TW201740959A - A pharmaceutical composition for increasing skeletal density and the using method thereof - Google Patents

Abstract

The present invention is related to a pharmaceutical composition for increasing skeletal density, which comprises a platelet rich fibrin (PRF) and an adipose tissue stem cells (ADSC) wherein the ADSC is adipose tissue mesenchymal stem cells.

Description

Pharmaceutical composition for increasing bone density and method of using same

The present invention relates to a pharmaceutical composition for increasing bone density and a method of using the same, and more particularly to a pharmaceutical composition comprising Platelet Rich Fibrin (PRF) and adipose mesenchymal stem cells (ADSC), which can be used to increase Bone density for the prevention and treatment of arthritis.

According to the National Nutrition and Health Change Survey Report (NAHSIT 2004-2008), the prevalence rate of osteoporosis in women over 50 years old in Taiwan is 10.7%, and that in women is 12.1%. If at least one part of the lumbar vertebrae, femoral neck and forearm meet osteoporosis The diagnosis of the disease is defined as the prevalence rate of 22.57% for men and 41.17% for women. If the hidden population is taken into account, the data published in the National Nutritional Health Change Survey Report is still underestimated. The actual number of people suffering from osteoporosis will be more than the number listed in the statistics.

Osteoporosis is a progressive disease characterized by abnormal mass and quantity of bone tissue, which in turn leads to impaired bone strength and increased risk of fracture. (Matsumoto et al., 2011) Although the detailed pathological mechanism needs further research, bone can be roughly Osteoporosis is attributed to a decrease in the secretion of estrogen, which causes a sharp imbalance in the balance between the original osteoblasts and osteoclasts, resulting in the occurrence of osteoporosis.

At present, the treatment of osteoporosis mainly focuses on reducing the rate of bone loss or promoting the formation of new bone. The drugs used include bisphosphonate, calcitonin, estrogen and hormone replacement therapy (HRT). Although these drugs are still used clinically to prevent or treat osteoporosis, they often have large side effects. For example, HRT can prevent osteoporosis, but these drugs can stimulate endometrial hyperplasia and increase the risk of endometrial cancer or breast cancer. Therefore, there is an urgent need to develop alternative methods for preventing and treating osteoporosis.

In view of the above, the present invention provides a pharmaceutical composition for increasing bone density, comprising a Platelet Rich Fibrin (PRF) and a fat stem cell.

The invention also provides a pharmaceutical composition for use in the manufacture of increased bone density, wherein the pharmaceutical composition comprises a Platelet Rich Fibrin (PRF) and a fat stem cell.

Preferably, the adipose stem cells are Adipose tissue stem cells (ADSC).

Preferably, the growth factor concentration in the platelet-rich cellulose is Platelet-derived growth factor-AB (PDGF-AB) 1738.7±582.24 pg/mL, and the transforming growth factor beta 1 (TGF) 405.7±82.9 pg/mL. Epidermal growth factor (EGF) 17.5±7.5pg/mL, Basic fibroblast growth factor (b-FGF) 7.6±3.2pg/mL, Nerve growth factor (NGF) 4.0±1.1pg/mL, Insulin-like growth factor-1 (IGF-1) 787.4±119.6pg/mL, Vascular endothelial growth factor (VEGF) 92.0±13.9pg /mL.

Preferably, the number of cells of the tissue stem cells is 4.8-5.0 x ^{10 5} live cells/mL.

SUMMARY OF THE INVENTION The Summary of the Invention is intended to provide a simplified summary of the disclosure of the invention. The basic spirit and other objects of the present invention, as well as the technical means and implementations of the present invention, can be readily understood by those skilled in the art.

1 is an observation result of body temperature change of each group of mice after injection according to an embodiment of the present invention; FIG. 2 is a result of bone density of each group according to an embodiment of the present invention; and FIG. 3 is a percentage of bone mass of each group of tibia bone according to an embodiment of the present invention. Figure 4 is a set of trabecular spacing of each group according to an embodiment of the present invention; Figure 5 is a number of trabecular bones of each group according to an embodiment of the present invention; Figure 6 is a serum calcium concentration of each group according to an embodiment of the present invention; Each group of H&E slice staining maps of the embodiments of the present invention.

In order to make the description of the present invention more detailed and complete, the following description of the embodiments of the present invention and the specific embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In addition, the singular noun used in this specification covers the plural of the noun in the case of no conflict with the context; the plural noun of the noun is also included in the plural noun used.

In this specification, the growth factor concentration in "Platelet Rich Fibrin (PRF)" is Platelet-derived growth factor-AB (PDGF-AB) 1738.7 ± 582.24 pg/mL, Transforming growth factor beta 1 ( TGF) 405.7±82.9pg/mL, Epidermal growth factor (EGF) 17.5±7.5pg/mL, Basic fibroblast growth factor (b-FGF) 7.6±3.2pg/mL, Nerve growth factor (NGF) 4.0±1.1pg/mL Insulin-like growth factor-1 (IGF-1) 787.4±119.6 pg/mL, Vascular endothelial growth factor (VEGF) 92.0±13.9 pg/mL.

In the present specification, "fatty stem cells" are Adipose tissue stem cells (ADSC). The number of cells was 4.8-5.0 x ^{10 5} live cells/mL.

The term "solution" is used in this specification to define its broad meaning, including the ingredients dissolved in a liquid and the ingredients suspended in a liquid.

The term "treating" includes a symptom, a secondary disorder, or a condition that partially or completely prevents, ameliorates, and/or treats osteoarthritis, which is promoted by Regeneration of cartilage and/or hard bone tissue to benefit individuals suffering from or suspected of having related signs, diseases or symptoms. The term "treating" as used in this specification also refers to a stent system to which the present invention is applied or administered to a body having associated symptoms, minor symptoms or symptoms of osteoarthritis to achieve partial or Completely reduce, slow, cure, delay the onset, inhibit the progression of the disease, reduce the severity of the disease, and/or reduce the occurrence of symptoms, symptoms, or secondary symptoms associated with one or more osteoarthritis. Symptoms, secondary signs and/or symptoms associated with osteoarthritis include, but are not limited to, pain, swelling, reduced range of activity, and inability to self-activity. Here, "treating" may also be administered to an individual having these early signs or symptoms to reduce the risk of the individual developing into a related symptom, secondary symptom and/or symptom of osteoarthritis. Here, "treating" is effective in reducing one or more signs or clinical markers. In other words, the treatment may also be to reduce, slow or terminate the progression of the disease course, symptoms or symptoms.

The term "subject" means an animal comprising humans which is capable of receiving the treatment of the stent system of the present invention in accordance with the teachings of the present invention. Unless specifically stated otherwise, the term "subject" means both male and female, and may be of any age, such as a child or an adult.

The present invention provides a pharmaceutical composition comprising Platelet Rich Fibrin (PRF) and adipose mesenchymal stem cells (ADSC), which can be used to increase bone density and prevent and treat osteoporosis.

The following examples are intended to be illustrative, and are not intended to limit the scope of the invention.

Example 1 Preparation of Platelet-rich Fibrin (PRF)

1.1 rabbit blood extraction

Before the blood is drawn, the rabbit should be mixed with Zoletil® (Zoletil® 50, Virbac, France) 50 mg/ml and Xylazine (Balanzine®, public medicine, Taiwan) 20 mg/ml 1:1, at 0.25 ml/ A dose of kg was administered intramuscularly for general anesthesia. The hair near the neck is then shaved and the neck skin is wiped from the inside out with 10% iodine and 75% alcohol for complete disinfection.

After disinfection, you can use a 10ml syringe with a 22G needle (DVR-3422, Terumo co, Philippines) without anticoagulant Under the conditions, fresh blood was collected from the external jugular vein of the rabbit neck.

1.2 Platelet-rich fibrin preparation

The freshly extracted rabbit blood was immediately transferred to an 8.5 ml BD Vacutainer® blood collection tube (REF 367988, BD Biosciences, USA) containing a clot activator. Since no anticoagulant was added, the blood collection tube was gently shaken by hand during the process. To avoid blood clotting. Finally, 500g of blood collection tube containing rabbit blood was centrifuged for 10 minutes. The platelet-rich fibrin in the middle of the blood collection tube was a mixture of red blood cells and other agglomerates and the top platelet-rich plasma PPP (Platelet Rich Plasma) was called PRF (Platelet Rich Fibrin). .

1.3 Platelet-rich fibrin release solution

The blood collection tube filled with platelet-rich fibrin is made to be sterilized with alcohol, and then the gelatin-rich fibrin and the gel at the bottom of the tube are separated in an aseptic workstation. The platelet-rich fibrin is then transferred to a clean and sterile centrifuge tube and placed at room temperature for 5 hours. The liquid released during the process is free of fibrin and is called a platelet-rich fibrin release solution. Store in a freezer at -20 °C.

Example 2 Fat extraction and stem cell culture

2.1 Mouse fat extraction

Select healthy mice without any obvious trauma, using Zoletil® (Zoletil® 50, Virbac, France) 50 mg/ml and Xylazine (Balanzine®, public drug, Taiwan) 20 mg/ml 1:1 mixed mixture, each intramuscularly injected at a dose of 0.25 ml/kg, and the mice were anesthetized. The hair of the mouse's abdomen was then shaved and the skin was wiped from the inside out with 10% iodine and 75% alcohol for complete disinfection.

The sterilized mice were subjected to fat extraction in a sterile environment. The abdomen of the mouse was first opened, and the adipose tissue was grasped in the pre-cooled PBS, and then the adipose tissue was taken from the abdominal cavity of the mouse and washed three times with PBS to remove excess substances such as blood. Subsequently, collagen type I was added, and after shaking for one hour at room temperature, it was centrifuged at 500 x g for 10 minutes. The precipitate was again dispersed and filtered through a 100 μm nylon mesh to remove residual bulk tissue pieces, and mixed with PBS and centrifuged at 500 g for 10 minutes. Finally, the supernatant was removed and the bottom cells were taken for cell culture. The cells taken out were primary bone marrow stem cells, and cell culture was carried out using α-MEM culture medium containing 10% fetal bovine serum and 1X three-in-one antibiotic. After the cells are cultured to a total of more than 1×10 ⁶ cells, the cell surface marker antibody can be prepared for flow cytometry screening.

2.2 Purification of adipose stem cells

After 1×10 ⁶ cells were thoroughly diluted with 1×0.25% Trypsin-0.02% EDTA, they were collected in a 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 minutes. After centrifugation, the supernatant in the centrifuge tube was removed, and the cell pellet was dispersed using 1 ml of pre-cooled PBS to completely clean the cells. After washing, it was centrifuged at 1000 rpm for 5 minutes to completely remove the supernatant in the tube.

After confirming that the cells are completely cleaned, the cells should be fixed with alcohol. The cells are dispersed by using 1 ml of PBS before fixation to prevent the cells from agglomerating when the alcohol is fixed. Then, 3 ml of 70% pre-cooled EtOH was slowly added, placed at -20 ° C for 1 hour or placed in a 4 ° C environment for 1 day, and centrifuged at 1000 rpm for 5 minutes. After clearing the supernatant, add 5 ml of PBS, and repeat the action once to clean the cells.

Then prepare 1:100 iso ab mouse anti human CD34 (BD553731, BD Biosciences, USA), CD45 (AB10558, abcam, USA), CD44 (AB119335, abcam, USA), CD73 (BD550738, BD Biosciences, USA) and CD90 ( BD554895, BD Biosciences, USA) and corresponding antibodies used to test antibody potency 1: 100 iso ab mouse IgG CD34 (BD553927, BD Biosciences, USA), CD45 (AB172730, abcam, USA), CD44 (BD553927, BD Biosciences, USA), CD73 (BD553927, BD Biosciences, USA) and CD90 (BD555746, BD Biosciences, USA), and then add 30 ul of antibody to 1 ml of cell solution, respectively, and then use 1 ml of PBS to break up the cell mass in the centrifuge tube. And placed in a 4 ° C environment for 1.5 hours. During the waiting period, 1:100 PE goat anti mouse ab (BD550589, BD Biosciences, USA) was prepared in the dark, and 30 ul to 1 ml of the cell solution was added and allowed to act for 30 minutes. Waiting After completion, the cells were centrifuged at 3000 rpm for 10 minutes and the supernatant was removed. Finally, after dispersing the cell pellets with 0.5 ml of PBS, it was ready to be screened using a flow cytometer (FACScan, Becton Dickinson, USA).

The surface antigens on the mesenchymal stem cells were identified by primary antibodies such as CD34, CD45, CD44, and CD90, and the primary antibody was labeled with the secondary antibody FITC and PI fluorescence, and then analyzed and purified by flow cytometry. The purified cells showed positive CD44 and CD90, and the fluorescence intensity was 48.6% and 99.6%, respectively. CD34 and CD45 showed negative reactions, and the fluorescence intensity was 0.5% and 2.0%, respectively. All are below 5%.

After the cells were screened by flow cytometry, a 15 ml centrifuge tube containing 10 ml of α-MEM culture solution was prepared in advance, and the cells containing the CD90 antibody were screened by flow cytometry, and the α-MEM culture solution contained 10% of the fetus. Cell culture was carried out with bovine serum and 1X 3-in-1 antibiotic.

2.3 Adipose stem cell culture and cell counting

After confirming the cell culture to the required amount under the microscope, and the growth pattern was normal, the culture solution in the culture dish was extracted by a vacuum aspirator in an aseptic workstation, and 5 ml of PBS solution was added thereto, and uniformly shaken. To clean the entire Petri dish, and finally use a vacuum aspirator to remove the PBS waste.

Then add 1 ml of 0.25% Trypsin-0.02% EDTA, and shake evenly to make the cells in the dish reach 1 time 0.25% Trypsin-0.02% EDTA, then put the dish back to 37 ° C, 5% CO ₂ culture In the box. After 1~2 minutes, observe under the microscope, the cells will appear round and granular when separated from the culture dish. At this time, it is necessary to add an equal volume of serum-containing α-MEM culture solution to stop the 1-fold 0.25% Trypsin-0.02% EDTA reaction to avoid The cells are damaged. Then, the culture medium containing the cells was collected in a centrifuge tube, and uniformly suspended by a motorized pipette, and 100 μl of the cell liquid was mixed with an equal volume of 0.5% Trypan blue, and then dropped on a blood cell counting plate and counted under a microscope. . After about 1-2 weeks, after the cells are full or the number is 1×10 ⁶ cells/ml, the adipose stem cells can be completely collected and stored in liquid nitrogen.

Example 3 animal experiment

3.1 Experimental grouping

The mice required for the experiment were 30 mice in 5 groups. 16-week-old ICR mice were randomly assigned to the non-surgical group, the sham-assisted sham-operated group (SHAM), and the ovarian-extraction group. The non-surgical group was used as the normal control group, while the ovarian-extracted group received the mice. PRF release solution (PRF-IV), adipose stem cells (ADSC-IV), adipocyte + PRF release solution (ADSC-PRF-IV), adipocyte + PRF release solution + intraosseous injection (ADSC-PRF-IV-i) And do not receive any treatment (Neg. Control), in order to explore whether the ovarian removal surgery process will affect the bone growth of the mouse, so the back muscles of the mouse are opened, the incision is not sutured without ovarian removal surgery, for ovarian removal mock surgical group. Each group of mice After 8 weeks of treatment, the systemic blood was collected and centrifuged to separate the serum, and the leg bones were removed and fixed with 10% neutral fumarin.

3.2 Mouse ovarian ablation surgery

Sixteen-week-old mice, which were fasted for about 8 to 12 hours, were first taken out of the cage to observe the presence or absence of abnormalities or obvious trauma. The weight measurement was then determined on the scale. Thereafter, Zoletil® (Zoletil® 50, Virbac, France) 50 mg/ml and Xylazine (Balanzine®, public drug, Taiwan) 20 mg/ml were mixed 1:1 and then anesthetized by intramuscular injection for ovarian ablation.

The mouse was placed on the operating table with the tail facing the operator and covered with a sterile hole. The scalpel opened a 3 cm long wound on the back of the mouse in the middle of the mouse, moved to the muscle layer above the left kidney, and opened the ovary and fallopian tube with the axillary forceps. After the scalpel was burned red, the ovary was removed. The oviduct was cut at the junction of the fallopian tube. After no continuous bleeding, the remaining part of the fallopian tube was placed in place, and the muscle layer was sutured by continuous suture using a 3-0 suture.

After the left ovary is removed, the back skin gap can be moved to the muscle layer above the right kidney, and the ovary and fallopian tube can be clipped with the axillary forceps, and the ovary can be cut by the red scalpel from the ovarian posterior fallopian tube. After no continuous bleeding, the remaining part of the fallopian tube was returned, and the muscle layer was sutured by continuous suture method using 3-0 suture. Finally, the back skin gap was closed by intermittent suture method, and the iodine was inserted. And dressing the wound, that is, completing the mouse ovarian ablation operation.

3.3 mouse ovary removal sham surgery

After about 8 to 12 hours of fasting in about 16 weeks old mice, the anesthesia dose was anesthetized by intramuscular injection. After about 8 to 12 hours, 16-week-old mice were caught from the cage and observed for abnormalities or obvious. Trauma, followed by placing a weight on the scale has determined the amount of anesthetic. Thereafter, it was anesthetized by intramuscular injection of Zoletil® (Zoletil® 50, Virbac, France) 50 mg/ml and Xylazine (Balanzine®, public drug, Taiwan) 20 mg/ml recommended by the American Society of Laboratory Animals.

The mouse was placed on the operating table with the tail facing the operator and covered with a sterile hole. Use a scalpel to open a 3 cm long wound on the back of the mouse in the middle of the mouse, move it to the muscle layer above the left kidney, see the ovary and fallopian tubes, and check for any damaged bleeding. The line sutures the muscle layer incision by continuous stitching.

Complete the incision suture on the left muscle layer, continue to move the back skin gap to the muscle layer above the right kidney, see the ovary and fallopian tube, and check the muscle layer after continuous bleeding with 3-0 suture. Incision suture. Finally, the back skin gap is closed by the intermittent suture method, and the iodine wine is banded and the wound is inserted, that is, the mouse ovary removal sham operation is completed.

3.3 Stem cells and platelet-rich fibrin release solution

Before the treatment, the cultured adipose stem cells were taken out from the culture dish at 37 ° C, 5% CO ₂ incubator, and after confirming the normal growth pattern under the microscope, the cells were separated from the culture dish using 1 time 0.25% Trypsin-0.02% EDTA. Next, the cells were washed with PBS, excess trypsin was removed, and the waste was removed by centrifugation at 3000 rpm for 10 minutes. After centrifugation, 100 μl of the cell solution was mixed and stained with an equal volume of 0.5% Trypan blue, and then dropped on a hemocytometer disk and counted under a microscope to confirm the amount of cells injected.

Mice that completed ovarian ablation were observed for two weeks and were determined to have no adverse reactions due to ovarian ablation, or any physical pain or discomfort.

At the time of injection, the tail of the mouse was completely disinfected with alcohol cotton to avoid bacterial infection. Then, using a 1c.c syringe with a 26G needle, the pre-injection material in the centrifuge tube was taken, and the tail vein of the tail of the mouse was calibrated to the tail vein. Push the stem cell mixture at a speed of 10 ml to avoid instantaneous push-in of the stem cell mixture, causing transient coagulation and death. After injection, the mice were stopped by alcohol cotton to stop bleeding, and the alcohol was removed after confirming that there was no bleeding.

3.4 Body temperature measurement

The mice after injection were measured for temperature of the anus with an electronic thermometer (IB MT200, Microlife, Swiss) to observe whether the mice had fever or severe temperature changes to evaluate whether the injected substance was strong in mice. Intense allergic reaction. The measurement time points were one hour after the injection, two hours after the injection, and five times after the injection.

Mice injected with stem cells and platelet-rich release fluids were recorded for changes in temperature at the anus by an electronic thermometer (IB MT200, Microlife, Swiss) to assess whether stem cells or platelet-rich fibrin would cause adverse immune rejection in mice. A sharp change in body temperature. The measurement time was one hour, two hours, one day, two days and three days after injection, for a total of five time points. The body temperature measurement results are recorded in Figure 1.

In the second group, the body temperature of the mice in the treatment group did not change too much, and there was no significant difference compared with the Pos. Control positive control group without injection. Therefore, we concluded that the injection of stem cells or rich The platelet-containing fibrin release solution did not cause a serious allergic reaction to the mice themselves.

3.5 Observation of adverse reactions

The mice were observed to have adverse reactions after injection of stem cells and platelet-rich fibrin-releasing liquid, such as abnormal tremor, whitening of the tail part, loss of blood color of the eyeball, and the like. The observation time was one hour, two hours, one day, two days and three days after the injection, for a total of five time points. The results showed that the mice in the treatment group did not experience severe tremors and pains such as tremors and pains at the time after the injection and at the three time points after the injection. The eating and drinking conditions were normal and there was no abnormality. Therefore, injection of adipose stem cells and injection of platelet rich The fibrin release solution does not cause immunological rejection in mice, and does not cause death or immediate harm.

Example 4. Assessment of bone growth

4.1 mouse tibia micro computed tomography

The experimental mice sacrificed after 8 weeks and completely removed the rat tibia. After removing excess muscle tissue, they were fixed with 10% neutral formalin for at least one week. The completed mouse tibia sample was distilled water (sterilized rinse with distilled water, Was washed from the Yongfeng Chemical Industry Co., Ltd. to remove residual fumarin solution attached to the bone sample, and scanned with a microcomputer tomography scanner (Skyscan 1176, Bruker, Belgium Kontich). The scanning area was 0.9 mm below the humerus growth plate of the mouse. Where, the scanning conditions are: (1) Energy intensity: 40kev (2) Current: 600uA (3) Resolution: 9um (4) Scanning field of view: 180 degrees (5) Exposure time: 860ms (6) Rotation ratio: 0.3 degrees / Per pic. The scanning site was 0.5~1.5mm under the growth plate of the proximal tibia of the left leg of the mouse. The analyzed items included bone mineral density (BMD), bone mass/tissue mass ratio (BV/TV), and trabecular bone spacing ( Tb.Sp) and the number of trabecular bone (Tb.N) total 4 items.

BMD values can be used to assess whether the growing bones are healthy and have a general bone function. A BMD value of too low means that the bone contains a low proportion of minerals, and the bones are loose, fragile and prone to cracking. Figure 2 shows a significant improvement in the effectiveness of the four injections of the number of injections, four treatment groups: PRF-IV, ADSC-IV, The bone mineral density of ADSC+PRF-IV and ADSC+PRF-IV-i were 0.0662g/cm3, 0.0738g/cm3, 0.0766g/cm3 and 0.1016g/cm3, respectively, whether using PRF alone or BMSC alone. Compared with the non-treatment group Neg.Control, significant differences were achieved, and the bone density of PRF and ADSC group was significantly higher than other groups, reaching extremely significant differences.

The percentage of bone volume (BV/TV) is a percentage value. The higher the BV/TV value of the test sample, the higher the proportion of bone in the sample and the more bone under the overall observation. The average percentage of BV/TV in untreated Neg.Control mice was 4.895%, which represented a decrease in bone mass and a decrease in the number of trabecular bone after ovarian ablation, thus making the bone spacing wider than normal mice. Figure 3 shows that the bone mass in the tibia of the treated group was significantly increased. The percentage of PRF-IV and ADSC-IVBV/TV was 6.366% and 7.0641%, especially the two groups of PRF+ADSC. No, whether it is the recommendation of tail vein injection of ADSC+PRF-IV or intraosseous injection of ADSC+PRF-IV-i, the percentage of BV/TV is 12.0767% and 10.1365%, both of which have reached extremely significant differences.

The higher the value of trabecular spacing (Tb.Sp), the higher the bone depletion, resulting in a smaller number of trabecular bones in the tibia sample of the mouse, or a thinner trabecular thickness, resulting in a smaller bone in the leg bone. The beam spacing is large. In the negative control group Neg.Control mice, after 10 weeks of ovary removal, the average trabecular bone number of the mouse tibia was higher than that of the positive control group Pos. Control mice. Low, resulting in a large spacing of the trabecular bone, and significant bone loss can be observed. Figure 4 shows that the injection group (PRF-IV, ADSC-IV) has no significant decrease in the trabecular bone spacing compared with the untreated negative control group, so the trabecular bone spacing values are not significant. difference. The group that was injected four times began to show a therapeutic effect, and the trabecular bone spacing began to shrink, and significant and extremely significant differences were achieved compared with the untreated negative control group.

The number of trabecular bone (Tb.N) is a virtual line in the mouse leg bone sample, and the number of trabecular bones passing through the virtual line is calculated. The lower Tb.N value represents the inside of the sample leg bone. The average number of trabecular bones is small, and the higher the Tb.N value, the higher the average number of trabecular bones in the leg bones of the sample. Figure 5 shows a significant increase in the mean number of trabecular bone. PRF-IV, BMSC-IV, BMSC+PRF-IV and BMSC+PRF-IV-i were 21.32%, 26.66%, 120.18% and 58.2%, respectively.

Example 5. Serum biochemical assay

After receiving the stem cells and the PRF injection, the mice were sacrificed after 8 weeks of body weight of about 25 g, and blood was collected. The extracted blood was then centrifuged at 3000 rpm for 10 minutes to collect the separated serum from the blood, and finally the serum of each group of mice was sent to the Union Clinical Laboratory (Taiwan) for automated biochemical analysis. The instrument (ADVIA 1800, SIEMENS, Germany) was used to analyze serum calcium and phosphorus ion content.

Figure 6 shows that the Pos.Control positive control mice and the SHAM ovarian sham-operated mice had similar serum calcium levels, while the ovarian-negative Neg.Control negative control mice showed a significant decrease in serum calcium levels. Therefore, we confirmed that ovarian-extracted mice did develop osteoporosis, resulting in a decrease in serum calcium levels. After four injections of stem cells and mice rich in platelet fibrin-releasing solution, the serum calcium concentration increased significantly, and reached a significant difference compared with the negative control group.

Example 6. Tissue section staining

The mouse tibia after completion of the computed tomography analysis was decalcified with a 1:1 mixed decalcification solution of 22.5% Formicacid (6274H, MP Biomedicals, Taiwan) and 10% Sodium citrate (A0208003001, Acros, USA). The decalcified leg bone samples were cut in half from the middle, then trimmed with tissue knives and placed in an embedding box, then immersed in formalin for storage and sent to the Agricultural Science and Technology Research Institute for subsequent steps. The tissue sample fixed in the embedding cassette will be dehydrated and then embedded in heat-melted liquid paraffin. After cooling, the solidified paraffin block was removed, cut into about 4 μm slices to prepare tissue sections, and subjected to H & E stain. Finally, the internal morphology of the bone was observed under an optical microscope.

Figure 7 indicates that the number of trabecular bones in the bone marrow cavity of mice is sparse compared to the untreated group, especially below the growth plate, and the number of trabecular bone in the medullary cavity is sparse. And injection of four stem cells or platelet-rich The group of fibrin-releasing fluids: ADSC-IV, PRF-IV, ADSC+PRF-IV, and ADSC+PRF-IV-i, the number of trabecular bone in the medullary cavity was significantly higher than that in the untreated group. More phenomena. Among them, ADSC+PRF-IV and ADSC+PRF-IV-i were effective in the treatment of these two groups. The microscopic observation of the staining results revealed that there were many trabecular bone distributions in the bone marrow cavity.

Although the embodiments of the present invention are disclosed in the above embodiments, the present invention is not intended to limit the invention, and the present invention may be practiced without departing from the spirit and scope of the invention. Various changes and modifications may be made thereto, and the scope of the invention is defined by the scope of the appended claims.

Claims (11)

A pharmaceutical composition for increasing bone density, comprising a Platelet Rich Fibrin (PRF) and a fat stem cell. The pharmaceutical composition according to claim 1, wherein the adipose stem cells are Adipose tissue stem cells (ADSC). The pharmaceutical composition according to claim 1, wherein the concentration of the growth factor in the platelet-rich cellulose is Platelet-derived growth factor-AB (PDGF-AB) 1738.7±582.24 pg/mL, Transforming growth factor beta 1 (TGF) 405.7±82.9 pg/mL, Epidermal growth factor (EGF) 17.5±7.5 pg/mL, Basic fibroblast growth factor (b-FGF) 7.6±3.2 pg/mL, Nerve growth factor (NGF) 4.0±1.1 pg /mL, Insulin-like growth factor-1 (IGF-1) 787.4 ± 119.6 pg/mL, Vascular endothelial growth factor (VEGF) 92.0 ± 13.9 pg/mL. The pharmaceutical composition according to claim 1, wherein the number of cells of the fat cell is 4.8-5.0 x ^{10 5} cells/mL. The pharmaceutical composition according to claim 1, wherein the ratio of the platelet cellulose to the adipose stem cells is 1:1 to 1:2. A pharmaceutical composition for use in the manufacture of increased bone density, wherein the pharmaceutical composition comprises a platelet rich cellulose (Platelet Rich Fibrin, PRF) and a fat stem cell. The use of the sixth aspect of the invention, wherein the adipose stem cells are Adipose tissue stem cells (ADSC). The use according to claim 6, wherein the concentration of the growth factor in the platelet-rich cellulose is Platelet-derived growth factor-AB (PDGF-AB) 1738.7±582.24 pg/mL, and the transforming growth factor beta 1 ( TGF) 405.7±82.9pg/mL, Epidermal growth factor (EGF) 17.5±7.5pg/mL, Basic fibroblast growth factor (b-FGF) 7.6±3.2pg/mL, Nerve growth factor (NGF) 4.0±1.1pg/mL Insulin-like growth factor-1 (IGF-1) 787.4±119.6 pg/mL, Vascular endothelial growth factor (VEGF) 92.0±13.9 pg/mL. The application for the use according to item 6 patents range, wherein the number of cells of adipose stem cells 4.8-5.0x1 ⁰⁵ cell / mL. The use according to claim 6, wherein the ratio of the platelet cellulose to the adipose stem cells is 1:1 to 1:2. The use of claim 6, wherein the use comprises administering to the body a pharmaceutical composition continuously and/or discontinuously for more than 4 days by intravenous and/or intra-osseous injection.