham&#39;s F10 containing 1%-5% FCS, 2 mg glutamine, streptomycin and penicillin G at 50,000U/1, vitamin C 10 mg/ml, 0.12 g/l of NaHCO 3  and 10 mM Hepes. The medium was maintained at 37° C. and a pH of 7.1-7.3 for the total 14 days of the perfusion. The perfusion rate was 0.1 ml/minute and the medium was perfused using a 12 channel pump (Ismatec). The medium was changed at 12 hour intervals. The pH, PCO 2  and PO 2  were measured hourly for the first 5 hours then 12 hourly thereafter.  
         [117]    117. A series of FCS batches was tested for biological effect on the trabecular bone cores using alkaline phosphatase, cell viability and osteocalcin production. A sufficient quantity was retained from the most suitable batch of FCS to maintain a reproducible medium for the performance of the experimental program contemplated by the investigators. It is important to note that frozen FCS (−80°C.) has a maximum storage life of 3 years.  
         [118]    118. The flow rate through the explanted bone sample must be fast enough to maintain cell viability but not so fast that a shear force greater than 3 dynes/cm 2  is induced. When the flow rate is too slow, cells are inadequately oxygenated and lactate builds up. When the flow rate is too fast, the shear force, itself, causes increases in PGE2 and IGF-1. The flow rate of 0.1 ml/minute selected was determined as optimal by prior experiment with differing flow rates in order to provide sufficient effluent medium volume for sampling and analysis of PGE2, cAMP and IGF-1 and also in order to maintain PO 2  and PCO 2 . PO 2  was monitored at each flow rate in these experiments to ensure adequate oxygenation of the cells in the bone explant perfusion/loading system.  
       Experiment 3  
     Injury Response Time (Establishment of Rest Period)  
       [119]    119. Cells placed in culture require time to adapt to their changed environment and this time period varied with the type of cell and the type of research we conducted. The necessary rest period for explanted trabecular bone samples in the instant system was determined. In our preliminary experiments, trabecular bone samples were perfused with culture medium plus 10% FCS. Under these conditions, IGF-1 increased from 5 to 14 hours and appeared to decline in the 15th hour, at the time the experiment was terminated.  
         [120]    120. Based on those preliminary data, a rest period of at least 48 hours was accepted as appropriate for IGF-1 to return to baseline level, before any intervention (mechanical loading, hormones, etc) was imposed on the bone explant organ culture. However, the adaptation time required was then documented over a series of full 24 hour periods to determine when the cells had recovered from the surgical trauma in order to determine the stable baseline condition from which intervention could be started.  
         [121]    121. Studies have provided the equivalent data for each of the second messengers, IGF-1 and certain other growth factors.  
       Experiments To Investigate Cell Viability And Biomarkers Under Varying Conditions  
       [122]    122. Experiments were designed to investigate a variety of load magnitudes and frequencies, growth factors and applied active substances.  
         [123]    123. Specifically it was considered necessary to provide for the investigation of markers including the release of prostaglandin E2 (PGE2), cyclic-AMP (cAMP), inositol 1,4,5-trisphosphate (IP3) and insulin-like growth factor (IGF-1), in the perfusion effluent from explanted bone samples. These entities were to be studied during responses to stimuli including varying conditions of mechanical load and further, under the influence of biochemical stimulus with hormones, growth factors or drug substances.  
         [124]    124. The markers, produced by stimuli, immediately hereinbefore described, are important in the regulation of bone modeling and remodeling, at every age and nutritional level, in the adaptive response of the skeleton to such challenges.  
       Cell Viability  
       [125]    125. a. Cell viability in samples of cow trabecular bone was determined at rest, at a maintenance load (the load at which the bone neither atrophies or hypertrophies) and at microstrains which ranged from 500 to 5000.  
         [126]    126. b. Oxygen utilization of the bone explant perfusion/loading model was determined at rest, at varied flow rates, at a maintenance load and at microstrains ranging from 500 to 5000.  
         [127]    127. Having established the flow rate limits for the instant perfusion chamber, experiments were conducted to verify cell viability. Percent viability at various time intervals was assessed in order to determine the number of cells still alive at any given time.  
         [128]    128. Two methods are commonly used to assess cell viability in cell culture. Alamar Blue Assay indicates succinate dehydrogenase activity in the cells. It incorporates an oxidation-reduction indicator that causes the Redox indicator to change from oxidized (non-fluorescent, blue) form to reduced (fluorescent, red) form in response to the cell metabolism in the culture medium. This assay is a general indicator of the metabolic function of the system but it does not allow quantification of cell viability, that is, calculating the percentage and distribution of viable cells. The use of MTT (sigma, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), does permit measurement of the number or percentage of live cells. In this assay, active mitochondrial dehydrogenases convert the water soluble MTT into an insoluble purple formazan by cleaving the tetrazolium ring. Cells with intact mitochondria will show a dark reddish/purple stain when tissue is viewed under a light microscope. Thus, mitochondrial staining is indicative of live cell function at the time MTT is administered. Since MTT is toxic to the cells, it can be used only at the end of an experiment. We used this method to determine the viability of cells after 14 days. At the end of perfusion runs, samples were perfused with MTT (30 mg/ml) for 6 hours then stabilized to 4°and sectioned to between 100 and 180 microns thick, using a diamond saw (EXACT, Germany), in order that viability throughout the sample could be investigated.  
         [129]    129. Somewhat less than 5% of the cells in the cow trabecular bone core, taken from the distal ulnae of 24 month old cows, died because of the surgical extraction and disk preparation procedure and of the remaining cells, more than 95% remained active throughout the 14 day studies.  
       Histological Assessment  
       [130]    130. Before loading the explanted bone samples, it was necessary to verify whole explant tissue viability over time.  
         [131]    131. MTT (30 mg/ml) was used as a cell viability marker. In one series of our cell viability experiments, four 14-day runs were conducted. In the first two runs, sample cores were processed with MTT every two days after a baseline core had been run for 8 hours and then removed. All cores were compared to the baseline core. 12 bone cores were perfused at a rate of 0.1 ml/minute. A baseline positive control viability sample was obtained 8 hours after the start of the experiment by perfusing a core with 30 mg/ml of MTT for 6 hours. The baseline sample and all other samples were perfused with serum free medium for the first 24 hours in order to collect 1 ml of medium for IGF-1 and PGE2 analysis. At the end of the 6 hour perfusion with MTT, the bone core was maintained at 4° C. at which time sections were cut using a diamond band saw. Sample sections were cut to a thickness between 100 and 180 microns in order that cell viability could be determined throughout the sample.  
         [132]    132. The base line sample (8 hours) was used as a positive control for viability. The number of viable cells in the 14 day sample showed no difference when compared to the positive control which had 95% viable cells. The sample sections taken from the top to the bottom of the sample demonstrated no difference in the number of cells showing the presence of MTT and the centers of all of the cores were found to be fully stained. However, there were a few trabecular areas that demonstrated cell death with no MTT present. It was felt that the diamond tipped keyhole drill used to excise the bone samples may have resulted in some damage in the outer few trabecular segments, resulting in tissue damage and cell death. It is clear from our results in this study that the bone cores obtained using this method and using the perfusion chamber apparatus of the present invention, can be maintained in a viable for 14 days.  
       Bioassay  
       [133]    133. Medium from the perfusion chamber to be used for the bioassay was sampled at varied time intervals according to the biomarker we chose to investigate. Pig osteoblasts obtained from Crenshaw (U of WI Madison) were characterized by alkaline phosphatase, collagen type 1 and the ability to produce bone nodules. Cells were plated out in 96-well, Nunclon, cell-culture grade, assay plates at a density of 45,000 cells per cm 2  in 100 ml per well of one of the following media:  
         [134]    134. Dulbecco&#39;s MEM  
         [135]    135. Dulbecco&#39;s BGJ (as used for the organ culture)  
         [136]    136. Ham&#39;s F-10  
         [137]    137. HI growth enhancement medium (Gibco)  
         [138]    138. The specific medium was chosen through trial and error depending on the best response of the markers we investigated (e.g. good for alkaline phosphatase and collagen). To the selected basic medium was added 10% FCS, ascorbic acid-2phosphate at 5 mg/l plus L-glutamine (or the stable analogue) for the first 24 hours. For the assay, the FCS is reduced from 10% to 1%, for 24 hours before the medium is replaced with medium from the perfusion culture. The control is unused medium used for the perfusion culture. Eight replicate wells were used for each sampling point. The cells were grown for 48 hours and then assayed for growth using the MTT method to measure succinate dehydrogenase activity. The MTT methods were calibrated against a known number of cells in a similar growth state; this was a control experiment using an agar plate and counting the cells with a cell counter. The presence of growth factors released from the perfusion culture were then assayed.  
       Loading  
       [139]    139. In loading experiments, the maximum compressive strain applied was 0.5% (5,000 μE) at 1 Hz sine wave. This equates to 20 μm compression at up to 50,000 μE sec −1 .  
         [140]    140. The bone explant perfusion/loading system we have developed has allowed us to assess bone cellular response to specific stimuli under controlled conditions. An understanding of these mechanisms allows for their manipulation and in turn may lead to the possible alleviation or control of osteoporosis and other skeletal changes which result in the loss of skeletal integrity and function. The instant system provides investigators, for the first time, with effective means to study morphological changes in the skeletal tissue. In addition, the instant system permits the study of the physiological responses of the bone tissue under clearly defined and specified experimental conditions that can be set up to reflect the human activities of daily living and life style. The present invention also for the first time, permits the study of human bone biopsies in a controlled environment. This will not only enable investigators to identify morphologic changes that occur with different bone disease but will also permit the determination of the physiologic and possibly genetic determinants in such conditions.  
         [141]    141. It will be apparent to those skilled in the art that numerous modifications or changes may be made without departing from the spirit or the scope of either the present invention or its method of use. Thus the invention is only limited by the following claims.