Abstract:
A method and apparatus for stimulating the growth of chondrocytes as part of a bone healing process includes a loudspeaker that applies audio frequency acoustic energy to the cells. A 1 kHz square wave at a 20 percent duty cycle is used and it is applied for a period of 20 minutes on each of a series of consecutive day.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application claims the benefit of U.S. Provisional patent application Ser. No. 60/669,116 filed on Apr. 7, 2005 and entitled “Sonic Activation Of Strain Sensitive Cells”. 
     
    
     BACKGROUND OF THE INVENTION  
       [0002]     The invention generally relates to the field of stimulating tissue growth and healing, and more particularly to apparatus and methods for stimulating chondrocytes that lead to accelerated healing of bone fractures.  
         [0003]     When tissues in a human body such as connective tissues, ligaments, bones, etc. are damaged they require time to heal. Some tissues, such as a bone fracture in a human body, require relatively longer periods of time to heal. The healing process for a bone fracture in the human body may take several weeks and may vary depending upon the location of the bone fracture, the age of the patient, the overall general health of the patient, and other factors that are patient-dependent. Depending upon the location of the fracture, the area of the bone fracture, the patient may have to be immobilized to encourage complete healing of the bone fracture. Immobilization of the patient for extended periods of time may have other adverse health consequences.  
         [0004]     Promoting bone growth is important in treating bone fractures, and it is important in the successful implantation of medical prostheses, such as those commonly known as “artificial” hips, knees, vertebral discs, and the like, where it is desired to promote bony ingrowth into the surface of the prosthesis to stabilize and secure it. Numerous techniques have been developed to promote healing of bone fractures. For example, it has been proposed to treat bone fractures by application of electrical voltage or current signals (e.g., U.S. Pat. Nos. 4,105,017; 4,266,532; 4,266,533, or 4,315,503). It has also been proposed to apply magnetic fields to stimulate healing of bone fractures (e.g., U.S. Pat. No. 3,890,953). Application of ultrasound to promoting tissue growth has also been disclosed (e.g., U.S. Pat. No. 4,530,360).  
         [0005]     It has been shown that a 1.5 MHz ultrasound signal, consisting of a 200 μs tone burst repeating at 1 kHz intervals, can stimulate chondrocytes and lead to accelerated bone fracture healing. Double-blind placebo-controlled clinical studies have shown that such pulsed ultrasound exposure is able to shorten the time to normal bone strength in both radius and tibial fractures. In vitro, such ultrasound exposure increased aggrecan mRNA and proteoglycan synthesis in chondrocytes. In animal studies, the same ultrasound exposure invariably increased mRNA expression from fracture callus. In addition, histological analysis of the fracture callus showed increased cartilage area. These findings suggest that pulsed ultrasound may have an effect on chondrocytes and may be able to modulate chondrogenesis and, following bone formation, these effects may eventually develop bone union at the fracture gap.  
         [0006]     An ultrasonic device is available that exploits this treatment method. This device uses a 1.5 MHz ultrasound carrier signal having a 200 μs tone burst repeating at 1 kHz intervals for treating fractures 20 minutes per day. This is an in vitro device which consists of a frame holding 6 transducers, one under each well of a 6-well plate. Ultrasound gel is placed between the transducers and the plate. This device has demonstrated increased proteoglycan synthesis in chondrocytes. A disadvantage, of the ultrasound treatment method, however, is the effect of heating. It has been found that after 20 minutes of treatment with the device, there is a 2-3° C. rise in temperature of the media.  
       SUMMARY OF THE INVENTION  
       [0007]     The present invention is a method and apparatus for stimulating the growth of chondrocytes as part of the bone healing process. More specifically, it has been discovered that growth of chondrocytes is stimulated by the periodic treatment with 1 kHz sound waves with no resulting increase in temperature. Preferably, the waveform of the applied sound waves is not sinusoidal such that higher frequency harmonics are also produced and applied during treatment.  
         [0008]     A general object of the invention is to provide a method and an in vitro apparatus that stimulates chondrocytes and causes them to produce extracellular matrix which leads to accelerated bone fracture healing. By administering a treatment with 1 kHz sound each day for a number of days, a highly significant increase in chondrogenesis occurs.  
         [0009]     Another object of the invention is to stimulate bone fracture healing without generating heat and with an inexpensive apparatus. The apparatus needed to practice the present method is little more than a loudspeaker driven by a 1 kHz signal source. The resulting audio frequency pressure waves produced in the treated bone do not produce any significant heating. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0010]      FIG. 1  is a top view of the treatment tray used in the preferred embodiment of the invention;  
         [0011]      FIG. 2  is a view in cross-section taken along the plane  2 - 2  indicated in  FIG. 1   
         [0012]      FIG. 3  is a circuit diagram of speakers used in the treatment tray of  FIG. 1 ;  
         [0013]      FIG. 4  is a graph indicating the results of treatment using the present invention in terms of number of nodules grown; and  
         [0014]      FIG. 5  is a graph indicating the results of treatment using the present invention in terms of nodule size. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0015]     Referring particularly to  FIG. 1 , the treatment tray is formed around a molded plastic, 6-well cell culture plate  10 . The culture plate  10  includes six circular recesses, or wells  12  in its top surface. The dimensions of the 6-well plate  10  are 12.8 cm×8.6 cm×2 cm. Each well  12  has a surface area of 9.6 cm 2 . As shown best in  FIG. 2 , a maintenance medium  20  is disposed in each of the wells  12  and the cells to be treated  22  are disposed on the bottom of each well  12 .  
         [0016]     The cell culture plate  10  is stacked on top of an identical plate  10 ′ with the six wells  12  in the culture plate  10  aligned directly above corresponding wells  12  in the lower plate  10 ′. Six loudspeakers  14  are mounted in the respective six wells  12  of the lower plate  10 . The speakers  14  face upward and are bonded to the bottom surface of each well  12 . An enclosed air space is formed between each speaker  14  and the bottom wall of the well  12  directly above it. As a result, the acoustic energy produced by the loudspeakers  14  is efficiently coupled to the well bottom walls and the cells  22  which they support. The stacked cell plates  10  and  10 ′ enable the cells  22  and medium  20  to be easily removed and then reinstalled in the exact same alignment with the loudspeakers  14 . The speakers  14  are commercially available from Panasonic as the model EAS2P104H. The active surface area of each speaker is 6.2 cm 2 .  
         [0017]     As shown in  FIG. 3 , the speakers  14  are connected in series-parallel and driven by a function generator  18 . The speakers  14  are driven with a 1 kHz square wave at 18 mV peak to peak with a 20% duty cycle. A scanning laser vibrometer was used to examine the motion produced by each speaker  14 , and the bottom of each well  12  was found to move an average of 1 nm with a drum-like motion, where the center of the well  12  moves more than the periphery. For the 1 kHz apparatus, the voltage used to drive the speakers was adjusted to give an average displacement of 2 nm. A square wave was used because the harmonics it produces enhances a drum-like motion of each well bottom.  
         [0018]     As shown in  FIG. 2 , the cells to be treated  22  are plated in the wells  12  such that they are disposed directly above one of the speakers  14 . ATDC5 cells, a chondrogenic clonal cell line, were cultured in a maintenance medium  20  consisting of a mixture of Dulbecco&#39;s modified Eagle medium and Ham&#39;s F-12 medium, supplemented with 5% fetal bovine serum, 1% penicillin-streptomycin, 10 μg/ml human transferrin, and 3×10 −8  M sodium selenite. ATDC5 cells are chondrocyte precursors, which can be differentiated into chondrocytes with the addition of insulin. ATDC5 cells that do not receive insulin remain chondrocyte precursors. Cells were maintained at 37° C. in a humidified atmosphere of 5% CO 2  in air. The cells were allowed to remain in culture for three days before sonic treatments.  
         [0019]     Starting the third day after plating the cells  22 , sound treatments were administered for 20 minutes each day for 11 days. Variations of this regimen are possible (i.e., starting treatments 5 or 7 days after plating and treating for 7 or 9 days, etc.), but this regimen is preferred. The treatments were performed in a 37° C. incubator. Each well  12  of the six-well plate  10  had 3 ml media and the media was changed every other day during the treatment process.  
         [0020]     Several treatment regimens have been tried, but the regimen of 3 days plated and 11 days of ultrasound treatments gave the best response to 1 kHz acoustic energy. There were 6 treatment plates in this experiment. Each treatment plate received 1 kHz squarewave, 20% duty cycle for 20 minutes per day. The treatments were performed in a 37° C. incubator. Treatment regimen varied for each plate, in order to determine the effect of start time of treatments and the number of treatments received. The treatment regimens were as follows: 
        14 days total in culture: 8 days plated, then 6 days of 1 kHz treatments     17 days total in culture: 5 days plated, then 12 days of 1 kHz treatments 8 days plated, then 9 days of 1 kHz treatments 11 days plated, then 6 days of 1 kHz treatments     20 days total in culture: 8 days plated, then 12 days of 1 kHz treatments.        
 
         [0024]     Each plate was observed under the microscope every day over the course of the experiment. During the process of chondrogenesis, chondrocytes produce extracellular matrix proteins, including proteoglycan and collagen II. These proteins condense to form nodules. The earliest day that nodules of cartilage were observed in the control plates was the sixteenth day after plating. On the other hand, all of the treated plates except for one (5 days plated, 12 days of treatments) had nodules visible on the eleventh day. This suggests treatment with 1 kHz vibration accelerated the date of visible formation of cartilage nodules. We found similar results in quantitative optical spectrometry. In previous experiments, Wang, S-J., D. G. Lewallen, M. E. Bolander, E. Y. S. Chao, and J. F. Greenleaf: Low Intensity Ultrasound Treatment Increases Strength In A Rat Femur Fracture Model, Journal of Orthopaedic Research 12(1):4047, 1994; Yang, K-H., J. Parvizi, S-Y. Wang, D. G. Lewallen, R. R. Kinnick, J. F. Greenleaf, and M. E. Bolander: Exposure To Low-Intensity Ultrasound Increases Aggrecan Gene Expression In A Rat Femur Fracture Model, Journal of Orthopaedic Research 14(5):802-809, 1996; and Parvizi, J., C-C. Wu, D. G. Lewallen, J. F. Greenleaf, and M. E. Bolander: Low Intensity Ultrasound Stimulates Proteoglycan Synthesis In Rat Chondrocytes By Increasing Aggrecan Gene Expression, Journal of Orthopaedic Research 17(4):488-494, 1999, the acceleration of aggrecan production or collagen production as we see with the 1 kHz treatment was associated with accelerated bone fracture healing.  
         [0025]     The treatment regimen strongly affected the number and size of nodules. Referring to  FIGS. 4 and 5 , when treatments were begun on the same day, more treatments led to an increased number and size of nodules. Average nodule size refers to the average number of pixels for one nodule. A larger number of nodules corresponds to a larger number of differentiation events, indicating that 1 kHz vibration increased differentiation of ATDC5 clonal chondrogenic cells. A larger area of nodules corresponds to increased proliferation, indicating that 1 kHz vibration not only increases differentiation but also proliferation of ATDC5 cells.  
         [0026]     Results show that 1 kHz vibration induces chondrogenesis as much as 1.5 MHz pulsed ultrasound in ATDC5 clonal chondrogenic cells, but without the generation of heat and resulting temperature increase. Experiments focusing on 1 kHz treatments show that 1 kHz vibration not only increases chondrogenesis but also increases differentiation and proliferation of ATDC5 cells.