120 results about "Osseous Differentiation" patented technology

The invention discloses a method for inducing osteoblasts by using mesenchymal stem cells, and belongs to the technical field of biology. The method for inducing the osteoblasts comprises the step of adding brucella outer membrane protein into a conventional osteoblast induction culture medium to promote the dental pulp stem cells to be differentiated into the osteoblasts. According to osteoblast differentiation metaphase differentiation marker detection, the cell ALP activity of a group added with 800ng / mL of brucella outer membrane protein is 2.36 times that of a conventional osteoblast induction culture medium group; according to osteoblast differentiation end-stage differentiation marker detection, the calcium deposition amount of brucella outer membrane proteome cells added with 800ng / mL is 481.54 mu g / mg protein, and is 2.55 times of that of a conventional osteoblast induction culture medium group. By adding the brucella outer membrane protein into a conventional osteoblast induction culture medium, the osteogenic differentiation efficiency of the dental pulp stem cells can be effectively improved.

The invention discloses application of a Chibby1 (CBY1) inhibitor in preparation of an anti-osteoporosis medicine for promoting bone formation, and belongs to the technical field of biological medicines. It is found for the first time that CBY1 can inhibit bone marrow stromal cells from differentiating into osteoblasts, and bone marrow stromal cells with CBY1 gene silencing are increased in osteoblast differentiation. According to the invention, it is proposed for the first time that osteoblast differentiation can be promoted by reducing CBY1 expression or inhibiting the CBY1 function, and the CBY1 inhibitor has the potential of increasing osteogenic activity and preventing and treating osteoporosis by inhibiting CBY1 expression or inhibiting the CBY1 function.

The invention relates to a time sequence degradable orthopedic implant material which comprises a base material and a biodegradable coating formed on the surface of the base material, the surface of the base material is provided with a nanocone array microstructure, the biodegradable coating takes a biodegradable polymer as a matrix and loads a metal element for promoting bone repair, and the metal element is a metal element for promoting bone repair. And the surface of the biodegradable coating is also connected with an anti-inflammatory active factor. According to the invention, grafted anti-inflammatory active factors are matched with the biodegradable coating loaded with metal elements for promoting bone repair, and the sequential active regulation and control of the biological effect of the micro-morphology on the surface of the base material cope with effector cells in different repair stages; the time sequence regulation and control on the macrophage inflammation phenotype and bone marrow mesenchymal stem cell osteogenic differentiation in different stages of bone repair are realized, so that each stage of bone tissue repair is matched, and bone repair and osseointegration are promoted.

The invention discloses a long-chain non-coding RNA (Ribonucleic Acid) and application thereof in regulating osteogenic differentiation of bone marrow mesenchymal stem cells. It is found that overexpression of long-chain non-coding RNA (lncRNA Gm20628) with the nucleotide sequence shown in SEQ ID NO.1 in bone marrow mesenchymal stem cells can effectively promote osteogenic differentiation of the bone marrow mesenchymal stem cells, and then bone defect repair of a mouse with skull injury is promoted. Therefore, the invention provides application of the expression promoter of the lncRNA Gm20628 in preparation for enhancing bone regeneration, repairing bone defects and treating osteoporosis and bone formation disorder related diseases. The method is not only beneficial to treatment of diseases related to the bone marrow mesenchymal stem cells, but also beneficial to construction of bone organs.

The invention belongs to the technical field of biotechnology and tissue engineering, and particularly relates to application of pilose antler bud cell secretions in preparation of anti-osteoporosis drugs. The pilose antler bud cell secreta is obtained by adopting gradient centrifugation, and cell experiments show that the pilose antler bud cell secreta provided by the invention has the effect of promoting osteogenic differentiation of cells; animal experiment results show that the cornu cervi pantotrichum bud cell secreta provided by the invention can significantly improve the bone mineral density, bone volume fraction, bone trabecula number, bone trabecula thickness, bone trabecula separation degree and cortical bone mineral density of mice, increase the bone trabecula area of primary osteogenesis and secondary osteogenesis centers of the mice, improve osteoporosis symptoms of the mice, and improve osteoporosis of the mice. The effect is obviously better than that of bone marrow mesenchymal stem cell secretions, part of indexes are equivalent to the treatment effect of diphosphates, and the antler bud cell secretions provided by the invention can be used for preparing medicines for preventing, relieving or treating osteoporosis.

The invention discloses an application of an AMPK (adenosine monophosphate kinase) activator or a PPAR (peroxisome proliferator activated receptor) gamma inhibitor in preparation of a medicine for preventing or treating diabetic osteoporosis. The invention discloses a key effect of a brand new signal channel, namely an AMPK-PPAR gamma-ERS axis, in osteogenic differentiation disorder of diabetes mellitus, and provides a method for preventing and treating diabetic osteoporosis by regulating and controlling the signal axis by taking metformin as a core. The invention provides new guidance for research and development of medicines for treating osteoporosis, and has potential clinical application and mechanism research values.

The invention discloses long-acting high-dynamic hydrazone bond hydrogel as well as a preparation method and application thereof, and particularly relates to the technical field of biomedical materials. The preparation method comprises the following steps: taking 1-adamantane acetic acid and adipic dihydrazide modified sodium hyaluronate and benzaldehyde-terminated eight-arm polyethylene glycol as carrier raw materials; the hydrazone bond hydrogel with relatively high stability is prepared through a coupling reaction of an aldehyde group of benzaldehyde-terminated eight-arm polyethylene glycol and a hydrazide group of 1-adamantane acetic acid and adipic dihydrazide modified sodium hyaluronate. The beta-cyclodextrin modified m-phenylenediamine catalyst is dynamically combined into the carrier through reversible host-guest complexation between beta-cyclodextrin and 1-adamantane acetic acid, so that the long-acting high-dynamic hydrazone bond hydrogel is obtained, and the requirement of three-dimensional culture of stem cells on mechanical properties can be met; and network dynamics can be maintained for a long time, so that stem cell spreading is supported, mechanical signal transduction is activated, osteogenesis-related gene expression is promoted, and stem cell osteogenesis differentiation capability is enhanced.

The invention provides a mesenchymal stem cell composition as well as a preparation method and application thereof in knee joint products, and belongs to the technical field of medicines. The invention relates to application of a mesenchymal stem cell combined naringin derivative in preparation of medicines for treating osteoporosis and repairing knee joint injury and arthritis. The naringin derivative is prepared from naringin through an ether esterification reaction, the metabolic cycle of the naringin derivative is prolonged and the water solubility is improved while the original activity is maintained, so that the free drug concentration and tissue infiltration capacity in bones are improved, the bioavailability of the naringin derivative is improved, and the bioavailability of the naringin derivative is improved. The prepared mesenchymal stem cell composition can induce mesenchymal stem cell osteogenic differentiation and improve bone repair capacity, has a good intervention effect on osteoporosis, has a sustained and controlled release effect and prolongs medicinal time.

The invention provides application of an FZD4 gene. The FZD4 gene is used for regulating osteogenic differentiation and mineralization of chicken bone marrow mesenchymal stem cells. The FZD4 gene in the chicken bone marrow mesenchymal stem cells is over-expressed and is used for promoting osteogenic differentiation and mineralization of the chicken bone marrow mesenchymal stem cells; or / and the FZD4 gene in the chicken bone marrow mesenchymal stem cells is knocked down to inhibit osteogenic differentiation and mineralization of the chicken bone marrow mesenchymal stem cells; the nucleotide sequence of the coding region of the FZD4 gene is as shown in SEQ ID NO. 1. According to the invention, the positive regulation effect of the FZD4 gene in osteogenic differentiation of chicken bone marrow mesenchymal stem cells is determined for the first time, the blank of a bone regulation mechanism of the poultry FZD4 gene is filled, and a new biological preparation target is provided for prevention and treatment of poultry bone diseases; the FZD4 gene can also be used as a molecular marker for breeding chicken varieties with high bone strength.

The application provides a kind of basic phosphatase induced T1 and T2 signal switching response type magnetic resonance imaging contrast agent and its preparation method and application, the response type magnetic resonance imaging contrast agent includes superparamagnetic nanoparticle, and polyethylene glycol phosphoric acid monoester modified to the surface of superparamagnetic nanoparticle;The superparamagnetic nanoparticle includes ferroferric oxide nanoparticle.The contrast agent of the application can realize T1 and T2 signal switching, is used for stem cell osteogenic differentiation monitoring, not only improves the detection sensitivity of magnetic resonance imaging to microstructure and function, also provides new tools and methods for stem cell treatment in biomedical research and clinical practice.

The application discloses application of microRNA-32-5p in preparation of a medicine for preventing and treating osteoporosis, and a medicine composition taking microRNA-32-5p as an active ingredient. It is proved through experiments that knocking out the microRNA-32-5p gene significantly aggravates osteoporosis induced by estrogen deficiency; the microRNA-32-5p plays a bone protection role by promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Therefore, the microRNA-32-5p can be applied to prevention and treatment of osteoporosis as a microRNA medicine.

The invention discloses a surface modified titanium stent with a magnetic hydrogel coating and a preparation method and application of the surface modified titanium stent, and belongs to the technical field of biomedical materials. The surface modified titanium scaffold with the magnetic hydrogel coating comprises a titanium substrate, a polydopamine coating and a hydrogel precursor solution coating, wherein the polydopamine coating and the hydrogel precursor solution coating are compounded on the titanium substrate; the polydopamine coating is positioned between the titanium substrate and the hydrogel precursor solution coating; the hydrogel precursor solution is prepared from freeze-dried GelMA, Fe3O4 nanoparticles and ammonium persulfate. The prepared surface modified titanium scaffold with the magnetic hydrogel coating has excellent mechanical properties and stability, and can improve the ALP activity level of cells, improve the collagen secretion level and promote mineralization in the stem cell osteogenic differentiation process through an external magnetic field with certain strength, so that the bone regeneration process is accelerated, and the osteogenic differentiation effect is improved. The method has a wide application prospect in preparation of bone graft materials and bone repair materials.

The invention belongs to the technical field of biological materials, and particularly relates to a nano composite material for bone repair as well as a preparation method and application thereof. One of the purposes of the present invention is to provide a nano composite material for bone repair, the nano composite material comprising anthraquinone derivative molecules and metal ions forming a nano network structure, the nano network structure being formed by a carbonyl group and an alpha-phenolic hydroxyl group structure in the anthraquinone derivative molecules on the basis of the coordination of adjacent oxygen atoms to the metal ions. The nanocomposite not only shows good biocompatibility, but also can effectively promote proliferation of rat bone marrow mesenchymal stem cells (rBMSCs) and accelerate the osteogenic differentiation process, thereby having important application value in bone tissue engineering and repair medicine.

The application provides a DNA tetrahedron-based complex and a preparation method and application thereof, and belongs to the field of medicines. The application designs a functionalized DNA tetrahedron complex modified bioactive composite hydrogel scaffold containing BMP-2, combines the functionalized BMP-2 binding peptide with the tetrahedron framework structure with unique three-dimensional spatial structure and fine programmability through a chemical clicking mode, and further prepares the composite hydrogel scaffold by combining with methacrylated hyaluronic acid. The functionalized DNA tetrahedron complex realizes the performance of efficiently capturing BMP-2, and the composite hydrogel scaffold realizes the targeted capture of endogenous BMP-2 in the early stage of bone defect and stimulates the osteogenic differentiation of stem cells to promote in-situ osteogenesis, avoids a series of complications caused by the delivery of exogenous bone morphogenetic protein-2, and solves the problems of poor in-vivo stability, safety and high cost, and has a good application prospect.

The invention discloses a rehmannia neutral homogeneous polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine preparations. The polysaccharide mainly comprises galactose, glucose and fructose, the molar ratio of the galactose to the glucose to the fructose is 2.72: 1.17: 1.11, and the weight-average molecular weight of the polysaccharide is 2152 Da. The rehmannia neutral polysaccharide can play a role in treating osteoporosis by increasing bone mineralization amount and bone mineral density; the proliferation and directional osteogenic differentiation of the bone marrow mesenchymal stem cells BMSCs can be promoted, and the obvious application effect is achieved in the aspect of preparing the bone defect treatment medicine through the stem cells. The rehmannia neutral homogeneous polysaccharide shows a huge application prospect in the aspects of preparation of medicines for treating osteoporosis and stem cell treatment (especially bone regeneration treatment based on mesenchymal stem cells). A new candidate substance and a research direction are provided for developing an efficient and safe bone metabolism disease treatment strategy derived from traditional Chinese medicines.

This invention belongs to the field of biomedical technology and relates to the application of OSM inhibitors in the preparation of drugs for the prevention or treatment of calcific aortic valve disease. This invention reveals for the first time that OSM drives osteogenic differentiation and calcified nodule formation of valvular interstitial cells through the OSM-OSMR-JAK2 / JAK3-STAT3-RUNX2 signaling axis, which is the core pathogenic mechanism leading to calcific aortic valve disease. This invention utilizes anti-OSM neutralizing antibodies or sulforaphane to effectively block this signaling axis, significantly inhibiting macrophage infiltration, valvular calcification, and improving hemodynamic abnormalities. This invention provides a precise target and a novel strategy for the modification therapy of calcific aortic valve disease, solving the technical problem of the ineffectiveness of traditional broad-spectrum anti-inflammatory therapy in valvular calcification.

This invention relates to the application of 5-(tetradecoxy)-2-furoic acid (TOFA) in the preparation of drugs that inhibit osteoclast differentiation and function. Using a RANKL-induced mouse bone marrow macrophage osteoclast differentiation model, experiments demonstrated that TOFA can effectively inhibit osteoclast differentiation and function by inhibiting ROS and downregulating the expression of key osteoclast differentiation and function proteins such as Nfatc1, Src, and Ctsk. Furthermore, TOFA at concentrations of 40 μM and below showed no significant cytotoxicity, proving that TOFA can serve as a candidate compound for developing drugs that inhibit osteoclast differentiation and function, and for treating diseases caused by excessive activation of osteoclasts, thus possessing significant clinical application value. Additionally, by constructing a mouse ovariectomized osteoporosis model, the preventive and therapeutic effects of TOFA on estrogen-deficiency-induced osteoporosis were demonstrated.

A process for the production of a composition comprising extracellular vesicles, comprising the following steps:  Culturing of stem cells, preferably adipose-derived stem cells;  Differentiating the stem cells, preferably osteogenically or chondrogenically differentiating the stem cells;  Adding of a particulate material, preferably gelatin beads, to obtain a scaffold-free multi-dimensional cell culture;  Collecting the liquid containing extracellular vesicles from the scaffold-free multi-dimensional cell culture;  Subjecting the liquid containing the extracellular vesicles to tangential flow filtration (TFF). The obtained composition comprising extracellular vesicles is useful for the treatment of cancer, in particular for the induction of apoptosis in cancer cells.

The invention discloses application of H-151 in preparation of drugs for preventing and treating calcified aortic valve diseases, relates to the technical field of biological medicines, and is characterized in that the invention provides application of an STING inhibitor H-151 or pharmaceutically acceptable salts thereof in preparation of drugs for preventing and / or treating calcified aortic valve diseases. The invention reveals for the first time that H-151 can inhibit the increase of the expression level of osteogenic differentiation markers ALP and RUNX2 in aortic valve interstitial cells induced by an osteogenic culture medium in an in-vitro experiment, and inhibit osteogenic differentiation and calcium salt deposition of the aortic valve interstitial cells; in addition, aortic valve thickening, fibrosis and calcium salt deposition of mice induced by an aortic valve injury model are inhibited in vivo. Based on the biological effect, the application of H-151 in developing the medicine for treating the calcified aortic valve disease is provided for the first time, and a new technical path and a new medicine choice are provided for preventing and treating the calcified aortic valve disease.

The invention relates to a fixing device for loading low-intensity pulsed ultrasound by taking water as a medium and application of the fixing device in promoting osteogenic differentiation of mesenchymal stem cells. According to the device, a double-base type probe fixing structure consisting of a main body fixing base and an accessory fixing base is arranged in a main body structure; and a culture vessel fixing structure on the inner wall of the main body structure is matched, so that the problems that a probe is easy to shift, the heights of culture vessels are inconsistent and the like in the prior art are effectively solved, and a standardized and high-repeatability loading environment is provided for an LIPUS in-vitro experiment. The device is further based on the device, water is used as an ultrasonic conduction medium to be applied to osteogenic differentiation induction of bone marrow mesenchymal stem cells (BMSCs), the efficient and stable osteogenic stimulation effect can be achieved, and the core defects that in the prior art, a probe makes direct contact with a culture vessel, consequently, cells fall off and are curled, morphological damage is caused, and sound field distribution is uneven are overcome; and meanwhile, the stability and repeatability of an experimental result are remarkably improved.

The invention belongs to the technical field of cell culture, and particularly relates to a bone marrow mesenchymal stem cell culture medium and a preparation method and application thereof. The mesenchymal stem cell culture medium comprises a basic culture medium, and betaine, glycyrrhizin, insulin, EGF (Epidermal Growth Factor), L-propyl dipeptide and naloxicol which are added into the basic culture medium. The bone marrow mesenchymal stem cell culture medium can effectively improve the multiplication capacity of bone marrow mesenchymal stem cells, delay cell senescence and improve the osteogenic differentiation capacity of the bone marrow mesenchymal stem cells, fetal calf serum is not added, the risk of introducing heterologous serum to carry viruses is avoided, and the safety of clinical application is improved.

The application relates to application of a medicine taking ACLY gene as an action target in preparation of a medicine for preventing and treating vascular calcification, and belongs to the technical field of biological medicine. In order to solve the problem that effective clinical means for treating vascular calcification is lacked at present, the application provides application of a medicine taking ACLY gene as an action target in preparation of a medicine for preventing and treating vascular calcification, the transcription or translation of the ACLY gene of vascular smooth muscle cells is efficiently and specifically inhibited, or the expression or activity of the ACLY protein of the vascular smooth muscle cells is efficiently and specifically reduced, the osteogenic differentiation of smooth muscle is reduced, the deposition of hydroxyapatite in the vascular media is reduced, and the progress of vascular calcification is alleviated. The medicine for preventing and treating vascular calcification is prepared on the basis of inhibition of the ACLY of vascular smooth muscle, has important clinical application potential for vascular calcification treatment, and can provide a new treatment option for patients with vascular calcification.

The invention discloses an application of loperamide in preparation of a medicine for treating osteoporosis, and belongs to the field of development of new application of medicines. Loperamide has the effects of inhibiting aging, increasing osteogenic differentiation of bone marrow mesenchymal stem cells and preventing osteoporosis of aged and postmenopausal mice; meanwhile, at the cellular level, loperamide can reduce expression of senescence factors in mouse mesenchymal stem cells, promote senescence cell apoptosis and inhibit cell senescence; and a new choice is provided for the treatment of senescence-related osteoporosis drugs.

The application provides an application of isoxazole 9 (ISX-9) in promoting mesenchymal stem cell osteogenic differentiation and bone injury regeneration repair. ISX-9 is a small molecule compound, and has the characteristics of easy to obtain, controllable structure and low cost. The in vitro experiment of the application shows that ISX-9 has the ability to induce mesenchymal stem cell osteogenic differentiation and promote the formation of mineralized nodules; the in vivo experiment shows that the hydrogel wrapped with ISX-9 can promote the regeneration repair of mouse skull defects, indicating that ISX-9 has good osteogenic induction and is a potential bone regeneration repair drug. Therefore, ISX-9 can promote mesenchymal stem cell osteogenic differentiation and be applied to bone tissue regeneration repair. The application also provides a direct action target and action mechanism of ISX-9 in promoting osteogenic differentiation. The application further provides a new drug sustained release delivery system of ISX-9, which is helpful for the application from theoretical research to clinical application.

The invention relates to the technical field of biological medicines, in particular to a galangal-sourced nano-vesicle as well as a preparation method and application thereof. The method specifically comprises the following steps: crushing rhizoma alpiniae officinarum rhizomes, mixing the crushed rhizoma alpiniae officinarum rhizomes with a buffer solution, soaking in a water bath, and filtering to remove solids so as to obtain a crude extract; and sequentially carrying out centrifugal treatment on the crude extract for four times, so as to obtain the galangal-derived nano-vesicles. The invention provides a method for extracting nano-vesicles from galangal, and finds for the first time that the obtained vesicles can promote osteogenic differentiation of human bone marrow mesenchymal stem cells with high efficiency and low toxicity, the effect of the vesicles is obviously superior to that of known active component galangin, and the cytotoxicity is obviously lower; the invention provides raw materials and methods with great potential for developing novel and safe anti-osteoporosis drugs or inducers.

The invention discloses a culture method for improving osteogenic differentiation capacity of bone marrow mesenchymal stem cells. The method comprises the following steps: S1, adding a certain amount of protopanoxadiol into a culture medium; s2, the culture medium containing protopanoxadiol is used for flushing and suspending the bone marrow mesenchymal stem cells, and cell suspension is obtained; and S3, adjusting the cell density in the cell suspension, inoculating the cell suspension into a cell culture device, and placing the cell culture device in an incubator for multiplication culture. According to the culture method, the culture medium containing the protopanoxadiol with the concentration of the protopanoxadiol being 5-50 [mu] M is used for culturing the mesenchymal stem cells, so that the cell osteogenic differentiation related gene expression of the mesenchymal stem cells is effectively increased, and the osteogenic differentiation capability of the mesenchymal stem cells is improved.

The invention provides an embryonic stem cell differentiation technology and application of the embryonic stem cell differentiation technology in screening of unknown risk alternative toxicity of a novel biological breeding product, and Cry1Ab protein is used as a test substance to evaluate the developmental toxicity of the Cry1Ab protein. The EBs are generated through hanging drop culture, and bone differentiation induction substances (beta-glycerophosphate, ascorbic acid and vitamin D3) are added to promote differentiation of the EBs. After bone cell induced differentiation is finished, the differentiation condition of bone cells is observed through alizarin red S dyeing and absorbance value detection, the cell growth condition is observed through cell total protein concentration and alkaline phosphatase activity detection, and then the influence of a test substance on the bone differentiation process is analyzed through the gene expression condition of osteogenic differentiation related markers (Runx2, SPARC and I-type collagen). The invention solves the problem of evaluating the unknown risk of a novel biological breeding product in vitro, creates an embryonic stem cell bone differentiation test technology, and uses the embryonic stem cell bone differentiation test technology as a screening method for the unknown risk alternative toxicity of the novel biological breeding product to be combined with an embryonic stem cell myocardial differentiation experiment for use. And the accuracy of predicating the developmental toxicity of the test substance is improved.

The invention provides a cell-loaded bionic bone scaffold with oxygen self-production and bacteriostasis effects as well as a preparation method and application thereof, and belongs to the technical field of bone tissue engineering. The bionic bone scaffold provided by the invention is formed by stacking and assembling a plurality of cylindrical bone scaffold units, has a natural bone simulated Hauses tube structure, has good biocompatibility and excellent mechanical properties, and can accurately simulate the pore and structural characteristics of a natural bone. The functional GelMA hydrogel filled in the grooves is loaded with different cells, biological factors and antibacterial drugs, so that osteogenesis and vascularization effects at specific positions of the stent are realized, formation of a vascularization network and bone repair by simulating a natural bone structure of a human body are facilitated, and a good antibacterial effect is achieved. According to the invention, the cylindrical bone scaffold unit is loaded with the self-oxygen-producing MOFs particles, and the self-oxygen-producing effect of slowly releasing oxygen can be achieved after the cylindrical bone scaffold unit is implanted in vivo, so that bone marrow mesenchymal stem cell osteogenic differentiation and vascularization network construction are promoted.

The present invention relates to a composition for promoting the osteogenic differentiation efficiency of adipose-derived stem cells. The present invention has revealed that treating Adipose-Derived Stem Cells (ADSCs) from older donors with reduced differentiation efficiency with FGF2 and / or HGF during the early stages of differentiation can restore damaged paracrine function and maximize differentiation efficiency. Therefore, it can be utilized as an in vitro process technology for promoting stem cell differentiation efficiency before or during adipose-derived stem cell transplantation.

The invention belongs to the technical field of biomedicine, and particularly relates to application of an IRF1 gene as a target spot in preparation of a preparation for regulating osteogenic differentiation of bone marrow mesenchymal stem cells. The invention provides application of an IRF1 gene as a target spot in preparation of a preparation for regulating and controlling osteogenic differentiation of bone marrow mesenchymal stem cells. The nucleotide sequence of the IRF1 gene is shown as SEQ ID NO.1. The invention also provides application of the IRF1 gene in preparation of a preparation for regulating and controlling osteogenic differentiation of bone marrow mesenchymal stem cells. The invention provides a brand new regulation and control target for enhancing mesenchymal stem cell transplantation treatment of osteoporosis, verifies the treatment effect and biological safety, and lays a solid foundation for subsequent clinical application.