Abstract:
A device including a pressure generating assembly and a chamber for generating hydrostatic pressure in the said chamber. In particular, the device is useful for cell mechanical stimulation and tissue regeneration by generating hydrostatic pressure in a chamber receiving samples such as cell cultures, biological tissues, and cell seeded biomaterial constructs for the purpose of treatment with hydrostatic pressure. The pressure generating assembly of the device comprises a piston and a cylinder generating and relieving pressure by moving the piston in the cylinder using an actuating system for quick placement and removal of the piston in and out of the cylinder. The device is portable and autoclavable and can be hand operated without any tool to generate pressures at high physiological levels up to at least 10 MPa. The device has a venting system allowing vital gasses to reach the samples under treatment when the device is placed in an incubator.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims an invention which was disclosed in Provisional Application No. 61/684,729, filed on 18 Aug. 2012, entitled “Hydrostatic Pressure Generator Device”. The benefit under 35 USC §119(e) of the U.S. provisional application is hereby claimed, and the aforementioned application is hereby incorporated herein by reference. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     Not Applicable 
     REFERENCE TO SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM LISTING COMPACT DISK APPENDIX 
     Not Applicable 
     BACKGROUND 
     1. Field of the Invention 
     The invention relates to a device comprising a pressure generating assembly and a chamber for generating hydrostatic pressure in the said chamber, more particularly, a portable and autoclavable device for generating hydrostatic pressure in a fluid filled chamber receiving samples such as cell cultures, biological tissues, and cell seeded biomaterial constructs for the purpose of treatment with hydrostatic pressure. 
     2. Description of the Prior Art 
     Musculoskeletal disorders such as age-related degenerative changes of cartilage, intervertebral disc, collagen and bone contribute to some of the most common causes of impairment and disability for middle-aged and older persons, such as back pain, osteoarthritis, and degenerative joint and bone diseases. With an increasing aging population, it is therefore important to have a means for regeneration of articular cartilage, intervertebral discs as well as collagen and bone remodeling. Musculoskeletal disorders are multifactorial phenomenon and both mechanical and biological factors have been implicated in cases of accelerated degeneration. 
     During normal daily activities, the cartilage cells, the chondrocytes, in the cartilage of a diarthrotic joint experience levels of hydrostatic pressures in the order of 7 to 10 MPa (Hall et al., 1996) and increased cartilage thickness occurs in joint regions exposed to high intermittent hydrostatic stress (Wong et al., 1990). The intervertebral disc is routinely subjected to compressive loads that alter with posture and muscle activity and can produce pressures greater than 2 MPa in human lumbar discs in vivo (Wilke et al., 1999). 
     It has been shown by many studies that mechanical stimulation such as hydrostatic pressure is a major factor in maintaining load bearing tissues by influencing different biological factors at cellular level. Different studies have confirmed that hydrostatic pressure influences cellular response such a synthesis rate and increased collagen secretion by cells in major load bearing tissues such as cartilage (Smith et al., 2000) and intervertebral disc (Kasra et al. 2001, 3003, 2006). 
     In mechanical stimulation of intervertebral disc cells, Kasra et al. (2003) showed that within physiological levels of hydrostatic pressure up to 5 MPa, the magnitude of hydrostatic pressure was the dominant factor, and the higher the pressure the higher was the rate of protein synthesis by cells. The regenerative advantage of intermittent hydrostatic pressure at physiological levels 5-10 MPa applied to cartilage cells in vitro is also shown in U.S. Pat. Application Publication No. 20030133915A1. These studies suggest that for cell and tissue mechanical stimulation purposes related to tissue regeneration and in vitro studies of cell cultures, using hydrostatic pressures at high physiological levels of up to 10 MPa would be beneficial. 
     In most of research labs and commercial systems, hydrostatic pressure has usually been generated in an incubator by pressurizing the incubator gas in a vessel using a compressor. In an ordinary laboratory environment by pressurizing a gas in an incubator, it can be difficult to generate a high hydrostatic pressure, more than 1 MPa, and loading frequency is usually low and less than 1 Hz. For generating high hydrostatic pressures at high frequencies, Kasra et al. (2001) introduced a system for in vitro studies of cell cultures using a fluid filled cylindrical chamber receiving a cell culture and a piston moving in the said chamber to generate pressure, subjecting the cell culture to dynamic hydrostatic pressure. This system was operated by a servo-hydraulic external actuator and could generate pressures up to 5 MPa and 20 Hz frequencies. This method has been used in other studies such as the study reported by Le Maitre et al. (2009) related to therapy of intervertebral disc degeneration. A similar method was used in U.S. Pat. Appl. Pub. No. 20030133915A1 using a hydraulic loading instrument in fluid communication with the pressurizing chamber which could generate a dynamic hydrostatic pressure of 10 MPa at 1 Hz frequency. These fluid filled chambers operated by hydraulic systems are expensive, have a large size, not portable, and having difficulty of keeping a sanitary environment. 
     The concept of using a fluid filled cylindrical chamber receiving a biological tissue and a piston moving in the said chamber as reported by Kasra et al. (2001, 2003, 2005) was also used for generating very high static hydrostatic pressures up to 200 MPa for cryopreservation of cells and tissues described in U.S. Pat. Appl. Pub. No. 20070087321A1. 
     Ease of operation is also a major factor which has not been a priority in designing of the aforementioned hydrostatic pressure devices. For example, in U.S. Pat. Appl. Pub. No. 20070087321A1, the pressurizing chamber has two openings, one receiving a pressurizing piston and the other a pressure gauge. The pressure chamber is equipped with a cap for retaining and moving the piston inside the chamber by attaching the cap to the chamber using multiple screws. For relieving pressure in the chamber either the pressure gauge or the piston has to be removed. Removing and reinstalling the pressure gauge is not practical for multiple uses, especially if the chamber is of plastic type material, compromising the pressure gauge sealing. It is also time consuming to adjust the piston for inserting and removing the piston in and out of the chamber by screwing and unscrewing multiple screws on the cap. The system described in U.S. Pat. Appl. Pub. No. 20070087321A1 does not have any venting system and cannot be placed in an incubator and therefore not suitable for treating cell cultures in an incubator. 
     In most of the hydrostatic pressure devices, including the device of the U.S. Pat. Appl. Pub. No. 20070087321A1, a piston is driven directly into a pressurizing chamber containing the sample to be treated. Having the piston and the chamber of the same diameter has the disadvantage of having a limitation on the size of the chamber if the device is to be hand operated. The required load for pushing the piston in the chamber increases with the square power of the inner chamber diameter and as the inner chamber diameter increases, it increasingly becomes more difficult to operate the device by hand without any tool to generate a high hydrostatic pressure at 10 MPa level. 
     Sealing method is also a very important factor in the design of hydrostatic pressure devices, and it can influence the range of motion of the piston in the chamber and holding time of the pressure in the chamber as well as maintaining sealing quality after autoclaving. But there is hardly any details on sealing methods used in the disclosure of the aforementioned hydrostatic pressure devices. 
     Considering the advancement of the field of tissue regeneration and increasing use of hydrostatic pressure for cell and tissue mechanical stimulation, there is a need for a light, portable and easy to operate hydrostatic pressure device which can generate pressures at high physiological levels up to at least 10 MPa. This device needs to be autoclavable, quick and easy to operate by hand without any tool, and have a venting system which allows the device to be placed in an incubator without having to remove the samples from the chamber after each treatment. The ease of use and affordability of such a device is expected to provide most of laboratories with the opportunity of having an important tool for performing research in an important area of tissue regeneration and studying catabolic and anabolic responses of different cells to hydrostatic mechanical stimulation. 
     SUMMARY OF THE INVENTION 
     It is the object of the present invention to provide a device comprising a pressure generating assembly and a chamber for generating hydrostatic pressure in the said chamber. The pressure generating assembly is preferably a separate part and can be connected to the chamber. The pressure generating assembly comprises means such as a piston and a cylindrical passageway defining a cylinder, which can be placed in connection with the chamber for generating and relieving pressure in the chamber by pushing and pulling the piston in the cylinder. The terms pressure and hydrostatic pressure are used interchangeably. 
     The chamber and the cylinder can be filled with a fluid and a gas, generating higher pressure with more fluid volume and having the maximum pressure when the chamber and the cylinder are fully filled with a fluid. 
     The systems, devices and methods of the invention are particularly suited for use in treatment of cell cultures, biological tissues, and cell seeded biomaterial constructs in a sterile condition for the purpose of treatment with the hydrostatic pressure preferably at physiological levels up to at least 10 MPa. 
     The system and method of the invention provide many advantages and new features including the following:
         (1) The device is light and portable.   (2) Depending on its size, the device can be operated by hand without any tool generating hydrostatic pressures up to at least 10 MPa, or it can be operated by hand using a tool such as a wrench. The device can also be operated using an external actuator for applying hydrostatic pressures within a wide range of amplitudes and frequencies.   (3) The device is autoclavable and can be used in a sterile condition.   (4) The device comprises a venting system allowing the vital gases to reach the device chamber when placed in an incubator.   (5) The pressure generating assembly of the device comprises self adjusting means for quick placement and removal of its piston in and out of its cylinder for generating and relieving pressure in the device chamber, making the use of the device very quick and easy.   (6) Having the device with its pressure generating assembly as a separate part from its chamber, the piston and cylinder of the pressure generating assembly can have a smaller cross-sectional area than that of the chamber cavity where the pressure is generated. Having a a smaller piston cross-sectional area than that of the chamber cavity, the device can generate pressures with much less effort compared to a device with equal piston and chamber cavity cross-sectional areas.       

     In a preferred embodiment, the device comprising:
         (a) A pressure generating assembly comprising a main body, an actuating body, a piston and its sealing member such as an oring, a central stud, and a shield.       

     The main body comprises a cavity at its top and a cylindrical passageway, namely cylinder, at its lower part extending between the main body top cavity and the exterior. There are a plurality of openings in the wall of the main body extending between the main body top cavity and exterior for venting purposes. 
     The actuating body comprises an internal passageway for receiving the piston and the central stud. The central stud can be threadebly mounted in the passageway of the actuating body and used for restraining the head of the piston in the passageway. The actuating body moves threadebly inside the main body pushing and pulling the piston in the cylinder of the main body for generating pressure. 
     The shield is placed on the main body in front of the venting openings of the main body to prevent the direct flow of air or gas into the device and reduce the chance of contamination.
         (b) A chamber comprising of a cavity (chamber cavity) wherein a sample is received and pressure is generated and an opening (chamber opening) connecting the chamber cavity to the exterior of the chamber. The main body of the pressure generating assembly can be threadebly mounted and sealed to the chamber opening using a sealing member. After mounting the main body of the pressure generating assembly at the chamber opening, the cylinder of the main body will be in connection with the chamber cavity and by moving the piston in the cylinder, pressure is generated in the chamber cavity. The chamber cavity comprises another opening for receiving a pressure gauge and its sealing member for monitoring the generated pressure. The seal type of connections of the pressure generating assembly and the pressure gauge to the chamber is preferably a face seal comprising preferably of an oring as the sealing member placed in a gland preferably in a shape of a half groove.       

     In another embodiment of the present invention, there is no cylinder in the main body and the piston moves directly in the chamber cavity to generate pressure. 
     Still in another embodiment of the present invention, when a hydrostatic pressure generator device is mainly operated by an external actuator, there is no central stud and the piston passes through and outside the internal passageway of the actuating body for actuation thereof by an external actuator, and a breading passageway connects the main body top cavity to the cylinder of the main body allowing passage of air or gas into chamber during dynamic movement of the piston. 
     Still yet in another embodiment of the present invention, a hydrostatic pressure generator device is used for generating negative pressure, lower than atmospheric pressure, by suction, and the main body comprises a bleeding passageway connecting the chamber cavity to the outside of the chamber allowing passage of air or a gas out of the chamber cavity when pushing the piston in the cylinder of the main body, and after closing and sealing the passageway with a nut and a sealing member, the suction is generated by pulling the piston out of the cylinder. 
     Still yet in another embodiment of the present invention, the main body is mounted and fixed in the chamber opening using a plurality of screws, and the central stud is mounted and fixed in the internal passageway of the actuating body using a pin. 
     In the described embodiments of the present invention, the hydrostatic pressure generator device may be used without any pressure gauge and instead the pressure can be calculated by measuring the applied load or torque on the actuating body using a formula relating the applied load or torque to the pressure. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       A fuller understanding of the nature and objects of the present invention will become apparent upon consideration of the following detailed description taken in connection with the accompanying drawings, wherein: 
         FIG. 1  is a cross-section view of a preferable embodiment of the hydrostatic pressure generator device in pressurizing position; 
         FIG. 2  is a cross-section view of the same embodiment of the hydrostatic pressure generator device shown in  FIG. 1  in venting position; 
         FIG. 3  is a cross-section view of another embodiment of the hydrostatic pressure generator device wherein the piston moves directly in the chamber cavity; 
         FIG. 4  is a cross-section view of another embodiment of the hydrostatic pressure generator device which is mainly used to operate with an external actuator; 
         FIG. 5  is a cross-section view of another embodiment of the hydrostatic pressure generator device for generating negative pressure, lower than atmospheric pressure, by suction; and 
         FIG. 6  is a cross-section view of another embodiment of the hydrostatic pressure generator device showing alternative methods for mounting the main body of the pressure generating assembly to the chamber using a plurality of screws, and the central stud in the actuating body using a pin. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The invention will now be described in detail with reference to the accompanying drawings. 
       FIGS. 1 and 2 , show a hydrostatic pressure generator device  10  according to an embodiment of the present invention. As hereinafter described, the hydrostatic pressure generator device  10  is operated between a pressurizing position and a retracting or venting position. The pressurizing position is shown in  FIG. 1  and the venting position is shown in  FIG. 2 . 
     In accordance with a preferred embodiment illustrated in  FIGS. 1 and 2 , hereinafter referred to as the first embodiment, the hydrostatic pressure generator device  10  comprises a pressure generating assembly  12  and a chamber  14 . The pressure generating assembly  12  comprises a main body  16 , an actuating body  18 , a piston  20  and its sealing member  22  such as an oring, a central stud  24  such as a bolt, and a shield  26  in front of a plurality of venting openings  28  in the wall of the main body  16  with a gap  30  preferably of about 1-2 mm. 
     The chamber  14  comprises of a chamber cavity  32  and a chamber opening  34  located at the bottom and top parts of the chamber  14  respectively. The chamber cavity  32  receiving materials to be exposed to hydrostatic pressure including a sample, a fluid, and air, having a higher maximum pressure with a higher fluid to air volume ratio. Preferably, the sample placed in the chamber cavity  32  can be a biological tissue or a cell culture dish and the liquid can be a cell culture media. The chamber opening  34  has a threaded wall and is where the main body  16  of the pressure generating assembly  12  is mounted and sealed to the chamber  14  using a sealing member  36 , such as an oring, that sits in a half grove on a step  38  above the chamber cavity  32 . A step  40  above the step  38  makes the sealing surface for a face seal between the main body  16  of the pressure generating assembly  12  and the chamber  14 . 
     Main body  16  comprises a cylindrical passageway defining a cylinder  42  at its lower part and a cavity  44  (main body cavity) with an opening on top. The cylinder  42  extends between the main body cavity  44  and the exterior of the main body  16 . The main body  16  is threaded internally on the wall  46  of the main body cavity  44  as well as externally on its outer surface  48 . 
     The pressure generating assembly  12  can be threadebly mounted and sealed to the chamber  14  by turning the main body  16  into the chamber opening  34 . There is preferably a smooth surface  50  with a reduced section at the lower end of the main body  16  that allows the full travel of the main body  16  into the chamber opening  34  until it pushes against the step  40  and the sealing member  36  making a sealed connection. 
     At its upper end, the piston  20  has a flange  52 , with a head  54 , preferably rounded, and a step  56 . The flange  52  is loosely secured in the actuating body  18  for actuation thereof, and the lower end  58  of the piston  20  moves in the cylinder  42  of the main body  16  to generate pressure. 
     Through the actuating body  18  there is an internal passageway  60  which is threaded on its upper part  62  and having a step  64  at its lower end above which the piston flange  52  is fitted for actuation thereof. The actuating body  18  is threaded on its outer surface  66  and moves threadably inside the cavity  44  of the main body  16 . 
     The actuating body  18  provides an actuating means for holding and moving the piston  20 . The piston  20  can move down and up in the cylinder  42  of the main body  16  by screwing and unscrewing rotation of the actuating body  18  in and out of the main body  16 , which pushes and pulls the piston  20  in the cylinder  42 . There is a taper  68  at the top of the cylinder  42  to provide a smooth entry of the piston  20  into the cylinder  42 . 
     The central stud  24  can be screwed in the threaded portion  62  of the internal passageway  60  of the actuating body  18 . The lower wall  70  of the central stud  24  can touch the head  54  of the flange  52  of the piston  20  by screwing the actuating body  18  into the main body  16  pushing the piston  20  into the cylinder  42  of the main body  16  generating pressure in the chamber cavity  32 . 
     Self adjusting means for entering the piston  20  inside the cylinder  42  is provided by having the flange  52 , with the rounded head  54 , loosely secured above the step  64  in the actuating body  18 , and having the taper  68  at the top of the cylinder  42 , which allows a smooth entry of the piston  20  into the cylinder  42 , prevents jamming of the piston  20  inside the cylinder  42 , and improves sealing performance of the sealing member  22  between the piston  20  and the cylinder  42 . 
     The amount of pressure generated in the chamber cavity  32  can be monitored by using a pressure monitoring device  72 , preferably an autoclavable or a sanitary pressure gauge. The term pressure gauge represents any pressure monitoring device such as a mechanical pressure gauge as well as different types of pressure transducers, pressure sensors, and pressure transmitters. 
     The pressure gauge  72  is connected to the chamber  14  via a passageway including two sections of an outer hole  74  and an inner hole  76 . The outer hole  74  is threaded receiving the pressure gauge  72 , and the inner hole  76  connects the outer hole  74  to the chamber cavity  32 . 
     There are two steps  78  and  80  between the outer hole  74  and inner hole  76  providing a sealing surface and a half groove gland respectively for a sealing member  82  such as an oring. The pressure gauge  72  can be threadably mounted in the outer hole  74 . There is preferably a clearance groove or undercut  84  at the end of threaded portion of the outer hole  74  before the step  78  allowing the full travel of the stud  86  of the pressure gauge  72  to the end of the outer hole  74  pushing against the step  78  and sealing member  82  making a face seal between the pressure gauge  72  and the chamber  14 . 
     As shown in  FIG. 2 , by unscrewing the actuating body  18  out of the main body  16 , the wall of the step  64  of actuating body  18  can touch the wall of the step  56  of the piston  20 , pulling the piston  20  out of the cylinder  42  of the main body  16  relieving pressure in the chamber cavity  32 . 
     The actuating body  18  can be unscrewed and moved upward in the main body  16  until it uncovers the venting openings  28  from inside of the main body  16  in the cavity  44 . With the venting openings  28  uncovered and the piston  20  out of the cylinder  42 , the vital gases required for survival of cells or biological tissues placed in the chamber cavity  32  can then be provided when the assembly is placed in an incubator. To reduce the chance of contamination, the shield  26  is placed in front of the venting openings  28  with a gap  30  preferably of about 1-2 mm. The shield  26  can be threaded on its inner surface  88  and installed threadably on the main body  16  in front of the venting openings  28 . In an alternative method, instead of the shield  26 , filters may be installed in the venting openings  28 . 
     In operation, referring to  FIG. 1 , a sample to be treated with the hydrostatic pressure is placed in the chamber cavity  32 , then the main body  16  is mounted and sealed in the chamber opening  34 . Then, from the top opening of the cylinder  42  in the main body  16 , the chamber cavity  32  and the cylinder  42  can be filled with a fluid, such as a cell culture media. Finally, the actuating body  18  is mounted in the cavity  44  of the main body  16  and hydrostatic pressure is generated in the chamber cavity  32  by rotating and screwing the actuating body  18  in the main body  16  as described. The chamber cavity  32  and the cylinder  42  can be filled with fluid and air, generating higher pressure with more fluid volume and having the maximum pressure when the chamber cavity  32  and the cylinder  42  are fully filled with liquid. 
     Depending on the size of the piston  20 , hydrostatic pressure magnitude, and static or dynamic choice of hydrostatic pressure, rotating action for screwing and unscrewing of the actuating body  18  into and out of the main body  16  can be done by hand without any tool, by hand with a tool such as a wrench, or by using a coupling mechanism to a rotary actuator. For turning and holding purposes, handles may be installed on the actuating body  18 , the main body  16 , and the chamber  14 . 
     A prototype of the first embodiment shown in  FIGS. 1 and 2  was made and tested successfully in all of its operating procedure as described. Using the prototype with a piston of 8 mm, pressures up to 10 MPa could easily be generated by rotating the actuating body  18  simply by hand and without any tools. 
     In a hydrostatic pressure generator device, such as the hydrostatic pressure generator device  10 , other sealing systems for sealing of the pressure gauge  72  and the main body  16  to the chamber  14  may be used. For example, a gasket or an oring with different gland types can be used to seal the main body  16  or the pressure gauge  72  to the chamber  14 , or a Teflon tape can be used to seal the pressure gauge  72  to the chamber  14 . However, the sealing system shown in  FIGS. 1 and 2  is preferable. 
     Preferably, the chamber  14 , the chamber cavity  32 , the chamber opening  34 , the actuating body  18 , and the main body  16  have cylindrical shapes as shown in  FIGS. 1 and 2 . The size of the hydrostatic pressure generator device can vary according to the size of its chamber cavity  32  which may be a miniature chamber to hold a small piece of a tissue, a small chamber to hold one culture dish, or a large chamber to hold a cell culture plate. The preferable sizes for the chamber cavity  32  are those which can receive a small commercially available single culture dish such as a 35 mm diameter cell culture dish or single cell culture dish inserts of diameters equal or less than 30 mm, plus 2-5 mm extra space around and on top of a dish for handling. The dishes can also be placed in the chamber cavity  32  in a stack and the height of the chamber cavity  32  can be adjusted according to the number of dishes in the stack. 
     The construction details of the invention, such as the hydrostatic pressure generator device  10  shown in  FIGS. 1 and 2 , are that the hydrostatic pressure generator device  10  preferably is made from materials which are autoclavable, resistant to corrosion, and do not cause any destructive reaction with the cell culture media. The preferable materials are polycarbonate, polyamide, Teflon, and stainless steel. The materials should also be sufficiently rigid and strong to hold a hydrostatic pressure of interest according to the size of the hydrostatic pressure generator device  10 . Stainless steel is preferable for miniature sizes which strength is of prime importance. Polyamide and Teflon may be used for large sizes when having a low weight becomes important. More preferably polycarbonate and still more preferably a combination of polycarbonate and stainless steel may be used for having an optimum strength and size with a low weight. 
     Reference will now be made to  FIG. 3  which shows another embodiment of the present invention, generally denoted by the reference  90 , comprising a pressure generating assembly  92  and a chamber  94 . In  FIG. 3 , for simplicity and brevity, like components are given the same reference numeral as in the first embodiment shown in  FIGS. 1 and 2 , and the description is not repeated. In the embodiment of  FIG. 3 , the chamber  94  and a main body  96  respectively replace the chamber  14  and main body  16  of the first embodiment shown in  FIGS. 1 and 2 . 
     In the embodiment of  FIG. 3 , the main body  96  includes a threaded passageway  98  (main body passageway) without any cylinder at its lower part, and the piston  20  moves directly in the chamber cavity  32  to generate pressure. There is a taper  100  at the top of the chamber cavity  32  to provide a smooth entry of the piston  20  into the chamber cavity  32 . The pressure generating assembly  92  can be mounted to the chamber  94  by screwing the main body  96  into the chamber opening  34  without using any sealing. In this embodiment, the main body  96  can be a separate part and to be connected to the chamber  94  as described and shown in  FIG. 3 , or the main body  96  can be integral with the chamber  14  wherein the main body  96  is an extension of the chamber opening  34 . For generating pressures up to 10 MPa by hand without any tools, this embodiment is preferable for a small hydrostatic pressure generator device with a cylindrical chamber cavity  32  of a diameter of less than 10 mm and more preferably less than 8 mm. 
     Reference will now be made to  FIG. 4  which shows another embodiment of the present invention, when a hydrostatic pressure generator device, generally denoted by the reference  102 , is mainly operated by an external actuator. The hydrostatic pressure generator device  102  comprises a pressure generating assembly  104  and the chamber  14 . In  FIG. 4 , for simplicity and brevity, like components are given the same reference numeral as in the first embodiment shown in  FIGS. 1 and 2 , and the description is not repeated. In the embodiment of  FIG. 4 , a main body  106 , an actuating body  108 , and a piston  110  respectively replace the main body  16 , the actuating body  18 , and the piston  20  of the first embodiment shown in  FIGS. 1 and 2 . 
     In more detail, still referring to the invention of  FIG. 4 , the actuating body  108  comprises a central passageway  112  with a smooth surface throughout its length wherein the piston  110  passes through and outside the actuating body  108 . The piston  110  comprises a head  114  which can be engaged with an external actuator. There is a flange  116  on the piston  110  with an upper surface  118  which is located under the lower surface  120  of the actuating body  108 . By screwing the actuating body  108  into the main body  106 , the lower surface  120  of actuating body  108  touches the upper surface  118  of the piston flange  116  pushing the piston  110  in the cylinder  42  of the main body  106  generating a hydrostatic pressure as a baseline static hydrostatic pressure preload. From the baseline pressure, using an external actuator, applying a static or dynamic load at the piston head  114  can then generate a static or dynamic hydrostatic pressure. 
     In further detail, still referring to the invention of  FIG. 4 , there is a passageway, which maybe in a form of a vertical hole  122  connected to a horizontal hole  124 , connecting the cavity  44  of the main body  106  to the inside of the cylinder  42  at its upper end allowing passage of air or incubator gas for breathing during dynamic movement of the piston  110 . This embodiment can also work independent of an external actuator for generating hydrostatic pressure as described for generating a static hydrostatic pressure preload. Unloading of the hydrostatic pressure can be done by means pulling the piston  110  out of the cylinder  42  such as by unscrewing the actuating body  108  out of the main body  106  until it pushes against a pin  126  which can be fit in a hole  128  at the top of the piston  110  and pulling the piston  110  out of the cylinder  42  of the main body  106 . 
     Reference will now be made to  FIG. 5  which shows another embodiment of the present invention, when a hydrostatic pressure generator device generally denoted by the reference  130 , is used for generating negative pressure, lower than atmospheric pressure, by suction. The hydrostatic pressure generator device  130  comprises a pressure generating assembly  132  and a chamber  134 . In  FIG. 5 , for simplicity and brevity, like components are given the same reference numeral as in the first embodiment shown in  FIGS. 1 and 2 , and the description is not repeated. In the embodiment of  FIG. 5 , a main body  136  and the chamber  134  respectively replace the main body  16  and the chamber  14  of the first embodiment shown in  FIGS. 1 and 2 , and a nut  138  with its sealing assembly is added. 
     In more detail, still referring to the invention of  FIG. 5 , the sealing systems of the main body  136  and the pressure gauge  72  to the chamber  134  are preferably face seals with groove glands suitable for a negative pressure in the chamber cavity  32 . The sealing of the main body  136  to the chamber  134  includes the sealing member  36 , such as an oring, fitted in a groove  140  in the step  142  at the top of the chamber cavity  32 . The sealing of the pressure gauge  72  to the chamber  134  includes the sealing member  82 , such as an oring, fitted in the groove  144  in a step  146  between the outer hole  76  and the inner hole  74 . In the main body  136 , there is a bleeding passageway which may consist of a vertical section  148  and a horizontal section  150  connecting the chamber cavity  32  to the outside of the chamber  134  through the lower part of main body  136 . The horizontal section  150  can be sealed with a face seal assembly consisting of the nut  138  and a sealing member  152  such as an oring, sitting in a groove  154 . 
     In the embodiment of  FIG. 5 , the negative pressure in the chamber cavity  32  can be generated by removing the nut  138  from the horizontal section  150 , then screwing the actuating body  18  into the main body  136  pushing the piston  20  into the cylinder  42  of the main body  136  until the fluid in the chamber rises up to the horizontal section  150 , then screwing back the nut  138  in the horizontal section  150  and seal the passage, and finally generating negative pressure by unscrewing the actuating body  18  out of the main body  136  and pulling the piston  20  out of the cylinder  42 . 
     Reference will now be made to  FIG. 6  which shows another embodiment of the present invention generally denoted by the reference  156  comprising a pressure generating assembly  158  and a chamber  160 . In  FIG. 6 , for simplicity and brevity, like components are given the same reference numeral as in the first embodiment shown in  FIGS. 1 and 2 , and the description is not repeated. In the embodiment of  FIG. 6 , the chamber  160 , a main body  162 , an actuating body  164 , and a central stud  166  respectively replace the chamber  14 , main body  16 , the actuating body  18 , and the central stud  24  of the first embodiment shown in  FIGS. 1 and 2 . 
     In  FIG. 6  an alternative connecting means to that of the first embodiment shown in  FIGS. 1 and 2 , for connecting the main body  162  to the chamber  160  is shown, which uses a plurality of screws  168  to attach the main body  162  to the chamber  160 . The main body  162  comprises a lower part  170  with a smooth surface which fits in the chamber opening  172  which also have a smooth surface. There is a flange  174  on the main body  162  with a plurality of openings  176  where the screws  168  can pass. On the top edge surface of the chamber  160 , there are plurality of threaded holes  178  matching the treads of screws  168 . The screws  168  can pass through the openings  176  of the flange  174  and be screwed in the threaded holes  178  in the chamber  160  connecting the main body  162  into chamber  160  pushing the lower surface of the main body  162  against the step  40  and the sealing member  36  sealing the connection. 
     Still referring to  FIG. 6 , in case of using a material such as Teflon which may not be strong enough for the threaded holes  178  to hold the screws  168 , the holes  178  in the chamber  160  may be extended as a passageway to the of the chamber  160  where the screws  168  with a long stem can pass and be fixed with a bolt at the bottom from outside of the chamber  160 . The main body  162  is preferably used with the chamber cavity  32  of a diameter preferably larger than 40 mm. The main body  164  can also be used with the actuating body  18  and the central stud  24  of the first embodiment shown in  FIGS. 1 and 2 . 
     In the embodiment of  FIG. 6 , the central stud  166  has the same function as the central stud  24  of the first embodiment, shown in  FIGS. 1 and 2 , in restraining the flange  52  of the piston  20 . The central stud  166  comprises a smooth surface  180  and a hole  182  across its upper end. The actuating body  164  also comprises a smooth passageway  184  with a hole  186  at its upper end. The central stud  166  can fit in the passageway  184  and fixed with a pin  188  which passes through the holes  182  and  186 . The pin  188  can also be used as a handle for rotating the actuating body  164  pushing the piston  20  in the cylinder  42  of the main body  162  generating pressure. 
     Referring to  FIG. 6 , the actuating body  164  operated with the pin  188  as a handle is preferably used with the piston  20  of a diameter of less than 8 mm so that it could be rotated by hand to generate a hydrostatic pressure of at least up to 10 MPa. 
     The actuating body  164  with the stud  166 , shown in  FIG. 6 , can also be used with the main body  16  and the chamber  14  of the first embodiment, shown in  FIGS. 1 and 2 . 
     In the embodiment of  FIG. 6 , the cross sectional area of the chamber cavity  32  can have any shape which preferably matches the shape of the main body  162  cross sectional area. For example, both the chamber cavity  32  and the main body  162  preferably have the same shape of either circular or rectangular sections, but a circular section with a cylindrical chamber is preferable. 
     In the described embodiments, a hydrostatic pressure generator device, such as the hydrostatic pressure generator device  10  shown in  FIGS. 1 and 2  may also be used without any pressure gauge  72  and instead the pressure can be calculated by measuring the applied torque on the actuating body  18  using a formula relating the applied torque to the pressure. Similarly, a hydrostatic pressure generator device, such as the hydrostatic pressure generator device  102  shown in  FIG. 4 , may also be used without any pressure gauge  72  and instead the pressure can be calculated by measuring the axial load on the piston  110  using a formula relating the axial load to the pressure. 
     While the foregoing written description of the invention enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The invention should therefore not be limited by the above described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention as claimed. 
     REFERENCES 
     US Patent Documents 
     20030133915 A1 7/2003 Smith et al. 
     20070087321 A1 4/2007 Pribenszky and Molnar 
     Other Publications 
     Kasra et al., Abstract of a publication entitled “Effect of dynamic hydrostatic loading on rabbit disc cells”, American Society of Mechanical Engineers, Bioengineering Division (Publication) BED, 50:191-192, 2001. 
     Kasra et al., “Effect of Dynamic Hydrostatic Pressure on intervertebral disc cells: A rabbit model” Journal of Orthopaedic Research, 21:597-603, 2003. 
     Kasra et al., “Frequency response of pig intervertebral disc cells subjected to dynamic hydrostatic pressure”, Journal of Orthopaedic Research., 24(10):1967-1973, 2006. 
     Smith et al., “Time-dependent effects of intermittent hydrostatic pressure on articular chondrocyte type II collagen and aggrecan mRNA expression” Journal of Rehabilitation Research and Development, 37:153-161, 2000. 
     Hall et al., “The cellular physiology of articular cartilage”, J. Exp. Physiol, 81:535-545, 1996. 
     Wong M, Carter D R. Theoretical stress analysis of organ culture osteogenesis. Bone, 11:127-31, 1990 
     Wilke et al., “New in vivo measurements of pressures in the intervertebral disc in daily life” Spine, 24:755-762, 1999. 
     Le Maitre et al., “Development of an in vitro model to test the efficacy of novel therapies for IVD degeneration” J Tissue Eng Regen Med. 3:461-469, 2009.