Pharmaceutical composition containing mesenchymal stem cells and application

Abstract

The invention belongs to the technical field of biology, and relates to a pharmaceutical composition containing mesenchymal stem cells and application. By controlling the using time, the medication mode and the dosage of the mesenchymal stem cells and the vitamin D, the synergistic effect of the mesenchymal stem cells and the vitamin D is achieved, the cortical bone mineral density can be effectively improved, meanwhile, the bone trabecula density and the bone trabecula cell density can be improved, and the bone quality is improved. In addition, during formation and distribution of the bone trabecula, the thickness of the bone trabecula, the distance between the bone trabecula and the number of the bone trabecula can be improved, and the biological performance of thighbone and lumbar vertebra bones is effectively improved. According to the technical scheme that the MSC and the RANKL inhibitor are combined for use, the MSC and the RANKL inhibitor have a synergistic effect, various functional indexes of osteoporosis can be comprehensively improved, obviously, the MSC and the RANKL inhibitor are combined for use, the thickness of bone trabecula, the distance between the bone trabecula and the number of the bone trabecula can be effectively improved, the biomechanical property of tibia is effectively improved, and a good reference basis is provided for clinical application.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a pharmaceutical composition containing mesenchymal stem cells and uses thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cells with self-renewal and multiple differentiation potentials. They can differentiate into bone, adipocytes or cartilage. This multi-lineage differentiation is referred to as tri-lineage differentiation of mesenchymal stem cells in the art.

[0003] Based on the multi-lineage differentiation potential of mesenchymal stem cells, they have been applied in many fields. Using mesenchymal stem cells (MSC) to improve bone density is a new direction in the dimension of regenerative medicine. For example, the article "Mesenchymal Stem Cells: Cell Fate Decision to Osteoblast or Adipocyte and Application in Osteoporosis Treatment" published by Lifang Hu et al. in Volume 19, Issue 360 ​​of International Journal of Molecular Sciences discloses the possibility of using mesenchymal stem cells in the treatment of osteoporosis, and further points out that the therapeutic strategy of changing the differentiation direction of MSCs may be a potential method for the treatment of osteoporosis. However, the document does not further provide a specific scheme for the controllable induction of MSCs differentiation direction.

[0004] There are some literature reports on the induction of MSC differentiation in the prior art. The in vitro experimental data disclosed in these literatures show that vitamin D can stimulate mesenchymal stem cells to differentiate into osteoblast-like cells, either in a suitable osteogenic medium or based on fibronectin (FN) (Francesca Posa et al. "Vitamin D Effects on Osteoblastic Differentiation of Mesenchymal Stem Cells from Dental Tissues", Stem Cells International, vo1.2016, Article ID 9150819, 9 pages, 2016. https: / / doi.org / 10.1155 / 2016 / 9150819. Francesca Posa et al. "Vitamin D Promotes MSCOsteogenic Differentiation Stimulating Cell Adhesion and αVβ3 Expression", Stem Cells International, vo1.2018, Article ID 6958713, 9 pages, 2018 · https: / / doi.org / 10.1155 / 2018 / 6958713).

[0005] However, these induction experimental results are all in vitro cell experiments, and the induction results are based on directly adding these inducers into the MSC culture medium. For those skilled in the art, osteoblasts have four different forms in vivo, namely pre-osteoblasts, osteoblasts, osteocytes and formation cells. And osteoblasts will experience four stages of osteocyte proliferation, extracellular matrix maturation, extracellular matrix mineralization and osteoblast apoptosis during bone formation, and will be affected by many aspects such as hormone levels and microenvironment in the body in this process. Therefore, although in vitro cell experiments can reflect the impact of a certain factor or several types of factors on osteoblast development, it is unknown whether the same induction effect can be exerted in clinical practice.

[0006] Of course, more importantly, the structure of bones includes periosteum, bone, articular cartilage, bone marrow, blood vessels, nerves, etc. Bone is the main component of bone, which is divided into compact bone and spongy bone according to the density of its structure. Spongy bone mainly exists at the two ends of long bones, the surface of vertebrae and ribs. From the anatomical characteristics, compact bone is dense and hard, with strong resistance to compression and torsion; spongy bone contains more hematopoietic cells, fat, and blood vessels, is spongy, and has a loose structure. The main evaluation index of bone quality is bone density (BMD), the full name of which is bone mineral density, expressed in grams per cubic centimeter, which is an absolute value. BMD measurement has the value of predicting fracture risk.

[0007] Bone density includes both the density of cortical bone (the main component of compact bone) and the density of trabecular bone (the main component of cancellous bone), and the two together affect the load-bearing capacity of bones.

[0008] However, the current existing technology generally uses calcified bone cells as tracking targets, without distinguishing the ratio of cortical bone to trabecular bone in bone cells. This is somewhat different from the technical effect of improving bone performance in osteoporosis patients.

[0009] Osteoporosis (OP) is a systemic disease characterized by low bone mass, damage to bone microstructure, increased bone fragility and susceptibility to fractures. It is mainly divided into two categories: primary osteoporosis and secondary osteoporosis. Among them, primary osteoporosis is further divided into three categories: postmenopausal osteoporosis (type I), senile osteoporosis (type II) and idiopathic osteoporosis. The clinical manifestations of osteoporosis are reduced biomechanical properties of the femur, vertebrae and tibia, which is directly reflected in the reduced load capacity of the femur, vertebrae and tibia.

[0010] Therefore, the essence of osteoporosis treatment is to improve the biomechanical properties of the femur, vertebrae, and tibia. Summary of the invention

[0011] The purpose of the present invention is to provide a pharmaceutical composition with possible clinical application, so as to actually exert the effect of mesenchymal stem cells (MSC) on bone quality in vivo, and solve the problem of decreased load capacity of femur, vertebrae, tibia, etc. caused by estrogen deficiency in postmenopausal women or the elderly with aging.

[0012] In order to achieve the above-mentioned purpose of the invention, two problems must be solved. One problem is that allogeneic mesenchymal stem cells will pass through the lungs, spleen and liver and finally reach the bones after intravenous injection of MSC, and the residual amount is relatively small; and the intravenously administered MSC will immediately concentrate in the pulmonary capillaries and be phagocytosed by monocytes within 24 hours, which makes the MSC stay in the body for a short time. MSC cannot deliver the therapeutic payload to the host environment through the secreted paracrine factors in a short time and limits the cell homing to the target tissue (i.e., the bone marrow and nervous system) and the transformation to osteoblasts, or the transformation amount is small; another problem is that MSC has three differentiation modes in the body: adipogenesis, osteogenesis and chondrogenesis. The differentiation direction of MSC after injection into the body is uncontrollable.

[0013] In addition, the inventors found that existing MSC clinical trials could not make further progress in clinical trials than preclinical studies, achieve the expected results of clinical trials, and fundamentally solve the problem of decreased load capacity of femurs, vertebrae, tibias, etc.

[0014] In order to achieve the above-mentioned object of the invention, the present invention discloses a pharmaceutical composition containing mesenchymal stem cells, comprising a prepared concentration of 0.2×10 6 cells / mL-2×10 6 cells / mL of mesenchymal stem cell intravenous injection solution. After preparation, one dose of mesenchymal stem cell intravenous injection solution contains 3×10 6 and an oral vitamin D agent containing 400-800 IU; the solvent of the mesenchymal stem cell intravenous injection is composed of 0.9% sodium chloride injection and 20% human albumin injection in a volume ratio of (1-3): (3-1).

[0015] Preferably, the present invention further discloses a pharmaceutical preparation package containing mesenchymal stem cells, wherein one pharmaceutical preparation package contains four mesenchymal stem cell biological preparations, four mesenchymal stem cell intravenous injection liquid solvents and 28 vitamin D oral preparations, each mesenchymal stem cell biological preparation contains multiple (generally 50-80) mesenchymal stem cell biological preparations, each mesenchymal stem cell intravenous injection liquid solvent contains multiple small portions (generally 50-80 small portions) of mesenchymal stem cell intravenous injection liquid solvents, and one mesenchymal stem cell biological preparation contains 3×10 6 mesenchymal stem cells; a small portion of mesenchymal stem cell intravenous injection solvent includes 3.75mL of 0.9% sodium chloride injection and 1.25mL of 20% human albumin injection; the content of vitamin D in a vitamin D oral preparation is 400-800IU.

[0016] The present invention adopts a ready-to-use preparation method, firstly mixing sodium chloride injection and human albumin injection to form an intravenous injection solvent, and then injecting the mesenchymal stem cell biological preparation into the prepared intravenous injection solvent to form the prepared mesenchymal stem cell intravenous injection.

[0017] Since the dosage of mesenchymal stem cells in clinical application is related to body weight, 50-80 portions are provided to correspond to adults in the range of 50kg-80kg to select appropriate portions as needed. As needed, the number of portions can also be designed in the form of 2kg, 5kg, 8kg, etc. as weight increment units. Under the premise of not changing the total amount of mesenchymal stem cells in the mesenchymal stem cell biological preparation and the total amount of the solvent composition of the mesenchymal stem cell intravenous injection solution, these changes are essentially the same as the present invention.

[0018] Furthermore, in the present invention, we further disclose the use of the above-mentioned pharmaceutical composition and pharmaceutical preparation package containing vitamin D and mesenchymal stem cells in the preparation of drugs for treating osteoporosis and fractures caused by estrogen changes in postmenopausal women.

[0019] At the same time, the present invention also discloses another pharmaceutical composition containing mesenchymal stem cells, comprising a prepared concentration of 0.2×10 6 cells / mL-2×10 6 cells / mL of mesenchymal stem cell intravenous injection solution. After preparation, one dose of mesenchymal stem cell intravenous injection solution contains 3×10 6 and a RANKL inhibitor at a concentration of 0.3-0.5 μg / ml; the solvent of the mesenchymal stem cell intravenous injection is composed of 0.9% sodium chloride injection and 20% human albumin injection in a volume ratio of (1-3): (3-1).

[0020] Preferably, the present invention further discloses a pharmaceutical preparation package containing mesenchymal stem cells, wherein one pharmaceutical preparation package contains four mesenchymal stem cell biological preparations, four mesenchymal stem cell intravenous injection liquid solvents and 14 RANKL inhibitors, each mesenchymal stem cell biological preparation contains multiple (generally 50-80) mesenchymal stem cell biological preparations, each mesenchymal stem cell intravenous injection liquid solvent contains multiple small portions (generally 50-80 small portions) of mesenchymal stem cell intravenous injection liquid solvents, and one mesenchymal stem cell biological preparation contains 3×10 6 mesenchymal stem cells; a small portion of mesenchymal stem cell intravenous injection solvent includes 3.75mL packaged 0.9% sodium chloride injection and 1.25mL packaged 20% human albumin injection; a portion of RANKL inhibitor contains multiple (generally 50-80) RANKL inhibitor intravenous injections, and each RANKL inhibitor intravenous injection contains 0.5μg RANKL inhibitor drug.

[0021] Similarly, in this technical solution, the aforementioned method of preparing on-the-spot is still adopted. Specifically, the sodium chloride injection and the human albumin injection are first mixed to form an intravenous injection solvent, and then the mesenchymal stem cell biological preparation is injected into the prepared intravenous injection solvent to form a prepared mesenchymal stem cell intravenous injection.

[0022] When using, it is also understood that, since the clinical dosage of mesenchymal stem cells is related to body weight, 50-80 portions are provided to correspond to adults in the range of 50kg-80kg to select appropriate portions as needed. As needed, the number of portions can also be designed according to different weight increments such as 2kg, 5kg, and 8kg. Under the premise of not changing the total amount of mesenchymal stem cells in the mesenchymal stem cell biological preparation and the total amount of the solvent composition of the mesenchymal stem cell intravenous injection solution, these changes are substantially the same as the present invention.

[0023] Furthermore, in the present invention, we further disclose the use of the above-mentioned pharmaceutical composition and pharmaceutical preparation package containing RANKL inhibitor and mesenchymal stem cells in the preparation of drugs for osteoporosis and fractures caused by decreased sex hormone levels.

[0024] Further preferably, in the present invention, we also disclose that the aforementioned two pharmaceutical compositions containing mesenchymal stem cells further contain an oral vitamin C agent with a content of 50-300 mg.

[0025] Further preferably, in a pharmaceutical preparation package containing mesenchymal stem cells, which comprises four parts of mesenchymal stem cell biological preparations, four parts of mesenchymal stem cell intravenous injection solution solvents and 28 parts of vitamin D oral preparations, the vitamin C is 28 parts, and the content of vitamin C in each part of vitamin C oral preparation is 50-300 mg.

[0026] At the same time, it is further preferred that in a pharmaceutical preparation package containing mesenchymal stem cells, which comprises four parts of mesenchymal stem cell biological preparations, four parts of mesenchymal stem cell intravenous injection solvents and 14 parts of RANKL inhibitors, the vitamin C is 28 parts, and the content of vitamin C in each part of vitamin C oral preparation is 50-300 mg.

[0027] As an active ingredient, vitamin C can ensure that more mesenchymal stem cells can reach the bone surface through a daily dose of 50-300 mg. Its specific modes of action include: First, it regulates the immune environment in the body and prolongs the survival time of mesenchymal stem cells in the body; second, it can regulate blood pressure and prompt mesenchymal stem cells to quickly reach the bone surface. The medication cycle and frequency of vitamin C can be the same as those of vitamin D, and it can also be used after the injection of mesenchymal stem cells. The medication cycle is the same as the medication cycle of mesenchymal stem cell injection.

[0028] The technical solution of the present application achieves a synergistic therapeutic effect of mesenchymal stem cells and vitamin D by controlling the use time of mesenchymal stem cells and vitamin D, adjusting the dosage of mesenchymal stem cells and vitamin D, and designing the dosage of mesenchymal stem cells and vitamin D. It can not only effectively increase the bone density of cortical bones, but also increase the density of trabeculae and the density of trabecular cells. It can also improve the thickness of trabeculae, the spacing between trabeculae, and the number of trabeculae during trabeculae formation and distribution, thereby making the trabeculae cooperate with the cortical bones and effectively improving the biomechanical properties of the femur and lumbar vertebrae.

[0029] In the technical solution of the combined use of MSC and RANKL inhibitor disclosed in the present application, the two work synergistically to comprehensively improve various functional indicators of osteoporosis. Notably, the combination of MSC and RANKL inhibitor can effectively improve trabecular thickness, trabecular spacing and trabecular number, effectively improve the biomechanical properties of the tibia, and provide a good reference basis for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of trabecular bone density (Trabeculae Mean BMD, mg / cc) of distal femur in Example 1.

[0031] Figure 2 is the number of trabeculae in Example 1 (Tb.N.mm- 1 ) schematic diagram.

[0032] Figure 3 Schematic diagram of trabecular connection density (Conn.D, mm-3) in Example 1.

[0033] Figure 4 Schematic diagram of the percentage change (%) of femoral shaft bone density BMD in Example 1.

[0034] Figure 5 Schematic diagram of the percentage change (%) of femoral neck bone density BMD in Example 1.

[0035] Fig. 6A . Schematic diagram of the changes in distal femoral cortical bone density (CortexMean BMD, mg / cc) in Example 2.

[0036] Figure 6B . Schematic diagram of the changes in trabecular bone density (Trabeculae Mean BMD, mg / cc) of the distal femur in Example 2.

[0037] Figure 6C . Schematic diagram of the changes in trabecular tissue mean BMD (mg / cc) of the distal femur in Example 2.

[0038] Figure 7 This is a schematic diagram (cross section) of femur CT fluoroscopy after 6 weeks of drug administration in the experimental period of Example 2. Note: 3 animals were selected from each experimental group for the picture display schematic diagram.

[0039] Figure 8 This is a schematic diagram of femoral CT fluoroscopy (coronal plane) during the 6-week drug administration in the experimental period of Example 2. Note: 3 animals were selected from each experimental group for the picture display schematic diagram.

[0040] Fig.9A . is a schematic diagram of the change in the trabecular thickness (Tb.Th, mm) of the distal femur in Example 2.

[0041] Fig. 9B . is a schematic diagram of the change in the trabecular spacing (Tb.Sp, mm) of the distal femur in Example 2.

[0042] Fig. 9C . Schematic diagram of the change in the number of distal femoral trabeculae (Tb.N, mm-1) in Example 2.

[0043] Fig.10 Schematic diagram of femoral shaft bone density (CortexMean BMD, mg / cc) in Example 2.

[0044] Fig.11 Schematic diagram of lumbar vertebrae bone density (mg / cc) in Example 2.

[0045] Fig. 12A . Schematic diagram of the change in the maximum load (unit, N) of the femoral shaft three-point bending in Example 2.

[0046] Fig. 12B . Schematic diagram of the change in the maximum compression load (unit, N) of the femoral neck in Example 2.

[0047] Fig. 12C . Schematic diagram of the change in the maximum compression load (unit, N) of the lumbar vertebrae in Example 2.

[0048] Fig.13This is a statistical analysis chart of the changes in tibia cortical bone mineral density (Cortex-BMD, mg / cc); all individual data are expressed as mean ± standard deviation (SD). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 represent the statistical analysis results of different experimental groups and normal control group, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 represent the statistical analysis results of different experimental groups and model control group.

[0049] Fig.14 This is a statistical analysis chart of changes in tibial trabecular bone mineral density (Trabecular BMD, mg / cc).

[0050] Fig.15 This is a statistical analysis chart of changes in tibia-bone mineral density (Bone-BMD, mg / cc).

[0051] FIG16 is a schematic diagram of CT fluoroscopic view of the tibia at the end point of the test; Fig.16A is a perspective view of the normal control group; Fig. 16B This is a perspective view of the model control group; Fig. 16C A perspective view of the RANKL inhibitor group; Fig.16D A perspective view of a stem cell group; Fig.16E Rendering of the combined treatment group.

[0052] Fig.17 This is a statistical analysis chart of changes in trabecular thickness (Tb.Th, mm). Figure 5-7 All individual data of each group compared with the model group were expressed as mean (Mean) ± standard deviation (SD); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 represent the statistical analysis results of different experimental groups and normal control group, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 represent the statistical analysis results of different experimental groups and model control group.

[0053] Fig.18 It is a statistical analysis chart of the changes in trabecular spacing (Tb.Sp, mm).

[0054] Fig.19 is the number of trabeculae (Tb.N, mm -1 )Statistical analysis chart of changes.

[0055] Fig. 20 This is an analysis and comparison chart of the maximum tibial compression load (unit, N).

[0056] Fig.21This is a statistical diagram of the continuous analysis of the bone turnover indicator PINP.

[0057] Fig. 22 Schematic diagram of statistics for Osteocalcin analysis.

[0058] Fig.23 This is a statistical diagram of the analysis of the bone absorption indicator CTX-1.

[0059] Fig.24 Schematic diagram of Tracp-5b analysis statistics. DETAILED DESCRIPTION

[0060] The nouns used in this article are defined as follows:

[0061] Cortical bone: It is dense bone and distributed on the surface of the bone. Its counterpart is cancellous bone, which is composed of trabeculae and distributed inside the bone. Its evaluation index is bone mineral density (BMD).

[0062] Injection: refers to a suspension of mesenchymal stem cells injected into the human body. In this application, it can be injected into the bone marrow or intravenously, and intravenous injection is preferred in this application.

[0063] 0.9% Sodium Chloride Injection: means that 100mL of Sodium Chloride Injection contains 0.9g of sodium chloride.

[0064] 20% human albumin solution: refers to 10g of protein in 50mL of human albumin, such as human albumin products of China Resources Boya Biopharmaceutical Group Co., Ltd. and human albumin products of Shandong Taibang Biological Products Co., Ltd.

[0065] Mesenchymal stem cells: MSC for short. According to the definition of human mesenchymal stem cells in the T / CSCB0003-2021 Human Mesenchymal Stem Cells group standard issued by the Chinese Society of Cell Biology: a type of stem cell that has a fibroblast-like morphology (spindle-shaped and fusiform) after adherent culture, can self-renew in vitro, and has the ability to differentiate into osteoblasts, adipocytes, and chondrocytes. It can be isolated from human tissues (such as bone marrow, umbilical cord, placenta, fat, umbilical cord blood, etc.), or obtained through differentiation or transdifferentiation, and human mesenchymal stem cells from different sources differ in gene expression and differentiation ability.

[0066] ce11s / mL refers to the number of cells contained in each milliliter of injection solution.

[0067] IU is the international unit of vitamin D, 1IU = 0.025ug.

[0068] Sham operation group: In this group of rats, only the ovaries were exposed and a small amount of nearby fat tissue was removed and then returned to the abdomen, and sutured layer by layer.

[0069] Model control group: This group of rats were established with a female postmenopausal osteoporosis model by castration (bilateral ovariectomy, OVX).

[0070] The dosage of vitamin D is based on the conversion of rat and adult body weights, and the dosage for rat experimental medication is switched to that for adults.

[0071] Type 1 collagen propeptide (PINP) is a metabolite produced by osteoblasts after synthesizing type 1 collagen, the main component of bone. It is released into the blood and urine and can be detected. It can sensitively reflect the osteogenic activity and the synthesis rate of type 1 collagen.

[0072] Mean (MEAN): The sum of all data divided by the amount of data (the number of data points).

[0073] Standard Deviation (SD): It is the square root of the arithmetic mean of the squares of the deviations between the standard values ​​of each unit in the population and its mean. It reflects the degree of dispersion between individuals in the group. It is most commonly used as a measure of the degree of statistical distribution in probability statistics.

[0074] (I) A technical solution of the present application is described in detail below.

[0075] There is evidence in the prior art that MSCs can alleviate osteoporosis to a certain extent, but how to improve cortical bone density in a targeted manner based on the prior art is the main issue discussed in this application. Vitamin D (Vit D) has a protective effect on bones through its biologically active form, 1α, 25-dihydroxyvitamin D3 (1,25(OH)2D3), by acting on calcium homeostasis and bone formation. Based on in vitro studies, the technical staff of this application has developed a pharmaceutical composition suitable for in vivo use to improve cortical bone density, and provided its administration method and dosage.

[0076] The present application adopts two active ingredients, mesenchymal stem cells and vitamin D, which are administered in the same or different dosage forms, the same or different specifications, simultaneously or at different times, and ultimately achieves the effect of increasing cortical bone density through fine-tuning of the in vivo microenvironment.

[0077] In vivo medication, that is, in this application, vitamin D can be administered orally or intravenously, preferably orally. The preferred scheme is that the administration time of vitamin D is on the day of injection of mesenchymal stem cells or any day within the previous week. The main reason is that, for example, osteoporosis patients have an imbalanced microenvironment and immune regulation disorders in their bodies, and their direct impact on mesenchymal stem cells is homing disorder and impaired osteogenic differentiation ability. Based on the regulation of the microenvironment in the body of osteoporosis patients, this application is designed to use vitamin D in advance while injecting mesenchymal stem cells or before injecting mesenchymal stem cells, that is, before injecting mesenchymal stem cells, first adjust the patient's serum 1,25(OH)2D3 level.

[0078] Among them, mesenchymal stem cells exist in the form of intravenous injection, and the content of mesenchymal stem cells in the intravenous injection is 0.1×10 6 cells / mL-3×10 6 cells / mL. Example: 0.1×10 6 cells / mL, 0.2×10 6 cells / mL, 0.3×10 6 cells / mL、0.4×10 6 cells / mL、0.5×10 6 cells / mL、0.6×10 6 cells / mL、0.7×10 6 cells / mL、0.8×10 6 cells / mL、0.9×10 6 cells / mL, 1.0×10 6 cells / mL, 1.1×10 6 cells / mL、1.2×10 6 cells / mL、1.3×10 6 cells / mL、1.4×10 6 cells / mL、1.5×10 6 cells / mL、1.6×10 6 cells / mL、1.7×10 6 cells / mL、1.8×10 6 cells / mL、1.9×10 6 cells / mL, 2.0×10 6 cells / mL, 2.1×10 6 cells / mL, 2.2×10 6 cells / mL、2.3×10 6 cells / mL、2.4×10 6 cells / mL、2.5×10 6 cells / mL、2.6×10 6 cells / mL、2.7×10 6 cells / mL、2.8×10 6 cells / mL、2.9×10 6 cells / mL、3.0×10 6 cells / mL. During use, the above contents can meet the requirements.

[0079] The intravenous injection includes 0.9% sodium chloride injection, and the mesenchymal stem cells are resuspended in the 0.9% sodium chloride injection, which has the same osmotic pressure as the in vivo environment and can effectively ensure the activity of MSCs.

[0080] In order to increase the shelf life of the pharmaceutical composition of the present application, the intravenous injection comprises mesenchymal stem cells resuspended in a cell solvent, the cell solvent comprises 0.9% sodium chloride injection and 20% human albumin solution, the volume ratio of the two is (1-3): (3-1), and the optional ones are 1: (1-3), 2: (1-3), 3: (1-3) and exemplarily are: 1:3, 1:2, 1:1, 2:3, 2:1, 3:2, 3:1.

[0081] The daily dosage of vitamin D is 400IU-800IU, which has the effect of pre-adjusting the microenvironment in the body, such as regulating the immune environment in the body, and is assisted by improving the survival rate of mesenchymal stem cells in the patient or the conversion rate to osteoblasts. In the present application, vitamin D is administered simultaneously with mesenchymal stem cells, or the administration time of vitamin D is earlier than the time of injecting mesenchymal stem cells, such as vitamin D is within the day or the previous week when mesenchymal stem cells are provided. Its purpose is to ensure that vitamin D can play the fastest regulation of the microenvironment in the body, such as regulating calcium, phosphorus, 1,25(0H)2VD3 in the blood and the immune environment in the body, so that after injecting mesenchymal stem cells, mesenchymal stem cells can be promoted to osteogenic differentiation.

[0082] Alternatively, in actual medication, for the sake of medication safety and efficacy, the amount of each active ingredient in the pharmaceutical composition is calculated based on the dosage for rats: the amount of mesenchymal stem cells is 0.5×10 6 cells / kg-10×10 6 cells / kg; the daily dose of vitamin D is 2-4IU.

[0083] Calculated based on the dosage for adults: mesenchymal stem cells dosage 0.5×10 6 cells / kg-10×10 6 cells / kg; the daily dosage of vitamin D is 400IU-800IU. Regardless of the dosage for rats or the dosage for adults: the administration cycle of vitamin D is not limited by the administration cycle of mesenchymal stem cells: preferably, the administration cycle of vitamin D is not shorter than the administration cycle of mesenchymal stem cells.

[0084] In order to ensure that mesenchymal stem cells can fully differentiate into osteoblasts, the administration method of mesenchymal stem cells is: the frequency of administration is once every two weeks, once every four weeks, or once every eight weeks; the total administration cycle does not exceed thirty-two weeks. If it is administered once every two weeks, you can choose to administer 1-16 times, and the corresponding cycle is two to thirty-two weeks; if it is administered once every four weeks, you can choose to administer 1-8 times, and the corresponding cycle is four to thirty-two weeks; if it is administered once every eight weeks, you can choose to administer 1-4 times, and the corresponding cycle is eight to thirty-two weeks. In practical applications, it is preferably administered once every two weeks, four times, five times, six times, seven times or eight times; once every four weeks, three times, four times, five times or six times; once every eight weeks, it is given twice or three times or four times.

[0085] In order to ensure that more mesenchymal stem cells can reach the bone surface, the pharmaceutical composition of the present application also includes: the fifth active ingredient vitamin C, the daily dosage of vitamin C is 50-300 mg. First, it regulates the immune environment in the body and prolongs the survival time of mesenchymal stem cells in the body; second, it can regulate blood pressure and prompt mesenchymal stem cells to quickly reach the bone surface. The medication cycle and frequency of vitamin C can be the same as those of vitamin D, and it can also be used after the injection of mesenchymal stem cells, and the medication cycle is the same as the medication cycle of mesenchymal stem cell injection.

[0086] As an improved technical solution of the present application, the pharmaceutical composition can be used to prepare drugs for treating traumatic fractures and drugs for treating osteoporosis.

[0087] The technical solution of the present application is described in detail below in conjunction with specific embodiments.

[0088] Example 1: Verification of the effects of mesenchymal stem cells alone in the medium-dose group and of mesenchymal stem cells combined with vitamin D in the medium-dose group.

[0089] Experimental design: 80 3-month-old female SD rats were enrolled. In group 1, 13 animals were set up as the sham operation group, and only part of the fat around the ovaries was removed by laparotomy. All the remaining animals were set up as the model control group, and all of them were removed by laparotomy on both sides of the ovaries and part of the fallopian tubes. The animals in the model control group with significantly decreased bone density and bone microstructure compared with the sham operation group were randomly divided into two groups, each with 20 rats, set as group 2: model control group, group 3: MSC treatment group; group 4: medium dose 4 times treatment group. Three months later, 3 animals were randomly selected from the sham operation group and the model control group to obtain femurs for testing bone density and bone microstructure.

[0090] Group medication:

[0091] The animals in group 1 (sham operation group) and group 2 (model control group) were injected with 5 ml / kg of cytolytic enzyme via the tail vein once every eight weeks for three consecutive times.

[0092] Animals in group 3 (MSC-treated group) were injected via tail vein with 3×10 6 Cells / kg(3×10 6 cells / kg), once every eight weeks, for three consecutive times.

[0093] Animals in group 4 (medium dose 3 times treatment group) were injected via tail vein with 3×10 6 Cells / kg, once every eight weeks, for three consecutive times; at the same time, take vitamin D orally, 4 IU per day, for a total of eight weeks. The first use of vitamin D is on the same day as the first use of mesenchymal stem cells.

[0094] One week after the last administration, all animals were killed, and bone density was measured. The specific grouping is shown in Table 1.

[0095] The cell solvent was a mixed solution of 0.9% sodium chloride injection and 20% human albumin, and the volume ratio of 0.9% sodium chloride injection to 20% human albumin solution was 3:1.

[0096] The MSC treatment group and the medium dose 3 times treatment group received 0.6×10 6 cells / mL of MSCs were dispersed in cell solvent and named hMSC100.

[0097] Table 1 Animal grouping information in Example 1

[0098]

[0099]

[0100] Detection indicators

[0101] During the experiment, the behavioral state and food intake of animals in each group were observed once a day; the body weight of animals was measured once a week; 3 months after castration and 1 week after the last administration, the animals were sampled and the femoral bone density and bone microstructure of the animals were analyzed by micro-CT.

[0102] Three months (12 weeks) after castration, compared with the sham operation group, the trabecular bone density (Trabeculae Mean BMD) of the distal femur of the model control group showed a downward trend; the number of trabeculae (Tb.N) was significantly reduced (p<0.05), and the trabecular connection density (Conn.D) was significantly reduced (p<0.05). The results showed that the model control group was in line with the trend of osteoporosis. For specific data, see Figure 1 , 2 3. Trabeculae support hematopoietic tissue and increase bone strength. The number, quality, direction and thickness of trabeculae have a great impact on bone strength.

[0103] After the treatment period, the percentage of the difference between the individual BMD value of each group and the average BMD value of the sham operation group was analyzed: BMD change % = (individual BMD value of each group - average BMD value of the sham operation group) / average BMD value of the sham operation group × 100)%.

[0104] like Figure 4 As shown, the percentage change of femoral bone density (BMD) in the model control group was significantly lower than that in the sham operation group (*P<0.05); the percentage change of femoral bone density (BMD) in the MSC treatment group was significantly higher than that in the model control group (##P<0.01); the percentage change of femoral bone density (BMD) in the medium dose 3 times treatment group was significantly higher than that in the MSC treatment group.

[0105] like Figure 5 As shown, the percentage change of femoral neck BMD in the MSC treatment group was significantly higher than that in the model control group (*P<0.05); the percentage change of femoral neck BMD in the medium-dose 3-time treatment group was significantly higher than that in the MSC treatment group.

[0106] The results showed that the femoral shaft and femoral neck bone mineral density of SD rats induced by castration surgery increased significantly after 4 weeks of hMSC100 treatment, indicating that MSC has a good therapeutic effect on osteoporosis. The medium-dose three-time treatment group combined with vitamin D is more conducive to improving the femoral shaft and femoral neck BMD.

[0107] Example 2: Verification of the effects of mesenchymal stem cells combined with vitamin D in different dosage groups.

[0108] Group design: sham operation group, model control group, low-dose stem cell treatment group for 4 times, medium-dose stem cell treatment group for 4 times, high-dose stem cell treatment group for 4 times, and medium-dose stem cell treatment group for 2 times.

[0109] Animal enrollment: The week of surgery was defined as week 0 (0w), and the animals were divided into a sham operation group and a model control group. From the modeling animals, appropriate animals were selected according to their general conditions and body weight and randomly grouped.

[0110] Number of animals included: 30 in the sham operation group, 30 in the model control group, 20 in the low-dose stem cell treatment group for 4 times, 30 in the medium-dose stem cell treatment group for 4 times, 20 in the high-dose stem cell treatment group for 4 times, and 10 in the medium-dose stem cell treatment group for 2 times, for a total of 140 animals (excluding animals in the satellite group).

[0111] Among them, all the animals in the group took vitamin D orally, 4 IU per day, for four weeks. The first use of vitamin D was on the same day as the first use of mesenchymal stem cells.

[0112] In the low-dose stem cell treatment group for 4 times, the medium-dose stem cell treatment group for 4 times, the high-dose stem cell treatment group for 4 times, and the medium-dose stem cell treatment group for 2 times, the cell solvent used for MSC was a mixed solution of 0.9% sodium chloride injection and 20% human albumin, and the volume ratio of 0.9% sodium chloride injection and 20% human albumin solution was 3:1.

[0113] 1. Dosage Information

[0114] Grouping method: Animals were randomly grouped according to their body weight 8 weeks after surgery.

[0115] Dosage: 1×10 6 , 3×10 6 , 10×10 6 cells / kg;

[0116] Dosing volume: 5 mL / kg for each test product;

[0117] Dosing concentration: 0.2×10 6 , 0.6×10 6 , 2×10 6 cells / mL;

[0118] Administration route: Each test product was injected into the tail vein;

[0119] Administration site: tail vein;

[0120] Reason for selection: consistent with clinical medication route;

[0121] Dosing frequency and cycle: see Table 2 for details;

[0122] The day of surgery was defined as day 1 (D1) of the experiment.

[0123] 2. Dosage design basis

[0124] The maximum proposed clinical dose is 1.8×10 8 cells / person, calculated based on 60kg / person, the dose is 3×10 6 cells / kg, once a week, for 4 times.

[0125] Table 2 Animal grouping information of Example 3

[0126]

[0127]

[0128] 3. Analysis of bone density and bone quality parameters.

[0129] Measurement time: 6 weeks of administration;

[0130] Measurement method: Micro-CT was used to detect the bone density of the left distal femur, left femoral shaft, left femoral neck, lumbar vertebra L4, and the left distal femur and left femoral neck (including trabecular parameters, bone volume fraction, etc.);

[0131] Animals for testing: Animals were dissected as planned.

[0132] 4. Bone strength.

[0133] Measurement time: 6 weeks of administration;

[0134] Determination method: An electronic universal material testing machine was used to perform three-point bending tests on the left femur, compression tests on the right femoral neck, and compression tests on the lumbar vertebra L3 (correlation analysis of bone strength and bone mass parameters was then performed);

[0135] Animals for testing: Animals were dissected as planned.

[0136] Test result 1: Effect of the test article on distal femoral bone density.

[0137] At the end of the experiment, the left femur of the animal was taken for microCT examination (Voxel size: 50um, Scan mode: high resolution, 4min), and the analysis selected area ROI was the area 2mm above the distal femoral growth plate and 1mm above (50um×20 layers).

[0138] The changes of distal femoral bone density during the 6-week trial period are shown in Table 3 and Figure 6. Fig. 6A :DistalFemur cortical bone mineral density (Cortex Mean BMD) changes, Figure 6B :Changes in trabecular BMD of distal femur, Figure 6C :Changes in trabecular tissue BMD in distal femur.

[0139] Table 3 Distal femur bone density values ​​(unit, mg / cc) at 6 weeks of administration during the test period

[0140]

[0141] Note: Bone density analysis was performed on animals in each experimental group during the experimental period, and all individual data were expressed as mean ± standard deviation (SD). * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001 indicates the statistical analysis results of animals in different experimental groups and the sham operation group.

[0142] Schematic diagram of femoral CT fluoroscopy (cross section) after 6 weeks of drug administration during the trial period, as shown in Figure 7 Note: Three animals were selected from each experimental group for picture display.

[0143] Schematic diagram of femoral CT fluoroscopy (coronal plane) after 6 weeks of drug administration during the trial period, as shown in Figure 8 Note: Three animals were selected from each experimental group for picture display.

[0144] The above results suggest that after 6 weeks of administration, hMSC100 treatment has an improvement trend on the decreased femoral BMD in the castration-induced postmenopausal osteoporosis model, and the most obvious trend is in the group treated with 4 times of medium dose of stem cells.

[0145] Test result 2: Effect of the test article on the microstructure of distal femoral trabeculae in animals

[0146] Under the test conditions, as shown in Table 4 and Fig.9A , Fig. 9B , Fig. 9C .

[0147] Table 4 Changes in microstructural parameters of distal femoral trabecular bone during 6 weeks of administration (unit, mm)

[0148] Tb.Th Tb.Sp Tb.N Group mm mm / mm Sham operation group 0.208±0.04 0.29±0.07 2.34±0.22 Model control group 0.149±0.03 0.63±0.13 1.35±0.27 Medium dose of stem cells 4 times treatment group 0.2±0.05 0.24±0.04 1.92±0.25 Medium dose of stem cells 2 times treatment group 0.204±0.03 0.28±0.14 1.96±0.23 Low-dose stem cell treatment group 0.203±0.02 0.24±0.03 2±0.49 High-dose stem cell 4-time treatment group 0.208±0.02 0.23±0.04 1.9±0.24

[0149] Note: Bone microarchitectural parameters of animals in each experimental group were analyzed during the experimental period, and all individual data were expressed as mean ± standard deviation. Fig.9A , Fig. 9B , Fig. 9C middle * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001, indicating the statistical analysis results of animals in different experimental groups and the sham operation group.

[0150] Test result 3: The effect of the test article on the femoral bone density of animals.

[0151] At the end of the experiment, the left femur of the animal was taken for microCT examination (Voxel size: 50um, Scan mode: high resolution, 4min), and the analysis selected area ROI was the 1mm area above 1.35cm from the femoral epiphysis (50um×20 layers).

[0152] As shown in Table 5 and Fig.10. Under the conditions of this experiment, after 6 weeks of administration, the cortical bone of the left femoral shaft in the model control group showed a decreasing trend in bone mineral density (BMD). In terms of femoral shaft bone mineral density, the BMD of the sham operation group was 11274±48.97 mg / cc; the BMD of the model control group was 11167±77.56 mg / cc, which was lower than that of the sham operation group. The BMD values ​​of the medium-dose stem cell 4-time treatment group, the medium-dose stem cell 2-time treatment group, the low-dose stem cell 4-time treatment group, and the high-dose stem cell 4-time treatment group were 11321±124.6 mg / cc, 11299±82.78 mg / cc, 11308±66.22 mg / cc, and 11327±71.41 mg / cc, respectively, which were all higher than those of the model control group.

[0153] Table 5 Femoral bone density values ​​(unit, mg / cc) at 6 weeks of administration during the test period

[0154] Group Cortex BMD Sham operation group 11274±48.97 Model control group 11167±77.56 Medium dose of stem cells 4 times treatment group 11321±124.6 Medium dose of stem cells 2 times treatment group 11299±82.78 Low-dose stem cell treatment group 11308±66.22 High-dose stem cell 4-time treatment group 11327±71.41

[0155] Note: Bone microarchitectural parameters of animals in each experimental group were analyzed during the experimental period, and all individual data were expressed as mean ± standard deviation. Fig.10 middle, * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001, indicating the statistical analysis results of animals in different experimental groups and the sham operation group.

[0156] Fig.11 The lumbar spine bone density is shown, and the conclusion is the same as the femoral bone density. This article will use a diagram to show the effect. Specific data: Six weeks after administration, the BMD values ​​of the medium-dose stem cell 4-time treatment group, the medium-dose stem cell 2-time treatment group, the low-dose stem cell 4-time treatment group, and the high-dose stem cell 4-time treatment group were all higher than those of the model control group.

[0157] Test Result 4: Effect of the Test Article on Animal Femoral Biomechanics

[0158] At the end of the test, the femoral samples that had been administered for 6 weeks were subjected to a three-point bending test and a femoral neck compression test. The bone samples were thawed at room temperature 2 hours in advance, and the long axis (37mm) and short axis (5mm) of the femur were measured with a vernier caliper. The femur was then placed on a universal material testing machine (span = 20mm) and the mid-section of the femur was pressed down at a speed of 1mm / min. The femoral condyle was fixed with a neutral resin for 24 hours in advance to expose the femoral neck, and then the femoral neck was fixed on a universal material testing machine and pressed down at a speed of 2mm / min.

[0159] The results show that, as shown in Table 6 and Fig. 12AIn the three-point bending test of the femoral shaft of femoral samples after 6 weeks of drug administration, the maximum load force of the sham operation group was 123.13±7.28N, and that of the model control group was 117.61±4.85N, which showed a decreasing trend compared with the sham operation group. The maximum load values ​​of the medium-dose 4-time stem cell treatment group and the medium-dose 2-time stem cell treatment group were 129.19±13.34N and 122.84±18.61N, respectively, which showed an increasing trend compared with the model control group. Fig. 12B In the femoral neck compression test, the maximum load of the model control group was 95.19±19.97N, which showed a downward trend compared with the maximum load of 106.64±25.09N in the sham operation. The maximum load values ​​of the medium-dose 4-time stem cell treatment group and the medium-dose 2-time stem cell treatment group were 98.66±10.91N and 94.96±16.13N, respectively, which were not much different from the model control group. The maximum load value of the medium-dose 4-time stem cell treatment group was slightly higher than that of the model control group.

[0160] Table 6 Femoral biomechanical test during the 6-week trial period (unit, N)

[0161]

[0162] Note: All animals in each experimental group underwent bone biomechanical tests within the experimental period, and all individual data were expressed as mean ± standard deviation (SD).

[0163] Fig. 12A , Fig. 12B middle, * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001, indicating the statistical analysis results of animals in different experimental groups and the sham operation group.

[0164] Test Result 5: Effect of the test article on the biomechanics of the animal lumbar spine (L3)

[0165] At the end of the experiment, compression tests were performed on the lumbar vertebrae (L3) samples at 6 weeks and 12 weeks of administration. The samples were thawed at room temperature 2 hours in advance, the upper and lower ends of the lumbar vertebrae were ground flat, placed on a universal material testing machine and fixed, and the upper end of the lumbar vertebrae was pressed down at a speed of 2 mm / min.

[0166] The results are shown in Table 7 and Fig. 12C At 6 weeks of administration, the maximum load force of the compression test in the sham operation group was 313.37±±±39.87N, and that in the model control group was 313.43±42.18N, which were not much different from the sham operation group. The maximum load values ​​of the medium-dose stem cell 4-time treatment group and the medium-dose stem cell 2-time treatment group were 328.41±±±39.03N and 318.85±50.61N, respectively.

[0167] Table 7 Lumbar spine biomechanical test during the 6-week trial period (unit, N)

[0168]

[0169]

[0170] Note: All animals in each experimental group underwent bone biomechanical tests within the experimental period, and all individual data were expressed as mean ± standard deviation (SD).

[0171] Fig. 12C middle, * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001, indicating the statistical analysis results of animals in different experimental groups and the sham operation group.

[0172] The aforementioned pharmaceutical composition containing mesenchymal stem cells can be used in preparing a drug for treating fractures caused by decreased bone density or a drug for treating osteoporosis.

[0173] (II) Another technical solution of the present application is described in detail below.

[0174] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0175] Among them, RANKL inhibitor was purchased from Xinbosheng (catalog number: BE0191).

[0176] definition:

[0177] Normal control group: refers to the rat osteoporosis model not induced by retinoic acid.

[0178] Model control group: the model of osteoporosis in rats was successfully established by retinoic acid-induced rats, but the stem cell inhibitor or combined drug group was not administered according to the improved point of this scheme, so as to compare the treatment effects with the three groups, also known as the model control group.

[0179] Example 4. Animal model preparation, drug administration and tibia density test

[0180] SD rats (SPF grade) were selected. In addition to the normal control group, animals that needed modeling were gavaged with 80 mg / kg of retinoic acid once a day for 2 weeks, with a dosing concentration of 8 mg / mL and a gavage volume of 10 mL / kg. A preliminary test of the efficacy evaluation of the retinoic acid-induced rat osteoporosis model was conducted to establish a retinoic acid-induced rat osteoporosis model. After the rat model was successfully established, it was randomly divided into 4 groups, with 8 rats in each group. They were:

[0181] The RANKL inhibitor group was intravenously injected at a dose of 0.5-1 μg / kg per day. In this study, a dose of 0.5 ug / kg was selected for injection, once every two days, for 4 weeks.

[0182] Stem cell group: MSC cells were injected once a week at a dose of 3×10 6 -5×10 6 cells / kg, 3×10 6 Cells / Kg MSC cells were injected.

[0183] Combined treatment group: RANKL inhibitor group injected with 0.5ug / kg every two days and 3×10 6 Cells / Kg MSCs cells.

[0184] Model control group: 5% human albumin solution was injected weekly.

[0185] Normal control group: The group without modeling was defined as the normal control group: 5% human albumin solution was injected weekly. All groups were treated for 4 consecutive weeks, and follow-up related tests were performed in the 8th week after 4 weeks of medication.

[0186] The concentration of MSCs was 0.3×10 6 -2×10 6 cel1s / mL, the concentration selected in this protocol is 0.6×10 6 The concentration of the RANKL inhibitor is set to 0.3 μg / mL-0.5 μg / mL, and the concentration selected in this scheme is 0.3 μg / mL.

[0187] Bone analysis: The right tibia of the animal was taken for microCT detection (Voxel size: 72um, Scon mode: high resolution, 4min), and the analysis area ROI was the area 1mm below the tibial growth plate (20um×50 layers). The specific analysis results are as follows:

[0188] Under the experimental conditions, after induction with retinoic acid suspension, the model control group showed a decrease in bone density (Bone BMD) in the right tibia, which was significantly lower than that in the normal control group, with the Bone BMD of the normal control group at 12541.03±204.48mg / cc. The Bone BMD value of the RANKL inhibitor group was roughly the same as that of the model control group, and there was no obvious therapeutic effect. The BMD values ​​of the stem cell group and the combined treatment group were both higher than those of the model control group, with the combined treatment group having the most obvious effect, with the Bone BMD at 12420.55±207.77mg / cc. Compared with the use of stem cells alone, the combined treatment group had a more obvious effect than the stem cell group.

[0189] In terms of Cortex BMD index, the model control group was significantly lower than the normal control group. The Cortex BMD of the normal control group was 12965.94±186.76mg / cc; the Cortex BMD of the RANKL inhibitor group did not change much in the treatment effect of the model control group. The BMD values ​​of the stem cell group and the combined treatment group were higher than those of the model control group, and the effect of the combined treatment group was the most obvious, while the treatment effect of the stem cell group was lower than that of the combined treatment group.

[0190] In terms of trabecular BMD index, the model control group was significantly lower than the normal control group, with statistical significance. Similarly, the therapeutic effect of the RANKL inhibitor group was not much different from that of the model control group, with no significant difference. However, the BMD values ​​of the stem cell group and the combined treatment group were higher than those of the model control group, and the effect of the combined treatment group was also the most obvious. The therapeutic effect of the stem cell group was lower than that of the combined treatment group, and it was statistically significant compared with the model control group.

[0191] Specific visible Figure 13-16. It can be seen that the above results indicate that the improvement effect of the combined treatment group is the most obvious; the experimental effect of the RANKL inhibitor group is not much different from that of the model control group, because the dosage of the RANKL inhibitor is small when it is used, and the medication time is only 4 weeks. Compared with the treatment with the inhibitor alone, the dosage in this scheme is 0.5ug / kg, once every two weeks, and continuously used for 4 weeks, that is, the total dosage is 7ug / kg. Compared with the dosage of 0.1-1.0mg / kg injection in the prior art, the total intravenous injection volume under the same injection cycle is 1.4-14mg / kg, that is, 1400-14000mg / kg. The dosage is hundreds of times that of this application. Therefore, the dosage will be increased normally when taking the medicine, and the treatment will continue for 1-3 years, but long-term use will produce side effects. The dosage and cycle are greatly shortened, so there is no significant effect in treatment, but it is found that when it is used in combination with MSCs cells, its therapeutic effect is better than the treatment effect of MSCs alone.

[0192] Furthermore, the right tibia of the animal was taken for microCT examination. See Figure 16 for a schematic diagram of CT perspective of the tibia at the end point of the experiment; Fig.16A As the normal control group, Fig. 16B As the model control group, Figures 16C-16E The results showed that the treatment of stem cell group and combined treatment group had an improvement trend on the decrease of BMD in retinoic acid-induced osteoporosis model, and the effect of combined treatment group was the most obvious.

[0193] Example 5. Analysis of tibia bone parameters

[0194] After 4 weeks of treatment, the SD rats were killed and their tibiae were removed. Figure 17-20 shown.

[0195] Fig.17 In terms of trabecular bone thickness (Tb.Th), the Tb.Th value of the model control group was 0.18mm, and the TV value of the normal control group was 0.36mm. The values ​​of the stem cell group and the combined treatment group were both higher than those of the model control group, at 0.21mm and 0.34mm, respectively, with the highest value in the combined treatment group. The trabecular bone thickness index of the RANKL inhibitor group was unchanged from that of the model control group.

[0196] Fig.18 In terms of trabecular spacing (Tb.Sp), the Tb.Sp value of the model control group was 0.7mm, the Tb.Sp value of the normal control group was 0.39mm, the Tb.Sp value of the RANKL inhibitor group and the model control group was basically the same, and the Tb.Sp values ​​of the stem cell group and the combined treatment group were 0.51mm and 0.48mm respectively. Similarly, the effect of Tb.Sp was that the combined treatment group was better than the stem cell group.

[0197] Fig.19 In terms of the trabecular number (Tb.N) index, the Tb.N value of the normal control group was 2.5mm, and that of the model control group was 1.3mm. There was no difference between the (Tb.N) index of the RANKL inhibitor group and the model control group. The Tb.N values ​​of the stem cell group and the combined treatment group were 1.62mm and 2.0mm, respectively. The effect of the combined treatment group was better than that of the stem cell group.

[0198] It was further proved that the stem cell group and the combined treatment group had a therapeutic effect on osteoporosis caused by retinoic acid, among which the combined treatment group combined with the RANKL inhibitor group had a better therapeutic effect.

[0199] Example 6. Tibia biomechanical changes

[0200] See Fig. 20 The drug was administered for 4 weeks. Tibial bonemechanics test was performed on the 8th week after administration. The maximum tibial compression load index was analyzed and compared. A comparative experimental analysis was performed between the model control group and the treatment group.

[0201] In terms of biomechanical change indicators, the value of the normal control group was 110N, the model control group was 60N, and the biological value of the RANKL inhibitor group was similar to that of the model control group, with no significant improvement. The biological values ​​of the stem cell group and the combined treatment group were 70N and 100N, respectively, indicating that the combined treatment group of MSC and RANKL inhibitor had a better effect.

[0202] The model control group showed a decreasing trend compared with the normal control group. After the administration of stem cells and combined treatment groups, the tibial load increased, and the maximum tibial compression load was higher than that of the model control group. Among them, the combined treatment group had the best effect.

[0203] Example 7. Continuous detection of bone turnover indicators

[0204] Three treatment groups, a normal control group, and a model control group were used for continuous follow-up testing. The drug was administered for four weeks, and bone turnover indexes were analyzed in the eighth week after the end of the drug administration.

[0205] Peripheral blood was collected from rats and analyzed using Rat / Mouse PINP EIA, Rat-Mid TM Osteocalcin EIA, RatLaps TM (CTX-I)EIA, RatTRAP TM (TRAcP 5b) ELISA kits were used to detect PINP, Osteocalcin, CTX-I and TRAcP 5b in serum. Figure 21-24 shown.

[0206] Fig.21 , bone formation index: PINP, the content of the model control group was significantly lower than that of the normal control group, and the PINP content of the stem cell treatment group and the combined treatment group was higher than that of the model control group.

[0207] Fig. 22 , Osteocalcin, the content of model control group was significantly lower than that of normal control group, and the content of PINP in stem cell treatment group and combined treatment group was higher than that in model control group.

[0208] Fig.23 , bone resorption index: CTX-I, the content of the model control group was significantly higher than that of the normal control group, and the PINP content of the stem cell treatment group and the combined treatment group was lower than that of the model control group.

[0209] Fig.24 , Tracp-5b, the content of the model control group was significantly increased compared with the normal control group. Similarly, the content of PINP in the stem cell treatment group and the combined treatment group was reduced compared with the model control group.

[0210] After modeling, the changing trend of bone turnover indicators was consistent with the symptoms of osteoporosis. After stem cell and combined administration, the symptoms of osteoporosis were alleviated.

[0211] The mesenchymal stem cells obtained through this scheme, in combination with RANKL inhibitors, can enhance the optimal repair effect on osteoporosis and have good application prospects.

[0212] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A pharmaceutical composition containing mesenchymal stem cells, characterized in that: The concentration of the prepared 6 cells / mL-2×10 6 cells / mL of mesenchymal stem cell intravenous injection and 400-800IU of vitamin D oral preparation; after preparation, one dose of mesenchymal stem cell intravenous injection contains 3×10 6 The solvent of the mesenchymal stem cell intravenous injection is composed of 0.9% sodium chloride injection and 20% human albumin injection in a volume ratio of (1-3): (3-1).

2. A pharmaceutical preparation package containing mesenchymal stem cells, characterized in that: One drug preparation package contains four mesenchymal stem cell biological preparations, four mesenchymal stem cell intravenous injection solvents and 28 vitamin D oral preparations. Each mesenchymal stem cell biological preparation contains multiple mesenchymal stem cell biological preparations, and each mesenchymal stem cell intravenous injection solvent contains multiple small mesenchymal stem cell intravenous injection solvents. One mesenchymal stem cell biological preparation contains 3×10 6 mesenchymal stem cells; a small portion of mesenchymal stem cell intravenous injection solvent includes 3.75mL of 0.9% sodium chloride injection and 1.25mL of 20% human albumin injection; the content of vitamin D in a vitamin D oral preparation is 400-800IU.

3. Use of the pharmaceutical composition containing mesenchymal stem cells according to claim 1 in the preparation of a drug for treating osteoporosis and fractures caused by estrogen changes in postmenopausal women.

4. Use of the mesenchymal stem cell-containing pharmaceutical preparation package according to claim 2 in the preparation of a drug for treating osteoporosis and fractures caused by estrogen changes in postmenopausal women.

5. A pharmaceutical composition containing mesenchymal stem cells, characterized in that: The concentration of the prepared 6 cells / mL-2×10 6 cells / mL of mesenchymal stem cell intravenous injection solution, and a RANKL inhibitor at a concentration of 0.3-0.5μg / ml; after preparation, one dose of mesenchymal stem cell intravenous injection solution contains 3×10 6 The solvent of the mesenchymal stem cell intravenous injection is composed of 0.9% sodium chloride injection and 20% human albumin injection in a volume ratio of (1-3): (3-1).

6. A pharmaceutical preparation package containing mesenchymal stem cells, characterized in that: One drug preparation package contains four mesenchymal stem cell biological preparations, four mesenchymal stem cell intravenous injection solvents and 14 RANKL inhibitors. Each mesenchymal stem cell biological preparation contains multiple mesenchymal stem cell biological preparations, and each mesenchymal stem cell intravenous injection solvent contains multiple small mesenchymal stem cell intravenous injection solvents. One mesenchymal stem cell biological preparation contains 3×10 6 mesenchymal stem cells; a small portion of mesenchymal stem cell intravenous injection solvent includes 3.75mL of 0.9% sodium chloride injection and 1.25mL of 20% human albumin injection; a portion of RANKL inhibitor contains multiple RANKL inhibitor intravenous injections, and each RANKL inhibitor intravenous injection contains 0.5μg RANKL inhibitor drug.

7. Use of the pharmaceutical composition containing mesenchymal stem cells according to claim 5 in the preparation of a drug for osteoporosis and fractures caused by decreased sex hormone levels.

8. Use of the mesenchymal stem cell-containing pharmaceutical preparation package according to claim 6 in the preparation of a drug for osteoporosis and fractures caused by decreased sex hormone levels.

9. The pharmaceutical composition containing mesenchymal stem cells according to claim 1 or claim 5, characterized in that: It also contains an oral vitamin C dosage of 50-300 mg.

10. The pharmaceutical preparation package containing mesenchymal stem cells according to claim 2 or claim 6, characterized in that: The vitamin C is 28 portions, and the content of vitamin C in each portion of the vitamin C oral preparation is 50-300 mg.

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