CN111632147A - Application of bone cell Wnt activator in preparing medicine for accelerating fracture healing, preventing and treating bone nonunion and no movement or weightlessness bone loss - Google Patents
Application of bone cell Wnt activator in preparing medicine for accelerating fracture healing, preventing and treating bone nonunion and no movement or weightlessness bone loss Download PDFInfo
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- CN111632147A CN111632147A CN202010510724.6A CN202010510724A CN111632147A CN 111632147 A CN111632147 A CN 111632147A CN 202010510724 A CN202010510724 A CN 202010510724A CN 111632147 A CN111632147 A CN 111632147A
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Abstract
The invention discloses an application of a Wnt (Wnt activator of osteocytes) in preparing a medicament for accelerating healing of fracture, preventing and treating nonunion and no movement or weightlessness bone loss, and relates to the two fields of fracture healing and weightlessness bone protection. The invention firstly proposes that the activation of the Wnt signal of the osteoblast can greatly accelerate the healing speed of the fracture and protect the weight loss bone by 100 percent, avoids a plurality of complications brought by the activation of the Wnt signal of the osteoblast, and is beneficial to better clinical outcome conversion and output. Meanwhile, the Wnt signal for activating the osteocyte is provided to treat delayed union of fracture or nonunion of fracture and even nonunion of bone, and is not influenced by the activity of osteoclast. The invention provides experimental data and research directions for clinical treatment of delayed fracture healing and nonunion. The invention also provides that the Wnt signal for activating the osteocyte can prevent and treat the weight loss bone loss by 100 percent for the first time, is beneficial to astronauts to prevent and treat the weight loss bone loss, and has important clinical significance and strategic value.
Description
Technical Field
The invention relates to two fields of fracture healing and weightless bone protection, in particular to application of a bone cell Wnt activator in preparing a medicament for accelerating fracture healing, preventing and treating nonunion and nonmovement or weightless bone loss.
Background
Wnt signals serve as the first five major developmental signals, and air-conditioning controls osteogenic, chondrogenic and adipogenic differentiation, and osteoclast differentiation. Wnt signaling is the most popular and successful in research, and has a division into canonical and non-canonical pathways that control cell fate during embryonic and post-natal stages, including cell proliferation, differentiation, polarization, migration, etc. Beta-catenin is a canonical Wnt signal transmitter, and when Wnt binds to its receptor Frizzld and co-receptor low density lipoprotein (Lrp)5/6, it is stabilized in the cytoplasm and enters the nucleus, where it cooperates with the transcription factor Tcf/Lef to start the expression of Wnt target gene.
Since 2001, clinical work found that inactivation or enhancement of Lrp5 produced osteoporosis or high bone density syndrome, however, basic research results suggest: the efficacy of Wnt signaling is determined by cellular nature (cell context), resulting in different efficacy for cells at different developmental stages.
Early developmental Wnt signaling promotes osteogenic differentiation, including mesenchymal, osteoprogenitor, and preosteoblast stages. In the progenitor cell stage, the inactivated canonical Wnt signal arrests osteogenesis and no bone is formed; at the same time, the progenitor cells differentiate into chondrocytes. In the preosteoblast stage, immature osteoblasts are produced, and at the same time, the preosteoblasts become adipocytes.
Late developmental Wnt signaling functions have been reported to be: it promotes the expression of its target gene Opg (osteoprotegerin), competitively binds to RankL to inhibit osteoclast differentiation and bone resorption. Mouse model shows: inactivating canonical Wnt signals in osteoblasts and/or osteocytes, downregulating Opg, increasing RankL/Opg ratio, promoting osteoclast differentiation, increasing bone resorption, and reducing bone mass (Glass II, 2005; Mov mirare-Srtic, 2014).
Osteocytes are ancient cells, as early as found in skeletal fossils in fish and dinosaurs. The number of the bone-setting agent accounts for the absolute majority, accounts for more than 90 percent of the total number of cells of the largest organ-bone of a human body, has a remarkable service life, and survives for decades. However, because the bone mineral is embedded in mineralized bone matrix, the bone mineral is slow in metabolism and is like degraded tissues, so that the bone mineral is less researched, and the function of the bone mineral is not dug.
Fracture healing is referred to as postnatal bone development. Fracture healing is a process that reproduces embryonic bone development, including repairing damaged bone, restoring healthy cellular composition, load-bearing structure and biomechanical properties, and biological function prior to fracture.
After the fracture occurs, soft tissue inflammation and edema can occur, local and systemic inflammatory reaction is started, proinflammatory cytokines are released, and immune cells are recruited to reach the injury part. Releasing osteogenic factors such as bone morphogenetic protein, TGF beta, Wnt10b, etc., recruiting and stimulating the surrounding stem cells, progenitor cells and stromal cells to differentiate into osteoblasts and form new bones. Meanwhile, the osteoclast is activated to differentiate and mature, and damaged bone tissues are removed. Finally, scarless healing occurs.
The healing process involves two osteogenic modes, intramembranous ossification and endochondral osteogenesis. The former is the direct differentiation of progenitor cells into osteoblasts, the latter is the formation of a cartilage template, which is then reconstituted after calcification. Intramembranous ossification occurs at the periosteal surface of the fracture site, while endochondral ossification occurs mainly in the center of the fracture site. In an anaerobic environment lacking blood vessels, chondrocytes differentiate and form cartilage callus. Activates the HIFla molecular pathway, generates VEGF, induces angiogenesis and restores the circulatory system.
The formation of blood vessels affects fracture healing and provides a microenvironment for hematopoietic stem cells residing in the bone marrow. Osteoclast (chondroclast) precursor cells and osteoprogenitor cells are brought in through blood, and are differentiated into broken chondrocytes for removing calcified cartilage and osteoblasts for forming new bone, and exert respective functions to form new bone. Thus, the development of osteoblasts is self-evident for fracture healing.
However, there are different insights into the role of osteoclasts in fracture healing. Although it is essential for bone remodeling, little mention is made of bone formation. It not only plays a role in removing damaged bone tissue, but also has a function of recruiting vascular invasion and osteoprogenitor cells at the early stage of fracture. Research has already proposed that when osteoclasts work, TGF beta 1 is activated, stem cells are recruited to reach bone absorption sites, and the bone is differentiated in situ to match bone absorption and achieve bone homeostasis. Production of bone growth factors or inhibitors, regulating osteogenic differentiation, has also been reported, such as Wntl0b, sclerostin, Semaphorin 4D.
Fracture is a common injury of trauma, and normal life and work are affected to a certain extent and for a certain period of time due to the long healing time and the need of braking in the early treatment period. Delayed union and fracture nonunion place a significant burden on the patient and medical system. Typically, fractures heal spontaneously. However, in 5-10% of cases, delayed or non-union fractures (collectively DNFs) may occur in fracture healing. Recent data on open long bone fractures show that 17% of patients develop bone nonunion, and another 8% develop delayed healing. Notably, NDFs occur at a high rate, particularly in the elderly. In 2009, the Chinese white paper for osteoporosis mentioned that in China, the population of osteoporosis is about 8000 ten thousand. 40% of women and 10% of men will experience one fracture of the hip, vertebra or wrist. At present, the incidence of DNFs is increased due to the fact that the old population in China is increased, the number of osteoporosis patients is increased day by day, and the patients are younger. DNFs can cause a significant morbidity, resulting in decreased productivity and decreased quality of life, and thus, nonunion of long bones is highly disruptive to quality of life, comparable to or even exceeding the effects of type i diabetes, stroke or aids. Although operation is the main treatment means, the molecular mechanism influencing osteogenesis by various risk factors is unclear, and no ideal symptomatic intervention medicine exists.
Bone formation is a great need for the treatment and rehabilitation of many diseases in clinics in orthopedics. Research on bone damage and repair based on healthy bone reformation of bone development has been always a prelude in this field, and a large number of studies have been conducted in countries such as the united states, european union, japanese korea, and the like. Numerous studies have attempted to obtain osteogenic advantages in osteoblast proliferation, and previous basic studies have not focused on reports of Wnt signaling promoting osteogenesis in later stages of bone development, leading to the classic questions of the industry. Plus Kousteni team report at Columbia university, USA: activating the osteoblast canonical Wnt signal, up-regulating the expression of Notch (one of five developmental signals) ligand Jag1, increasing the Notch signal of hematopoietic stem cells, generating anemia and medullary cell expansion, acute leukemia and perinatal death, and making both academic and clinical doubts about the application of Wnt signal transformation.
At present, the aerospace industry is developed in a competitive way, and when astronauts experience weightlessness, bones can quickly degenerate and serious bone loss is caused; after returning, long-time (3-6 months) closed treatment and rehabilitation are needed.
Aging, ambulatory, weightlessness, etc. have long been known to increase osteoclast and bone resorption, thereby reducing bone mass. There are many drugs in clinical practice that reduce bone resorption, but few reports have reported that such drugs are suitable for protecting astronauts from performing tasks during aerospace flight. Research and development of medicines for preventing and treating weightlessness bone loss is a current research hotspot, researches on how to improve the bone mass of patients with osteoporosis and prevent the influence of astronauts on weightlessness are carried out, and the medicine has important clinical significance and strategic value.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide application of a Wnt (Wnt-activating agent) of osteocytes in preparing medicaments for accelerating healing of fracture, preventing and treating nonunion and no movement or weightlessness bone loss.
The invention is realized by the following steps:
an application of a bone cell Wnt signal path activator in preparing a medicament for accelerating fracture healing.
In the preferred embodiment of the present invention, the healing time of the above-mentioned bone fracture healing accelerating medicine is only half of the healing time of natural bone fracture.
In a preferred embodiment of the present invention, the above-mentioned Wnt pathway activator of bone cells activates Wnt signaling in bone cells by any drug or means, so as to achieve therapeutic effect.
Preferably, the bone cell Wnt signaling pathway activator is any drug or pathway to activate Wnt signaling in bone cells;
the fracture is at least one of a compression vertebral fracture, a hip fracture, a lumbar fracture, a femoral fracture, and a tibial fracture.
The inventor firstly suggests that the effector cells of Wnt/beta-catenin for generating bone anabolism are osteocytes in the previous research, and the established osteocyte classical Wnt signal model mouse does not have leukemia even if the mouse grows to the adult stage. After the research report, the physiological significance of the discovery and the potential application value thereof are continuously and highly evaluated in the journal of Nat RevEndocrinol 2015, 11: 192; Nat Rev Rheumatotol, 2015, 11: 128). Recently, the inventor originally found that the Wnt of the osteocyte can improve the proliferation and self-renewal capacity of the stem cell in vivo and in vitro.
Therefore, based on the innovative findings, the inventors propose a scientific hypothesis that the Wnt-activated osteocyte accelerates the healing of fracture, and the healing of fracture is evaluated by performing a conventional fracture operation on the Wnt-activated osteocyte mice and littermate WT mice. Research results show that the bone cell Wnt can greatly accelerate the healing speed of the fracture.
The inventor focuses on osteocytes, and researches physiological and pathological changes of rapid fracture healing generated by the Wnt signals of the osteocytes on the basis of avoiding various complications brought by the activation of the Wnt signals of the osteoblasts, so that the Wnt signals of the osteocytes are beneficial to better clinical outcome transformation and output.
Evaluation of the healing effect of osteocyte Wnt signaling on a normal physiologic fracture model confirmed the surprising rate of osteocyte Wnt signaling on fracture healing and the Wnt-activated fracture healing pattern in mice differed from the conventional endochondral osteogenesis pattern.
The third exon of the Catnb gene for coding the beta-catenin protein is deleted, the third exon codes a beta-catenin continuous phosphorylation site sequence, and the beta-catenin continuous phosphorylation site sequence is degraded and eliminated after being ubiquitinated. And the Catnb gene with the deletion of the third exon can continuously and stably express beta-catenin protein without degradation. The beta-catenin protein enables the expression of the Wnt signal channel to be continuously kept at a high level and becomes dominant activation, and the inventor finds and proves that the Wnt signal of the bone cell is activated to promote the osteogenesis.
DMP1 protein (dentin matrix protein 1) is a typical bone matrix acidic protein. In bone tissue, DMP1 is expressed primarily by osteocytes, while DMP1 expression is also present in osteoblasts and chondrocytes. DMP1 has direct regulation effect on the mature differentiation of osteoblasts and bone mineralization.
8kbDMP1 was linked to Cre and expressed specifically only in bone cells, with DMP1 as the promoter. The 8kb long DMP1 promoter sequence differs from the original 10kb DMP1 promoter sequence because the 10kb promoter facilitates gene expression in bone and osteoblasts.
The Wnt signaling of osteocytes promotes bone formation and bone resorption, resulting in anabolism greater than catabolism. Unlike the function of osteoblast Wnt, osteoblast Wnt does not promote osteogenic metabolism, but inhibits osteoclast differentiation, reduces bone resorption, and increases bone mass.
An application of a bone cell Wnt signal channel activator in preparing a medicament for treating delayed union or nonunion of fracture.
In the preferred embodiment of the present invention, the fracture is caused by the clinical non-union of open fracture or closed fracture; preferably, the bone fracture does not heal due to bone resorption inhibitor; preferably, the bone resorption inhibitor is an inhibition of bone resorption by a bisphosphonate drug; preferably, the bisphosphonate drug is at least one of alendronate sodium, risedronate sodium, ibandronate sodium and zoledronic acid.
In a preferred embodiment of the present invention, the above-mentioned Wnt pathway activator of bone cells activates Wnt signaling in bone cells by any drug or means, so as to achieve therapeutic effect.
Preferably, the osteocyte Wnt signal pathway activator activates the osteocyte Wnt signal by specifically expressing beta-catenin protein only in the osteocyte.
Clinically, there are different views on the use of bisphosphonates and their drug holidays. In the twenty th conference on orthopedics and the thirteenth COA international academic conference held in Shanghai in 2019, experts in the group of osteoporosis and fracture have been discussed strongly about drug holidays and fracture risks. Bisphosphonates are thought by the scholars not to affect fracture healing, and there are questions asked at the site and thought to reduce osteogenic function after osteoclast inhibition.
The inventor manufactures a fracture nonunion model caused by bone resorption inhibition by ZA (zoledronic acid), evaluates the fracture union condition of a mouse activated by the Wnt of the osteocyte in the fracture nonunion model caused by the bone resorption inhibition, and proves that the Wnt signal of the osteocyte can reverse and inhibit the fracture nonunion caused by the bone resorption.
The inventor finds out that the Wnt signal of the osteocyte has strong promoting effect on the fracture and simultaneously proves that the Wnt signal of the osteocyte has unprecedented superior effect on treating delayed union and even nonunion of the fracture. Provides experimental data and research direction for clinical treatment of delayed fracture healing and bone nonunion.
PTH and PTHrP (parathyroid hormone-related protein) are clinically very effective osteogenesis-promoting drugs. PTH and PTHrP are important regulators of bone metabolism. PTH was the first anabolic drug approved by the FDA for the treatment of osteoporosis. PTH intermittent injection mediates osteoblast anabolism, stimulates osteoblasts to secrete factors such as interleukin-1 and interleukin-6, activates osteoclasts, and promotes the synergistic effect of osteoblasts and osteoclasts. Thus, PTH is frequently used for the prevention and treatment of osteoporosis, bone fractures, bone defects and bone loss.
A large number of clinical and basic experimental studies confirm that PTH is effective in fracture repair, and the injection of rat PTH (1-34) subcutaneously at a concentration of 30ug/kg per day for closed femoral fractures was found to improve torsional strength, hardness, bone density, bone volume, cartilage size, etc. by day 21. Another report achieved very good results: PTH (1-84) treated 65-position menopausal, loose bone, pelvic closed fracture patient, injected 21 position 100 microgram daily, compared with 44 position, healing time was shortened from 12.6 weeks of control group to 7.8 weeks, 38% reduced healing time, and more improved function. These are all closed fracture healing data, indicating that PTH can treat and prevent bone nonunion or delay healing. Although the promotion of bone fracture by PTH has been demonstrated, the mechanism of promoting bone fracture healing has rarely been elucidated.
The effect of osteoblasts on PTH osteogenesis is remarkable due to the prominent osteogenesis effect of PTH and the good treatment effect on bone fracture. The inventors designed a control experiment to evaluate fracture healing in mice following routine fracture surgery with continuous daily PTH injections (clinically intermittent injection therapy). Meanwhile, a nonunion model of fracture due to bone resorption inhibition was made with ZA, and fracture healing of PTH in the nonunion model of fracture due to bone resorption inhibition was evaluated. In addition, clinical sequential therapy (PTH-ZA) was simulated to assess the persistence of the osteogenic effect of PTH at fracture.
The inventors evaluated Wnt signaling in osteocytes and the healing effects of pharmaceutical factors PTH (parathyroid hormone) and ZA (zoledronic acid) using osteocytes as target cells on a model of bone fracture nonunion caused by inhibition of bone resorption, and found that Wnt signaling in osteocytes is not affected by inhibition of bone resorption, and that Wnt signaling in osteocytes exhibits a potent healing therapeutic effect in a model of bone fracture nonunion caused by inhibition of bone resorption. Since the bone cell Wnt is independent of mechanical factors and does not need a special osteogenesis mode of mechanical stimulation, the Wnt is expected to become a gospel for a patient suffering from bed-ridden bone nonunion after clinical transformation is completed.
An application of a Wnt signal channel activator of a bone cell in preparing a medicament for preventing and treating bone loss.
Research on bone damage and repair based on healthy bone reformation of bone development has been always a prelude in this field, and a large number of studies have been conducted in countries such as the united states, european union, japanese korea, and the like. In the process of weightlessness, mechanical stimulation and osteogenic medicaments such as PTH, active vitamin D and the like are difficult to exert similar effects on the earth, and the bone loss is not protected enough; the sclerostin (competitive inhibitor protein of the Wnt receptor) antibody with the best bone increasing effect only inhibits the bone loss of the mice caused by partial weight loss, and provides good experimental data for near-earth flight. Continued production of sclerostin in weight loss, whether injection of sclerostin antibody can counteract weight loss of bone for long periods? Is long-term injection associated with side effects under weightlessness conditions? These problems all require systematic investigation.
The invention provides a scheme capable of preventing and treating the weight loss bone loss by 100 percent, and the model mouse is subjected to tail suspension simulation weight loss for only two weeks to cause a large amount of rapid bone loss, but the weight loss bone loss can be protected by 100 percent by activating the bone cell Wnt. After the wild control mouse loses weight, the expression of sclerostin is greatly increased, osteogenic differentiation is greatly reduced, and osteoclast differentiation and bone resorption are greatly improved; however, the osteocyte Wnt can maintain osteoblastic differentiation, and inhibit osteoclast differentiation and bone resorption improvement caused by weight loss. In international weight loss studies, it is rare to report a protective effect that can achieve this degree. Based on its complete prevention of bone loss due to weight loss, and strong bone regeneration capacity, we propose: the bone cell Wnt is independent of physiological action of reducing sclerostin expression to increase bone, and is suitable for preventing space bone loss.
In a preferred embodiment of the present invention, the bone loss is bone loss caused by an out-of-force stimulation; preferably, the bone loss is bone loss resulting from weight loss, no movement; preferably, the weight loss is simulated space weight loss, ground-approaching flight weight loss or space weight loss.
In a preferred embodiment of the present invention, the above-mentioned activator of Wnt signaling pathway in osteocytes activates Wnt signaling in osteocytes through any drugs or means, so as to achieve therapeutic effects.
The invention has the following beneficial effects:
the invention firstly proposes that the activation of the Wnt signal of the osteoblast can greatly accelerate the healing speed of the fracture and protect the weight loss bone by 100 percent, avoids a plurality of complications brought by the activation of the Wnt signal of the osteoblast, and is beneficial to better clinical outcome conversion and output. Meanwhile, the Wnt signal for activating the osteocyte is proposed to treat delayed union or nonunion of fracture and is not influenced by the activity of osteoclast. The invention provides experimental data and research directions for clinical treatment of delayed fracture healing and nonunion. Since the bone cell Wnt is independent of mechanical factors and does not need a special osteogenesis mode of mechanical stimulation, the Wnt is expected to become a gospel for a patient suffering from bed-ridden bone nonunion after clinical transformation is completed. The invention also provides that the Wnt signal for activating the osteocyte can prevent and treat the weight loss bone loss by 100 percent for the first time, is beneficial to astronauts to prevent and treat the weight loss bone loss, and has important clinical significance and strategic value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a technical roadmap for experimental study of a fracture part shown in example 4;
FIG. 2 is a graph showing the results of experiments in Experimental example 1 in which Wnt signaling activating osteocytes accelerates fracture healing;
FIG. 3 is a graph showing the experimental results of the effect on the healing of a fracture in a mouse after inhibiting bone resorption in Experimental example 1;
FIG. 4 is a graph showing the change of osteoclast during the healing of a fracture in a mouse in Experimental example 1;
FIG. 5 shows da β cat in examples 1 and 2OtResults of fracture healing pattern in mice;
FIG. 6 is a histological staining analysis chart of the right tibia specimen at the fourth week after fracture in each experimental group in Experimental example 4;
FIG. 7 shows the results of bone density measurement of right femur and 1-6 lumbar vertebrae and serum P1NP level of each experimental group of mice in Experimental example 4;
FIG. 8 is a graph showing the results of experimental example 5 of the change in the callus area, cartilage area, and cartilage ratio in the fracture healing process due to the inhibition of bone resorption;
FIG. 9 is a statistical chart of information on experimental animals in Experimental example 6;
FIG. 10 shows the bone loss-bone density and micro-CT detection caused by the Wnt protection simulated weightlessness of activated bone cells;
figure 11 is a graph of the results of the effect of activating osteocyte Wnt on osteoclast number and bone resorption;
fig. 12 is a graph showing the results of bone loss-bone histomorphometry and osteogenic differentiation assays due to Wnt-protective simulated weightlessness in activated bone cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
This example performs the model construction of experimental mice.
Constructing Dmp 1-Cre; catnb f/+ mice.
Mouse da β cat activated by osteocyte canonical Wnt signalOtThe mouse is established by applying Cre-loxP technology, and is produced by Catnb lox (ex3) × DMP1-8kb-Cre mating Catnb gene coding β -catenin protein inserts creloxp sequence at exon 3, is cut out after being recognized by Cre recombinase, is degraded after being ubiquitinated after being continuously phosphorylated because continuous coding site sequence of exon 3 coding 2-catenin is continuously phosphorylated, and β -catenin without exon 3 is not degraded and is stabilized in cytoplasm to be continuously expressed in a cytoplasm to be a high level of Wnt signal expression, and is expressed only on a transgenic bone marrow cell expressing DMp 64-8 kb-expressing a gene expressed on a table 16-expressing a gene expressing a protein of gene expressing a gene.
Table 1 animal model classification table.
Example 2
(1) Constructing Dmp 1-Cre; catnb f/+ mouse fracture model.
The fracture model is manufactured by adopting an operation method. Mice of 8-10 weeks, 20 in each of the control group and the experimental group were selected, and were anesthetized with an intraperitoneal injection of 5% chloral hydrate (0.4mg/g) at a concentration of 0.08ml/10 g. After 30 minutes, mice were operated with a continuous inhalation of buprenorphine (0.1 mg/kg). After the skin of the right leg of the mouse is prepared, 70% alcohol is used for disinfection, the right leg is parallel to the shinbone, the skin is cut open, then a 6mm incision is made above the patella and perpendicular to the skin by using a No. 11 surgical blade, the muscle is separated in a blunt manner, the upper 1/2 of the shinbone is exposed, then a incision is made perpendicular to the inner side of the patella for exposing the tibial plateau, a 0.4mm syringe needle is used for penetrating bone marrow from the tibial plateau, then a 0.26mm needle-free pin is used for inserting into a marrow cavity, the other end of the pin penetrates out from the far end of the shinbone, and the redundant needle is cut off to enable the pin to be flush with the tibial. The soft tissue in the upper part of the distal tibia is then blunt dissected, followed by gradual pressure testing perpendicular to the tibia using ophthalmic scissors until a transverse fracture develops. Finally, the muscle and the skin are sutured in sequence by absorbable suture. And (3) putting the mice subjected to the operation on a water bath kettle at 37 ℃ to wake up, and after waking up, returning the mice to the breeding room to continue breeding and observing.
(2) Constructing Dmp 1-Cre; catnb f/+ fracture model at the time of bone resorption inhibition in mice.
Dmp 1-Cre; the Catnb f/+ mice were injected with 0.1mg/kg zoledronic acid in the abdominal cavity one week before surgery, fracture models were made by conventional surgery, and the middle-upper part of the tibial shaft on the right side of the mice was selected as the fracture point for intramedullary fixation, and the specific method was the same as that in example 1. Mice were observed for fracture healing by X-ray weekly.
The statistical table of the information of the experimental mice is shown in the following table 2.
Table 2 statistical table of experimental mouse information.
Example 3
(1) Constructing a mouse fracture model when wild mouse bone resorption is inhibited.
0.1mg/kg zoledronic acid is injected into the abdominal cavity one week before the operation, a fracture model is manufactured by adopting the conventional operation, the middle part of the tibial shaft on the right side of the mouse is selected as a fracture point, and the intramedullary fixation is carried out, and the specific method is as shown in the example 1. Mice were observed for fracture healing by X-ray weekly. (2) Constructing a wild-type mouse fracture model intermittently and continuously injecting PTH under the condition of inhibiting bone resorption.
100ng/g PTH (bachem, cat 4011474.0005) was subcutaneously injected every day around the beginning of the surgery, 0.1mg/kg zoledronic acid (Nowa pharmaceutical) was intraperitoneally injected one week before the surgery, a fracture model was prepared by the surgery method shown in example 1, and the middle part of the tibial shaft of the right side of the mouse was selected as a fracture point to perform intramedullary fixation. Mice were observed for fracture healing by X-ray weekly.
Example 4
This example provides methods for tissue sample treatment of all groups of fractured mice, and the treatment and sampling time for each group is shown in FIG. 1.
Panel A in FIG. 1 is wild type and da β catOtThe mice were subjected to bone resorption inhibitor (ZA) sample treatment experimental design, and B in fig. 1 is an experimental design for intermittent continuous injection of PTH under bone resorption inhibiting conditions.
Panel B in figure 1 is a randomized assignment of 16 week old female C57 mice to five experimental groups (n >5) based on right femoral bone density values: PTH-PTH group, PTH-ZA group and PTH-PTH + ZA group were subcutaneously injected with PTH (100ng/g) daily; while the vehicle-vehicle group and the vehicle-ZA group were subcutaneously injected daily with an equal body weight ratio of PTH solvent (vehicle). Three weeks after the consecutive injections, vehicle-ZA group, PTH-ZA group and PTH-PTH + ZA group were intraperitoneally injected with ZA (0.1ug/g) once; equal body weight proportion saline was injected into the vehicle-vehicle group and the PTH-PTH group (C57 mice were purchased from experimental animals center of Chongqing university of medicine).
After ZA/saline injection, both of the PTH-PTH group and PTH-PTH + ZA group were subcutaneously injected with PTH (100ng/g) daily until harvest. While the vehicle-vehicle group, the vehicle-ZA group and the PTH-ZA group were subcutaneously injected with an equal amount of PTH solvent (vehicle) per day according to body weight until collection. Solvents (vehicles) for PTH include acetic acid, β -mercaptoethanol and NaCl. Acetic acid: chongqing chemical industry, beta-mercaptoethanol: sigmaM3148-25ML, NaCl: chongqing Chuandong chemical industry.
(1) Collection of mouse fracture samples
Samples were collected from mice sacrificed at days 7, 14, 21, 28, and 56, respectively, after surgical fracture of the mice. The fracture healing condition of the mice is observed by X-ray on the 7 th, 14 th, 21 th and 28 th days of postoperative fracture. New bones formed were double-labeled with calcein and alizarin red (alizarin red), 7 and 2 days before the mice were harvested, respectively, with reference to the previous method (Tu, x., ethyl. Blood was collected from the mice before sacrifice. The right tibia was isolated, the internally fixed needle was removed, and immediately fixed with 4% formalin. The contralateral tibia is immediately placed in liquid nitrogen for freezing storage after soft tissues are removed.
(2) The speed and quality of fracture healing in mice were determined.
A. Determination of bone mass, bone structure and strength in mice: the bone content, bone density of the total bone, lumbar vertebrae and femur, bone volume of dense/cancellous bone, number, thickness and gap distance of trabeculae of bone were measured by X-ray in the same manner as described in (Tu, X, 2015; Hilton, 2008; Tu, X, 2012), respectively.
B. Determination of mineralization and osteogenesis rates in mice: as described in our previous methods (Tu, X, 2015; Hilton, 2008; Tu, X, 2012), new bones formed with calcein and alizarin red (alizarin red) double markers were used 7 and 2 days before the mice were harvested, and the rate of formation of cortical and cancellous bone was determined and calculated using bone histomorphometry (bone osteometrics).
C. Determination of osteoclast and bone resorption transformation:
osteoclast number was counted by histological specific staining of osteoclasts as before (Tu, X, 2015; Hilton, 2008; Tu, X, 2012);
serological bone metabolism biochemical markers were determined by ELISA: CTX, osteocalcin, P1NP, alkaline phosphatase, PTH and the like, and analyzing the bone resorption transformation level; serum calcium and phosphorus levels were determined chemically and the effect on calcium and phosphorus metabolism was investigated.
D. Osteoblast and bone formation assays: the right tibia was fixed by conventional histology, i.e., after being fixed overnight in 10% neutral formalin, it was replaced with 70% alcohol and stored at 4 ℃ until use. Decalcified with 14% EDTA/PBS (pH7.4) for 1-2 weeks, dehydrated and embedded in paraffin, and sectioned at 6 μm. Immunohistochemical and in situ hybridization techniques (referred to Hu, H., et al., Sequential role of Hedgehog and Wnt signaling in osteoblast grade. and Tu, X., et al., Noncanic Wnt signaling through G protein-linked PKCdelta activating ligands bone formation.) were used to detect osteoblast series markers including Runx2, osterix, collagen 1, and osteocalcin.
(3) Measuring the expression of certain stimulating factors such as RANKL, sclerostin and Wnt signal path related genes and proteins at the fracture healing part. The right tibia was fixed by conventional histology, i.e., after being fixed overnight in 10% neutral formalin, it was replaced with 70% alcohol and stored at 4 ℃ until use. Decalcified with 14% EDTA/PBS (pH7.4) for 1-2 weeks, dehydrated and embedded in paraffin, and sectioned at 6 μm. The expression conditions of RANKL, sclerostin and Wnt signal path protein are detected by real-time fluorescent quantitative PCR technology.
Experimental example 1
Experimental study proves that the Wnt activating osteocyte accelerates fracture healing and can reverse fracture nonunion caused by bone resorption inhibition.
(1) The Wnt signal of the activated bone cell accelerates the healing of the fracture, and can shorten the healing time of the fracture by half.
Under the condition of no intervention, a fracture healing model of the right tibia of the mouse (shown in figure 2) is constructed by a conventional operation method, and one, two, three and four side tibia samples after fracture are respectively collectedOtThe fracture of the mouse is fuzzy and heals; while the fracture line of the wild-type mice in the control group remained clear and did not heal until four weeks (see FIG. 2). The result of the image analysis shows that the bone cell Wnt obviously accelerates the fracture healing.
Paraffin section HE staining results showed that fracture healing in control mice underwent four processes, with typical blood callus, remodeling and healing periods occurring at first, second, third and four weeks post fracture, however, da β catOtThe performance of the mice is completely different: at week one, the callus formed was small and by week two, complete healing occurred (see figure 2). Thus, it was clarified that the Wnt signaling of osteocytes has the function of shortening the healing time of half of the fracture in mice.
In fig. 2, blue arrows indicate blood callus, red arrows indicate healing, and green arrows indicate callus.
(2) Zoledronic acid delayed fracture healing in control mice, but did not affect da β catOtThe mice accelerated fracture healing.
Mice were monitored for bone density (BMD) weekly for 5 weeks following intraperitoneal injection of the bone resorption clinical drug Zoledronic Acid (ZA). Compared with mice without ZA injection, BMD of the mice after 1 week of injection does not change obviously, and BMD of the left femur, the right femur and the L1-6 lumbar vertebra are increased obviously at week 5 (shown in figure 3), which indicates that zoledronic acid plays a clinical role, inhibits bone absorption and increases bone mass.
The bone resorption index began to decrease significantly at the first week (i.e. before fracture surgery) and the levels of CTX decreased by 22.44% and 23.26% for the control wild type and da β catOt mice, respectively, before comparative injection. And the CTX values continued to decrease, with the levels of CTX decreasing by 28.86% and 26.25% in control wild type and da β catOt mice, respectively, at week 4 post ZA injection (i.e., week 3 post fracture) (see panel C in fig. 3). This fully suggests that ZA functions clinically and inhibits bone resorption.
ZA treatment, control mice had nonunion around the fracture, and we continued to observe nonunion around the eighth week, histological staining of tibial fracture specimens showed that ZA-treated wild-type mice had no union and increased callus around the fracture, and no tendency to union around the fracture at the eighth week, however, da β catOtWhile the mice were still in the second week, complete healing occurred, and fracture healing time was still half of the conventional healing time of the control mice (D-panel in fig. 3, red arrows indicate healings).
It can be concluded that bone resorption is important for natural healing of mouse fracture, and bone resorption is inhibited, resulting in nonunion of fracture. After the Wnt signal of the bone cells is activated, the fracture healing can not be influenced by the bone resorption height. This study finding indicates that: the bone cell Wnt has clinical transformation value and can heal bone nonunion.
Experimental example 2
This example was conducted to study the change of osteoclasts in the healing process of a fracture in a mouse.
As shown in FIG. 4, in a wild-type mouse, osteoclasts began to appear at the fractured site in the first week after fracture, and then a large amount of osteoclasts were produced, and the number of osteoclasts reached a peak in the third week and decreased to the original state before fracture when healing occurred in the fourth week, da β catOtThe number of osteoclasts in the fractured region of the mice increased stepwise like in the wild type mice, but at 1 and 3 weeks after fracture, the number of osteoclasts was lower and higher than in the wild type mice, respectively, and the number of osteoclasts per tissue area at the second peripheral fracture line (oc.n/t.ar) was equal in da β catOtThe overall tendency of osteoclast generation was higher when the mice were fractured than in wild-type mice (see FIG. 4).
Experimental example 3
The experimental example further studies the fracture healing mode of the mouse activated by the bone cell Wnt, and the result shows that the fracture healing mode of the mouse activated by the bone cell Wnt is different from the conventional endochondral bone formation mode.
The previous experiments demonstrate that the bone cell Wnt can accelerate fracture healing and heal nonunion. To determine whether it passed the conventional endochondral osteogenic healing mechanism, fracture samples were analyzed by ABH-OrangeG staining.
Staining results referring to FIG. 5, panel A in FIG. 5 is an Allen Blue Haematoxylin-Orange G (ABH/OG) staining pattern of right-side fractured tibia specimens after 1, 2, 3, 4 weeks of conventional fracture of mice. Wild type mouse forms the blood scab when 1 week after the fracture, sees that there is the regular chondrocyte of similar growth plate appearance arrangement, and week 2 osteogenic differentiation forms the callus, and corresponding osteocyte quantity reduces, and 3 week bone remodeling, chondrocyte disappearance completely, and a small amount of mesenchymal fibrous tissues appear in fracture healing department, remodels all around the fourth and accomplishes, and the fracture is restoreed completely.
However, da β catOtMice show relatively few chondrocytes in small eschars at fracture week 1 wild type and da β cat relative to the respective eschar areaOtRat produced similar cartilage area ratios at week 2 da β catOtThe rat osteogenic differentiation produces new bone, healing the fracture. A large amount of mesenchymal fibrous tissues can be seen at the fracture site, and chondrocytes are depleted in advance. At weeks 3 and 4, fracture healing was already completed and the mesenchymal fibrous tissue gradually became bone.
Panel B of FIG. 5 is an AlcianBlue Haematoxylin-Orange G (ABH/OG) staining pattern of right-side fractured tibia specimens after 1, 2, 3, 4 weeks of mice injection of zoledronic acid fractureOtRat callus and cartilage formation at fracture 1 week post fracture, wild type and da β catOtMice developed scabs and cartilage of similar size to those without each ZA treatment. However, in contrast, wild-type mice had clear fracture lines at 2 weeks post-fracture, no reduction in cartilage proportion and healing occurred.
Histomorphometry quantified cartilage area in callus and the results showed da β catOtThe area of cartilage in the mouse callus was significantly smaller than in the control mice (panel C in figure 5), and it can be seen that the osteocytes Wnt-activated miceThe mode of regenerative healing after fracture is different from the conventional endochondral osteogenesis mode. However, the fact that the healing of the mice is accelerated by the Wnt activation of the osteocytes still needs to be further explored.
Experimental example 4
The experimental example investigated the effect of bone resorption inhibition on fracture healing and the reversal of PTH on bone resorption inhibition-induced fracture nonunion. The results demonstrate that continuous intermittent injection of PTH can reverse the fracture nonunion resulting from bone resorption, but clinical sequential PTH-ZA therapy cannot reverse the fracture nonunion.
Since the fracture healing of the conventional mouse tibia fracture model can be completed in the fourth week after fracture (see the above experimental results), samples of the fourth week after fracture are taken for histological staining analysis, and the analysis results are shown in fig. 6.
FIG. 6 shows that the fracture healing process was completed 4 weeks after the blank control (vehicle-vehicle) mice had been treated, and the connection between the fractured ends was intact. It can be seen that after callus remodeling, the marrow cavity is unobstructed and returns to the normal form before fracture.
However, after the clinical drug for inhibiting bone resorption, Zoledronic Acid (ZA), was injected, the tibial bone fracture of the mice in the bone resorption-inhibiting group (vehicle-ZA) did not heal, the callus was not remodeled and disappeared, the shape of the callus became large, and no new bone ingrowth was observed between the fractured ends.
PTH is a bone-strengthening drug used clinically and has been confirmed to have a function of promoting fracture healing. After 4 weeks of fracture, the tibia fractures in PTH intermittent injection group (PTH-PTH) mice healed, but remodeling was not completed, the callus was still large, including many trabeculae growing between the fractured ends of the fracture where new bone connections had grown.
The PTH-ZA group, which was treated as a clinical sequential therapy for osteoporosis, did not heal, and appeared to have a similar appearance to the vehicle-ZA group, forming large callus. Trabeculae are newly grown in the marrow cavity at the two ends of the callus. And (4) prompting: PTH is short-lived and essentially loses osteogenic function after withdrawal. Therefore, after PTH withdrawal inhibits bone resorption, there is no supplementation of osteogenic factors at the fracture site, thus affecting bone remodeling.
However, when PTH was not discontinued (PTH-PTH + ZA), this deficiency was compensated, healing occurred, and the healing profile was consistent with that of the ZA-free, PTH-PTH group. Therefore, we initially conclude that: inhibition of bone resorption leads to nonunion, at which time, in combination with PTH treatment, nonunion can be reversed.
In FIG. 7, panels A and B show the results of bone density measurements of the right femur and 1-6 lumbar vertebrae of mice in each experimental group after ZA and PTH treatment. The bone density was measured at the time points: the first day (-4w) of intermittent PTH injection started 4 weeks before surgery, the day (-1w) of ZA injection, the beginning of surgery one week later (0w), 1 week after surgery (1w) and 4 weeks after surgery (4 w). Fig. 7 shows that both ZA and PTH increased femoral and lumbar vertebral density compared to the control group. The increase in bone density of mice in the PTH group began at week 3; the group ZA is a little earlier, beginning at week 2, after which the bone density continues to increase.
The determination of the level of the serum osteogenesis marker P1NP (see FIG. 7): the percentage increase in serum P1NP at week 1 post-fracture compared to pre-experiment (-1 week, i.e. one week prior to fracture, time point of ZA/saline injection) showed similar reductions in both the vehicle-vehicle group, vehicle-ZA group and PTH-ZA sequential treatment group, with significant increases in serum P1NP levels only in PTH-PTH group and PTH-PTH + ZA group.
The experimental example shows that: inhibition of bone resorption or treatment with osteogenic drugs (PTH) increased bone density in mice. The former does not increase bone formation, and only through continuous and intermittent injection of PTH, bone formation can be continuously increased. Clinical sequential therapy with PTH-ZA failed to increase bone formation in mice, demonstrating that PTH-ZA failed to complete part of the mechanism of fracture healing.
Experimental example 5
In order to investigate the mechanism of the effect of inhibiting bone resorption on fracture healing, the area of blood callus/callus, the area of cartilage and the area of cartilage occupying the blood callus/callus formed during and when bone resorption inhibition increased osteogenic factor PTH were analyzed.
FIG. 8, Panel A, is a graph of bone-cartilage staining of fractured tibia at week 1 and week 2, respectively, with Haematoxylin-Orange G (ABH/OG). Chondrocytes were stained blue and cortical bone and trabecular bone were stained orange.
Analysis by static histological analysis software found: there was no significant difference in the size of the scab area in each group at week 1 post-fracture (shown in panel B in fig. 8). The callus areas of the vehicle-ZA group and PTH-ZA group showed an increasing trend at 2 weeks after fracture compared to the control vehicle-vehicle group. The callus area of PTH-ZA group was increased from that at week 1 (p < 0.05).
Cartilage area measurements were similar to the change in blood or callus area (panel C in fig. 8). No significant difference in cartilage area was seen between groups at week 1 after fracture. At 2 weeks post-fracture, the cartilage areas of the vehicle-ZA group and PTH-ZA group were significantly increased compared to the control vehicle-vehicle group, and the cartilage areas of these two groups were significantly increased relative to their respective first week cartilage areas. The results show that: inhibit bone resorption, and increase cartilage area.
Comparing cartilage area to the area of blood and callus formed found: in the 1 st week after fracture, the area of the cartilage in each group accounts for the similar area of the blood crust, 17-24%, and no difference exists between groups. (diagram D in FIG. 8); at 2 weeks post-fracture, the soft bone area of the ZA-treated groups, including the vehicle-ZA group and PTH-ZA group, was significantly greater than the vehicle-vehicle group by 13% and 14%, respectively. And is significantly higher than the area of cartilage to blood crust at week 1. However, the PTH-PTH and PTH-PTH + ZA groups of the PTH continuous treatment group showed no significant change in the cartilage area to callus area ratio of the control vehicle-vehicle group or in the cartilage area to callus area ratio at week 1 of each.
The research result of the experimental example shows that: simple inhibition of bone resorption or simple PTH does not affect cartilage formation; simple inhibition of bone resorption leads to increased callus formation, increased cartilage area, and increased cartilage area over callus formation. While the continuous inhibition of bone resorption in combination with PTH reverses the above phenomenon. These data further suggest that cartilage retention may be a possible factor in bone resorption inhibition leading to non-union fractures.
Experimental example 6
Dmp1-Cre constructed in example 1 was used; constructing Dmp1-Cre by Catnb f/+ mice; catnb f/+ mouse tail suspension simulation bone weightlessness model.
A model of mouse bone weightlessness is constructed by hanging the tail by using a method of fixing the tail by sticking medical adhesive tapes. Selecting 14 weeks of mice, 10 mice in each of a control group and an experimental group, adhering and fixing 1/3 positions behind the tail of the mice in a ring shape by using medical cloth adhesive tapes, hanging the bridle on a cloth rope wound on a cage by using a clip hook, enabling the head of the mouse to incline downwards by 30-45 degrees, lifting the hind limb upwards, observing the suspension condition of the mice for 4 times every day at regular time, and adding food and water to the mice. It needs to be noted that the medical adhesive tape needs to adjust the elasticity, can not be too tight or too loose, and too tight can hinder mouse afterbody blood flow and hinder, leads to the afterbody swelling, and too loose then leads to medical adhesive tape landing easily. The statistical chart of the experimental animal information is shown in FIG. 9.
Experimental example 7
This example provides methods for bone weightlessness mouse tissue sample processing. Samples were collected from sacrificed mice 14 days after tail suspension. The condition of bone density is observed by X-ray before and after 14 days of tail suspension of the mouse. New bones formed were double-labeled with calcein and alizarin red (alizarin red), 7 and 2 days before the mice were harvested, respectively, with reference to the previous method (Tu, x., ethyl. Blood of the mice is collected before the mice are killed, thighbones and shinbones of the mice are separated, the left thighbones are immediately placed in liquid nitrogen for freezing after soft tissues are removed, the right thighbones are immediately fixed by 4% neutral formalin solution for 24 hours and then washed clean by PBS, and then the thighbones are placed in 70% alcohol and stored in a refrigerator at 4 ℃.
Experimental example 8
In this experimental example, the content of bone, the bone density of all bones, lumbar vertebrae and femurs, the bone volume of dense/cancellous bone, the number of trabeculae, the thickness and the gap distance were measured by X-ray, DEXA-PIXImus and micro-CT, respectively.
As shown in fig. 10, the results of the test show that the simulated weight loss reduced the body weight of the mouse in fig. 10. Panel C in figure 10 shows that weight loss decreased both the left and right femoral densities in the lumbar vertebrae of the wild mouse, while the left and right femoral densities were not decreased in the lumbar vertebrae of the mutant mouse. As can be seen from the graphs D and E in FIG. 10, the micro-CT detects cancellous bone, BV/TV is the volume/total volume of bone, Tb.N is the number of trabeculae, Tb.Th is the width of trabeculae, and Tb.Sp is the separation degree of trabeculae. Compared with a normal loading wild mouse, the bone volume of the wild mouse after weight loss simulation is reduced; compared with the mutant mouse with normal load, the bone volume of the mutant mouse after the simulated weightlessness is unchanged.
FIG. 10 is a graph F-G showing micro-CT examination of cortical bone, BA/TA being bone area/total area, da β catOtThe mice were activated for osteocytic Wnt and the results showed that neither wild type nor mutant mice reduced cortical bone area after simulated weightlessness.
Experimental example 9
Determination of mineralization and osteogenesis rates of mice, 7 and 2 days before harvesting mice, new bones formed by double labeling with calcein and alizarin red (alizarin red) were measured and calculated for the formation rates of cortical and cancellous bones of mice using bone histomorphometry (bone osteometrics), respectively, with reference to example 4, b.
Determination of osteoclast and bone resorption transformation:
osteoclast number was counted by histological specific staining of osteoclasts as before (Tu, X, 2015; Hilton, 2008; Tu, X, 2012);
serological bone metabolism biochemical markers were determined by ELISA: CTX, osteocalcin, P1NP, alkaline phosphatase, PTH and the like, and analyzing the bone resorption transformation level; serum calcium and phosphorus levels were determined chemically and the effect on calcium and phosphorus metabolism was investigated.
The results of the experiment are shown in FIG. 11, in which the graphs A-B in FIG. 11 show that the left four are wild type and the right four are mutant type; osteoclast staining of femoral (left) and lumbar (right) sections of normal-bearing and mock-weightless mice. The C-D plots in fig. 11 show that, after counting osteoclasts on the cancellous bone surfaces of the femur (left) and the lumbar (right), the results show that the weight loss increases the osteoclast number on the cancellous bone surface of the control mice, and the femur and the lumbar vertebrae are both significantly increased; but the weight loss maintains the quantity of osteoclasts on the surface of the femur of a mouse activated by the Wnt of the osteocyte, and reduces the quantity of the osteoclasts on the surface of the femur of the mouse activated by the Wnt of the osteocyte. N.oc./t.ar is the number of osteoclasts per bone area, n.oc/B.S is the number of osteoclasts per bone surface, and oc.s./B.S is the length of osteoclasts per bone surface.
Figure 11, panel E, shows the results of the serum bone resorption assay, with increased bone resorption by 40-fold in the weightless wild-type mice, while Wnt-activated bone cells in the weightless mock situation significantly reduced bone resorption.
Fig. 11F shows a graph of the osteoclast differentiation gene detection result, in which the weight loss reduces the osteoblast differentiation of the control mouse, but the weight loss not only does not activate the osteoblast Wnt-activated mouse to reduce the femur osteoblast differentiation, but also enhances the osteoblast differentiation, and inhibits the osteoclast differentiation and bone resorption increase caused by the weight loss.
The weight loss enables the control group mice to enhance the expression of the bone cell marker Sost, but the bone cell Wnt activated mice only maintain the expression level of the Sost gene and do not enhance the expression level. P <0.05 compared to control mice, # p <0.05 compared to normal load mice.
Experimental example 10
Osteoblasts and bone formation were measured by the osteoblast and bone formation measurement method described in example 4. The results of the experiments are shown in fig. 12, and as can be seen from fig. 12, the histological h.e. staining pattern (left) of the femoral sections of Normal negative recombination (Normal loading) and mock weightless mice (unloading) and the morphological examination of the bone tissue in the red box region (right) are shown in panel a. The detection result shows that the cancellous bone of the control group mouse has bone loss under weightlessness, and the cancellous bone of the mouse activated by the Wnt osteocyte has no bone loss under weightlessness.
Fig. 12, panels B-C, show histomorphometric findings of the femur and the lumbar vertebrae, showing that weight loss decreased the density and number of osteoblasts in cancellous bone of control mice, while Wnt-activated cancellous bone of mice did not experience a decrease in density and number of osteoblasts under weight loss.
Fig. 12D-E shows osteoblast and osteocyte marker gene detection results, and the results show that weight loss reduces osteoblast differentiation of control mice, but weight loss not only does not reduce femoral osteogenic differentiation of Wnt-activated mice, but also enhances osteogenic differentiation. The weight loss enhanced the expression of the control mouse bone cell marker Sost, but the expression of the mouse Sost was maintained in the Wnt-activated mice, which were bone cells, without enhancement. P <0.05 compared to control mice, # p <0.05 compared to normal load mice.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An application of a bone cell Wnt signal path activator in preparing a medicament for accelerating fracture healing.
2. The use of claim 1, wherein the fracture is at least one of a compression vertebral fracture, a hip fracture, a lumbar fracture, a femoral fracture, and a tibial fracture.
3. The use according to claim 2, wherein the osteocyte Wnt signalling pathway activator is through any drug or pathway to activate Wnt signalling in osteocytes.
4. An application of a bone cell Wnt signal channel activator in preparing a medicament for treating delayed union or nonunion of fracture.
5. The use of claim 4, wherein the fracture nonunion is due to a clinically surgically created nonunion of open or closed fractures; preferably, the fracture nonunion is fracture nonunion caused by bone resorption inhibitor; preferably, the bone resorption inhibitor is inhibition of bone resorption by bisphosphonates; preferably, the bisphosphonate drug is at least one of alendronate sodium, risedronate sodium, ibandronate sodium and zoledronic acid.
6. The use of claim 4, wherein the osteocyte Wnt signalling pathway activator is to activate Wnt signalling in the osteocyte by any drug or pathway.
7. An application of a Wnt signal channel activator of a bone cell in preparing a medicament for preventing and treating bone loss.
8. The use according to claim 7, wherein the bone loss is bone loss resulting from an out-of-mechanics stimulus; preferably, the bone loss is bone loss resulting from weight loss, no movement.
9. Use according to claim 7, wherein the weight loss is a simulated space weight loss, a near-ground flight weight loss or a space weight loss.
10. The use of claim 8, wherein the osteocyte Wnt signaling pathway activator is through any drug or pathway to activate Wnt signaling in osteocytes.
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| CN115770297A (en) * | 2022-12-19 | 2023-03-10 | 重庆医科大学 | Application of TGF beta signal agonist and inhibitor in preparation of medicines for promoting or inhibiting osteoclast differentiation and bone resorption |
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