Pharmaceutical products made from Wharton's jelly for promoting bone regeneration

Abstract

The present invention relates to a pharmaceutical product produced by Wharton's jelly of the umbilical cord, which promotes bone regeneration. The pharmaceutical product can be obtained by either of the following two methods: (1) treating the Wharton's jelly collected from the umbilical cord with a cryoprotectant, then storing it frozen and thawing it before use; and (2) Wharton's jelly collected from the umbilical cord should be directly freeze-dried and stored under dry conditions before use.

Description

[Technical field]

[0001] The present invention relates to a medicinal product made from Wharton's jelly of umbilical cord, which has the effect of promoting bone regeneration. [Background technology]

[0002] Bones may be damaged or removed due to trauma, fracture, infection, or tumor metastasis and invasion. When a bone defect exceeds twice the diameter of the bone shaft, it is known as a critical size bone defect because it will not heal naturally even if various bone regenerative materials are implanted. Furthermore, if the fracture site is too large, the weight-bearing capacity will be reduced and the risk of subsequent fracture will increase significantly. In addition, regeneration of compact bone defects is relatively difficult. Therefore, how to repair compact bone defects is a challenging problem in today's orthopedic clinical practice.

[0003] Currently, common clinical bone materials are classified into three types. The first type is autogenous bone grafting, in which bone tissue is surgically harvested from other body parts and used to fill the bone defect site. The disadvantages of this method are the long recovery period, the need for two invasive surgeries, and the amount / area of ​​bone grafting may not be sufficient. The second type is allogeneic bone grafting, i.e., the use of excess bone tissue harvested from other patients who require surgical treatment after post-processing. In addition to ethical issues, there is a risk of disease transmission. The third type is artificial bone material, which requires research and development of new synthetic bone materials for clinical application. Summary of the Invention

[0004] Therefore, the present invention provides a medical product produced from Wharton's jelly of the umbilical cord, which has the effect of promoting bone regeneration, and which is produced by either of the following two methods: method (1): treating the Wharton's jelly collected from the umbilical cord with a cryoprotectant, then cryopreserving it and thawing it before use; and method (2): directly freeze-drying the Wharton's jelly collected from the umbilical cord, and storing it under dry conditions before use.

[0005] According to the present invention, said pharmaceutical product, named Frozen Wharton's Jelly (WJF), possesses cell bodies, obtained by a method (1) comprising the steps of harvesting Wharton's Jelly from the maternal umbilical cord, treating it with a cryoprotectant, then freezing it, and keeping the product in a frozen state before use.

[0006] According to one embodiment of the present invention, the cryoprotectant is a solution containing 10% dimethyl sulfoxide (DMSO).

[0007] According to one embodiment of the invention, the cryopreservation is performed in liquid nitrogen.

[0008] According to a particular example of the invention, pharmaceutical products made from Wharton's jelly are first protected with a cryoprotectant (e.g., a 10% DMSO solution) and then stored frozen and thawed prior to use.

[0009] According to the present invention, said pharmaceutical product, named WJD (Decellularized Wharton's Jelly matrix), does not possess cell bodies, is obtained by a method (2) comprising the steps of harvesting Wharton's Jelly from the mother's umbilical cord, directly freeze-drying it, and storing it under dry conditions at room temperature before use.

[0010] According to an embodiment of the invention, the frozen product is stored at -80°C.

[0011] According to a particular embodiment of the invention, said pharmaceutical product is obtained by a method comprising harvesting fresh Wharton's jelly from the umbilical cord, freezing it directly at -80°C, and then freeze-drying it to remove the water from the tissue.

[0012] According to the present invention, the pharmaceutical products produced from Wharton's Jelly obtained by both of the above methods can improve alkaline phosphatase in osteoblasts, improve the expression level of bone differentiation genes in osteoblasts, promote osteoblast differentiation, enhance calcium deposition, effectively enhance bone production, enable bone formation in bone defects, enhance the activity of osteoblasts in bone defects, and induce the growth and development of new bone.

[0013] In one aspect, the present invention provides the use of a pharmaceutical product produced from Wharton's jelly in the preparation of a medicament for promoting bone regeneration.

[0014] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. [Brief description of the drawings]

[0015] The foregoing summary and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings, in which there is shown, for the purpose of illustrating the invention, embodiments which are presently preferred, it being understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

[0016] In the drawings:

[0017] [Figure 1]FIG. 1 shows histological and morphological analysis of fresh frozen or decellularized Wharton's jelly according to the present invention, in which FIG. 1A shows fresh frozen Wharton's jelly (WJF) from umbilical cord; FIG. 1B shows paraffin embedded and sectioned decellularized Wharton's jelly matrix (WJD). FIG. 1A1 and FIG. 1B1 show H&E staining, in which fresh WJF shows a dense structure and the tissue is rich in mesenchymal stem cells, whereas WJD shows a sparse and porous structure. FIG. 1A2 and FIG. 1B2 show Sirius red staining, in which fresh WJF collagen is tightly packed in bundles, whereas WJD collagen surrounds voids. FIG. 1A3 and FIG. 1B3 show PAS staining, in which polysaccharides fill the Wharton's jelly tissue. FIG. 1C and FIG. 1D show scanning electron micrographs of WJD showing different sizes of porous structures. Figure 1E shows that freeze-dried Wharton's jelly exhibits porous structures of different sizes, whether analyzed by paraffin-embedded sections, quantification by HE staining, or scanning electron microscopy, and furthermore, the distribution of pore sizes is mostly in the range of 10 to 70 micrometers.

[0018] [Diagram 2] Figure 2 shows that co-culture of rat osteoblasts (ROB) with two kinds of Wharton's jelly enhances the expression level of bone differentiation genes in osteoblasts, Figure 2A shows that ROBs are cultured alone or co-cultured with WJD or WJF in vitro for 14 days and subjected to RT-PCR assay of rat RUNX2, the results show that there is expression of rat RUNX2 in all groups; Figure 2B shows rat RUNX2 expression in ROB quantified by real-time RT-PCR. The results show that the rat RUNX2 expression level in osteoblasts of ROB / WJD group and ROB / WJF group is significantly increased compared to ROB group (p<0.05). *: significant difference compared to ROB group, p<0.05.

[0019] [Diagram 3] Figure 3 shows that co-culture with either WJD or WJF increases the expression level of alkaline phosphatase in ROB by quantification of alkaline phosphatase, where Figure 3A is a graph showing the model of ROB co-cultured directly or indirectly with WJD or WJF; Figure 3B shows the cell staining of alkaline phosphatase after 14 days of culture; Figure 3C shows the quantification of intracellular alkaline phosphatase concentration, where the level of alkaline phosphatase expressed in ROB of ROB / WJD group, ROB+WJD group, ROB / WJF group or ROB+WJF group is significantly increased compared to ROB alone group as shown by OD595 absorbance value (p<0.05). *: Significant difference compared to ROB group, p<0.05.

[0020] [Figure 4] Figure 4 shows that co-culture of ROB with either of the two Wharton's jelly products promotes the performance of calcium deposition in osteoblasts. Figure 4A shows that ROB was cultured alone or indirectly co-cultured with WJD or WJF in vitro for 21 days and stained with Alizarin Red S. After treatment with osteogenic differentiation medium, osteoblasts in all groups exhibit calcium deposition. Figure 4B shows the quantification of calcium ion concentration in each group. The OD540 absorbance value shows that the calcium deposition in both ROB / WJD and ROB / WJF groups is higher than that in the ROB group (p<0.05). *: Significant difference compared to the ROB group, p<0.05.

[0021] [Diagram 5]Figure 5 shows that the implantation of two types of Wharton's jelly in the treatment group effectively promotes bone formation in rats with skull defects, as observed via micro-computed tomography (micro-CT). The bone healing of rats with skull defects was tracked via micro-CT at 1, 2, 3, 4, and 5 months after surgery. Figure 5A shows that in the injury + saline group, only slight bone growth along the periphery of the defect is observed, while in the injury + WJD and injury + WJF groups, newly formed free bone growth is observed not only around the defect but also within the defect. Figure 5B shows the quantification of the percentage of new bone area within the defect area, with the border of the bone defect indicated by a red dashed line. The results show that the percentage of new bone is significantly increased in the injury + WJD and injury + WJF groups compared to the injury + saline group. *: Significant difference compared to the injury + saline group at the same age, p<0.05.

[0022] [Figure 6] Figure 6 shows that the percentage of new bone in rats with skull defects is effectively increased in either of the two Wharton's jelly product treatment groups, as observed by H&E staining. Figure 6A shows a low-magnification panoramic image of a horizontal plane tissue section of the skull stained with H&E (the area of ​​the bone defect is indicated by a white dotted line); Figure 6B shows the border area of ​​the bone defect at low magnification and gradually enlarged; Figure 6C shows the inner area of ​​the bone defect (scale bars are 1 mm and 200 μm, respectively) at low magnification and gradually enlarged; Figure 6D shows the percentage of new bone area quantified by H&E staining. The results show that the percentage of new bone area is significantly increased in the injury+WJD group and the injury+WJF group compared to the injury+saline group (p<0.05, n=5). *: significant difference compared to the injury+saline group, p<0.05.

[0023] [Figure 7]Figure 7 shows that implantation of either of the two Wharton's jelly products effectively increases the distribution of mature osteoblasts in rats with skull defects, as observed by ALP staining. Figure 7A shows low-magnification panoramic images of horizontal histological sections of the skull stained by ALP in each group; Figure 7B shows the boundary area of ​​the bone defect at gradually enlarged magnifications from low magnification; Figure 7C shows the inner area of ​​the bone defect; Figure 7D shows the percentage of the area of ​​mature osteoblasts quantified by ALP staining. The results show that the percentage of the distribution area of ​​mature osteoblasts is significantly increased in the injury + WJD group and the injury + WJF group compared to the injury + saline group (p<0.05, n=5). *: Significant difference compared to the injury + saline group, p<0.05.

[0024] [Figure 8] FIG. 8 shows that the implantation of either of the two Wharton's jelly products effectively increases the distribution of immature osteoblasts in rats with skull defects, as observed by immunohistochemical staining with anti-osteocalcin. FIG. 8A shows low-magnification panoramic images of horizontal histological sections of skulls stained with anti-osteocalcin in each group; FIG. 8B shows the boundary region of the bone defect at gradually enlarged magnifications from low magnification; FIG. 8C shows the inner region of the bone defect; FIG. 8D shows the percentage of the area of ​​immature osteoblasts quantified by anti-osteocalcin staining. The results show that the percentage of the distribution area of ​​immature osteoblasts is significantly increased in the injury+WJD group and the injury+WJF group compared to the injury+saline group (p<0.05, n=5). *: Significant difference compared to the injury+saline group, p<0.05.

[0025] [Figure 9]Figure 9 shows the trend of bone development in the new skull. Immunohistological staining with anti-osteocalcin was used to identify immature osteoblasts, ALP histological staining was used to identify mature osteoblasts, and H&E staining was used to identify mature bone. The quantitative data from the three types of staining were summed up to show different stages of bone development and comprehensively evaluate the trend of bone formation in the rat skull defect. The results show that new bone development is significantly higher when either fresh frozen Wharton's jelly or decellularized Wharton's jelly was implanted.

[0026] Detailed Description of the Invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0027] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "sample" includes a plurality of such samples and equivalents thereof known to those of skill in the art.

[0028] The terms "including" or "comprising" are generally used in the sense of including / including permitting the presence of one or more features, ingredients or components. The terms "including" or "comprising" are intended to encompass the terms "consisting of" or "essentially consisting of."

[0029] The term "individual" or "subject" includes humans and non-human animals, such as companion animals (dogs, cats, etc.), farm animals (cows, sheep, pigs, horses, etc.), and laboratory animals (rats, mice, guinea pigs, etc.).

[0030] As used herein, the term Wharton's jelly refers to Wharton's jelly in mammalian umbilical cord, which is known as interlamellar jelly, a mucous connective tissue substance found in the umbilical cord that consists of a large amount of extracellular matrix forming a three-dimensional colloid structure rich in collagen, glycoproteins and hyaluronic acid. The umbilical cord contains two umbilical arteries and one umbilical vein, and the connective tissue surrounding the blood vessels is known as Wharton's jelly.

[0031] A suitable source for preparing the products of the invention is "Wharton's jelly" from the maternal umbilical cord, which may be processed, for example, by dissection, mincing, washing, enzymatic digestion, cryoprotection, freezing, drying, baking, or any combination thereof.

[0032] The term "critical size bone defect" as used herein means that bone is destroyed or removed due to trauma, fracture, bacterial infection, or tumor metastasis and invasion. If the defect exceeds twice the diameter of the long bone shaft and does not heal naturally despite bone repair methods such as bone grafting, this phenomenon is called a critical size bone defect.

[0033] The following examples are provided to further illustrate the present invention and are intended to be illustrative only and not limiting. In light of the present disclosure, it will be understood by those skilled in the art that changes or modifications may be made therein and still obtain a like or similar result, but which do not depart from the spirit and scope of the invention. EXAMPLES

[0034] material and method

[0035] 1.1 Preparation of Wharton's jelly

[0036] Human umbilical cords were collected aseptically after delivery and kept in cold (4°C) Hank's Balanced Salt Solution (HBSS; Gibco 14185-052). Wharton's jelly was isolated within 24 hours after collection. All laboratory equipment was autoclaved, disinfected with 75% ethanol, and cooked before use. In a laminar hood, the umbilical cord was disinfected by immersion in 75% ethanol, placed in HBSS, and cut longitudinally. The blood vessels were dissected and pulled out, yielding Wharton's jelly, which appeared as milky white interstitial tissue.

[0037] The isolated Wharton's jelly was then processed in two ways: Methods (1). Frozen Wharton's Jelly (WJF): Freshly obtained Wharton's Jelly was immediately frozen in the presence of cryoprotectant (10% DMSO) to obtain WJF, which could be easily used after thawing.

[0038] Method (2). Decellularized Wharton's Jelly Matrix (WJD): The obtained fresh Wharton's jelly was stored in a refrigerator at -80°C, and then the moisture in the tissue was removed using a freeze-dryer to obtain WJD, which was sealed and stored in a dry box before use.

[0039] WJF and WJD were used for in vitro and animal experiments. Furthermore, WJF and WJD were embedded in paraffin and subjected to histochemical staining to examine the histological structure.

[0040] 1.2 Tissue fiber staining

[0041] WJF and WJD were embedded in paraffin. After deparaffinization and rehydration, the tissue sections were stained with 0.1% Sirius Red (Sigma 2610-10-8) in picric acid. The tissue sections were then washed by immersion in ddH2O. Next, the tissue sections were immersed in a series of increasing concentrations of alcohol, and then dehydrated by immersion in xylene. Finally, the section slides were mounted with mounting medium and observed under a light microscope.

[0042] 1.3 Tissue glycoprotein staining

[0043] After WJF and WJD were deparaffinized and rehydrated, the tissue sections were stained with Periodic Acid-Schiff (PAS) using a Schiff Stain kit. The tissue sections were stained with periodic acid solution for 5 minutes and washed with ddH2O three times for 2 minutes each time. Next, the tissue sections were stained with Schiff's reagent for 15 minutes and washed with running ddH2O for 5 minutes. After staining, the interstitial glycoproteins are pink-purple. The tissue sections were then dehydrated by immersion in increasing concentrations of alcohol, then immersed in xylene, and finally sealed for light microscopy and photography.

[0044] 1.4 In vitro culture of rat osteoblasts (ROB)

[0045] One-day-old Sprague Dawley (SD) rats were sacrificed by an overdose of anesthesia, and the rat skulls were obtained using sterilized instruments in a laminar flow cabinet and cut into cubes with sides of 1 mm, then washed with sterile HBSS to remove red blood cells. Next, the tissues were reacted with 0.1% trypsin and 0.1% collagenase for 2 hours at 37°C under shaking. Then, Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) was added to terminate the effectiveness of trypsin, and then centrifuged at 1500 rpm for 5 minutes. The cells attached to the bottom were resuspended in DMEM supplemented with 10% FBS. The cells were seeded in 10 cm culture dishes, and the medium was replaced every 4 days. After 10 to 14 days, when complete confluence was reached, the cells were passaged. In this example, osteoblasts of the second passage were used.

[0046] 1.5 Grouping of in vitro osteoblast cultures

[0047] The in vitro cultured ROBs were divided into the following five groups: (1) osteoblast group: 2x10 4 (1) Osteoblast / WJD group: 2x10 4ROBs / well were seeded into the lower chamber of a 24-well plate, and WJD was added to the upper chamber of the transwell; (3) osteoblast + WJD group: 2x10 4 (4) osteoblast + WJF group: 2x10 ROBs / well seeded in a 24-well plate and cultured with WJD; (5) osteoblast + WJF group: 2x10 4 ROBs / well were seeded in the lower chamber of a 24-well plate, and WJFs were added to the upper chamber of the transwell; (5) osteoblast + WJF group: 2x10 4 ROBs / well were seeded in a 24-well plate and cultured with fresh WJF. The medium (DMEM supplemented with 10% FBS) was replaced every 3 days. After 14 days of culture, alkaline phosphatase cell staining was performed.

[0048] 1.6 Reverse transcription-polymerase chain reaction (RT-PCR)

[0049] The cells were treated with 0.05% trypsin, and fresh skull tissue was reacted with Trizol (Sigma T9424) and centrifuged at 12000g for 15 minutes at 4°C to extract total RNA. The supernatant was mixed with an equal volume of isopropyl alcohol and then centrifuged at 12000g for 10 minutes at 4°C. The RNA was washed with alcohol and redissolved in DEPC water. The RNA concentration was measured before use. The purified RNA was then transcribed into complementary DNA (cDNA) by reverse transcription (RT). This cDNA was subjected to polymerase chain reaction (PCR) with primers and a master mix. The following primers were used: Rat-Runt-related transcription factor 2 (Rat-RUNX2) (212 bp) Forward: 5'-GCTTCTCCAACCCACGAATG-3' Reverse: 5'-GAACTGATAGGACGCTGACGA-3' Rat-GAPDH(160bp) Forward: 5'-CTCTACCCACGGCAAGTTCAAC-3' Reverse: 5'-GGTGAAGACGCCAGTAGACTCCA-3'

[0050] The final PCR products were analyzed by DNA electrophoresis using a 2% agarose gel and photographed under a UV transilluminator.

[0051] 1.7 Alkaline phosphatase cell staining

[0052] The cells were washed with saline solution, fixed with 2.5% glutardialdehyde for 15 min, and 5-bromo-4-chloro-3-indolylphosphate / nitro blue tetrazolium (BCIP / NBT, Sigma B5655) was dissolved in secondary water and then added to a 24-well plate. After 1 h of reaction in a 37°C oven in the dark (Ocarino et al., 2008), 10% in hydrogen chloride (HCl) was added and reacted in a 37°C oven in the dark for 12 h. The absorbance of the supernatant at 595 nm was measured to quantify alkaline phosphatase in each group. Higher absorbance indicates higher levels of alkaline phosphatase, which indicates more cells differentiate into osteoblasts.

[0053] 1.8 Alizarin Red S staining

[0054] ROBs from osteoblast, osteoblast / WJD and osteoblast / WJF groups were cultured for 21 days using osteogenic medium (R&D CCM007) (medium was replaced every 3 days). The cells were fixed with 2.5% glutardialdehyde for 15 min, then stained with 2% Alizarin Red S (ARS, designated as Sigma A5533) for 1 h in a 37°C oven in the dark. After that, 10% cetylpyridinium chloride (CPC, designated as Sigma C-0732) (Qiao et al., 2016) was added and reacted for 1 h in a 37°C oven in the dark. The absorbance of the supernatant at 540 nm was measured, and the red area of ​​each group was quantified. Higher absorbance indicates higher calcium precipitation levels.

[0055] 1.9 Experimental animals

[0056] In this experiment, 8-week-old male Sprague Dawley (SD) rats weighing 250-280 g were used as experimental animals. The animals were kept in a 45 x 24 x 20 cm transparent PC cage, with 12 hours of light per day (7:30 a.m. to 7:30 p.m.), constant temperature air conditioning (22 ± 2°C) to control the ambient temperature, and a sufficient supply of food and drinking water. The bedding was changed once a week.

[0057] 1.10 Establishment of a critical-size skull defect model in rats

[0058] SD rats were anesthetized by intraperitoneal injection of Zoletil 50 and xylazine hydrochloride (Sigma 23076359), the head of the rat was fixed in a stereotaxic apparatus, and the hair was cleanly shaved with a razor. The skin of the rat was disinfected with a cotton ball soaked in iodine solution, incised with a scalpel, and the subcutaneous tissue was pushed aside with surgical scissors to remove the periosteum. Then, the parietal bone was perforated with a surgical trephine burr (diameter 8 mm), and the skull was detached from the dura mater underneath to form a critical size bone defect animal model. Finally, the subcutaneous tissue and skin were sutured.

[0059] 1.11 Grouping of Experimental Animals

[0060] SD rats were divided into three groups: 1. Injury + saline group: After the skull defect, saline was administered without any treatment. 2. Injury + WJD group: Immediately after the skull defect, WJD dissolved in saline was administered to the defect area. 3. Injury + WJF group: Immediately after the skull defect, WJF dissolved in saline was administered to the defect area.

[0061] 1.12 Micro-computed tomography (micro-CT)

[0062] The micro-CT images were processed with AMIDE software (SOURCEFORGE), and three-dimensional X-ray images were reconstructed. UHRA 3D scanner (MI Labs) was applied in this study to scan the rats' live skulls on the first day after surgery, and then monthly at 1, 2, 3, 4, and 5 months to track the healing level of the defective skull.

[0063] 1.13 Sacrifice of experimental animals, perfusion fixation, decalcification and paraffin sectioning

[0064] SD rats were anesthetized by intraperitoneal injection of Zoletil 50 and xylazine hydrochloride (Sigma 23076359) and fixed by perfusion of 4% paraformaldehyde and 7.5% picric acid in 0.01M PBS. Skulls were obtained and placed in fixative for 24 hours. For decalcification, skulls were immersed in 20% EDTA in 9% NaOH solution for 9 days, with two changes of the solution. Decalcified skull tissues were washed with running water for 8 hours, then dehydrated in increasing concentrations of alcohol, and processed for paraffin embedding in xylene and pure paraffin. The samples were then cut horizontally into 7 μm-thick tissue sections on a paraffin microtome.

[0065] 1.14 Hematoxylin and eosin staining

[0066] The tissue sections were deparaffinized, rehydrated, stained with hematoxylin (Muto Pure Chemicals; No. 3008-1) for 5 min, then washed with running water for 30 min. After mounting in 70% alcohol for 3 min, the sections were placed in a ventilated environment to allow the remaining alcohol to evaporate. The tissue sections were then stained with eosin (Muto Pure Chemicals; No. 3008-2) for 1 min, then washed with running water for 15 min, dehydrated in alcohol solutions of increasing concentrations, and treated twice with xylene. Finally, the slides were mounted with mounting medium and examined under a light microscope and photographed. The section with the largest area of ​​skull tissue was obtained, and the circumference of the border between the old and new bone was marked to quantify the original area of ​​the bone defect. The amount of new bone within the bone defect area was then quantified and expressed as a percentage.

[0067] 1.15 Alkaline phosphate staining (ALP staining)

[0068] Skull tissue sections were deparaffinized, rehydrated, and reacted with an alkaline phosphatase kit (Sigma 85L2-1KT) in an oven at 37 °C for 1 h, then immersed in ddH2O for 2 min. Slides were mounted with mounting medium for examination under a light microscope, photography, and quantification. In the stained sections, purple stained cells were observed, indicating that the sections contained ALP, i.e., the cells were mature osteoblasts. The section with the largest area of ​​skull tissue was obtained, and the circumference of the border between old and new bone was marked to quantify the original area of ​​the bone defect. The new bone within the bone defect area was then quantified and expressed as a percentage of the osteoblast area.

[0069] 1.16 Anti-osteocalcin antibody immunohistochemical staining

[0070] After deparaffinization of the skull tissue sections, primary mouse anti-osteocalcin antibody (Abcam ab13740, 1:200) was added and reacted at 4°C for 16-18 hours. Secondary goat anti-mouse IgG-conjugated biotin (Millipore AP124B, 1:250) was then added and reacted at room temperature for 1 hour, and then reacted with VECTASTAIN® ABC-conjugated HRP kit (Vector Laboratoris PK-4000) at room temperature for 1 hour. Next, the sections were washed three times for 5 minutes with 0.01M PBS. Finally, DAB (5 mg DAB in 10 mL Tris-HCl, 35% H2O2 3.5 μL, pH 7.4) was added to develop the color. After staining was completed, the sections were air-dried at room temperature. The sections were dehydrated by immersing in increasing concentrations of alcohol, and then immersed in xylene. Finally, the slides were mounted with mounting medium and observed and photographed under a light microscope.

[0071] Histological sections of all the skulls were taken and the percentage of osteocalcin (marked in dark brown) within the defect area was quantified to indicate ossification activity.

[0072] 1.17 Statistical analysis

[0073] All experimental data were expressed as mean ± standard deviation. Comparisons between mean values ​​were performed by one-way or two-way analysis of variance, followed by Fisher's least significant difference (LSD) test for multiple comparisons. For all experimental data, p<0.05 was set as the minimum entry criterion for significant differences.

[0074] 2. Results

[0075] 2.1 Histological and morphological analysis of WJD and WJF

[0076] Freshly frozen Wharton's jelly (WJF) products (Fig. 1A) and freeze-dried Wharton's jelly (WJD) products (Fig. 1B) extracted from the umbilical cord were embedded in paraffin and serially sectioned. The cellular and tissue structure of Wharton's jelly was observed by H&E staining. The results showed that WJF had a large number of nuclei and a dense and well-arranged structure (Fig. 1A1). In freeze-dried Wharton's jelly, no nuclei were observed, the structure changed from dense to loose, and various pores were observed (Fig. 1B1). The main components of Wharton's jelly are collagen and glycoprotein, and the distribution of collagen and glycoprotein was observed by staining with Sirius red and PAS, respectively. The results showed that collagen and glycoprotein in Wharton's jelly were not destroyed by freeze-drying and still functioned as a supporting structure for Wharton's jelly (Figs. 1A2-A3, B2-B3). The cell morphology was observed by scanning electron microscopy (Figure 1C). Scanning images showed that WJD sections were established with densely distributed pores (Figure 1D). Multiple views were randomly selected and framed to quantify the pore size and calculate the average pore size distribution. The analysis showed that WJDs had multiple pore structures of various sizes, whether they were paraffin-embedded, sectioned, and quantified by H&E staining or analyzed by scanning electron microscopy, and the pore size distribution was mostly within the range of 10-70 microns (Figure 1E).

[0077] 2.2 Co-culture of rat osteoblasts with Wharton's jelly products increased the expression of bone differentiation genes in osteoblasts.

[0078] In vitro, rat osteoblasts (ROB) were cultured alone or co-cultured with WJD or WJF for 14 days, and then subjected to trypsinization. The substratum of cells was then harvested for RNA extraction. RT-PCR was performed using rat runt-related transcription factor 2 (RUNX2) to examine the osteogenic differentiation of ROB. The results showed that GAPDH was detected in the ROB, ROB / WJD, or ROB / WJF groups, and RUNX2 was detected in all groups (Figure 2A). Furthermore, a real-time quantitative polymerase chain reaction device was used to analyze the intergroup differences in gene expression levels. The results showed that the expression levels of rat RUNX2 in osteoblasts of the ROB / WJD and ROB / WJF groups were significantly increased compared to the ROB group (Figure 2B).

[0079] 2.3 Co-culture of rat osteoblasts with Wharton's jelly products increased the expression of alkaline phosphatase in osteoblasts.

[0080] In vitro, ROBs were cultured alone or co-cultured indirectly or directly with WJD and WJF, then treated with 10% FBS-supplemented DMEM for 14 days, then the cells were stained with BICP / NBT and the absorbance was measured at OD595 to observe the expression level of alkaline phosphatase in ROBs (Figure 3A, Figure 3B). The results showed that the intracellular expression of alkaline phosphatase in ROBs co-cultured indirectly or directly with WJD or WJF was significantly increased compared to osteoblasts cultured alone (p<0.05, Figure 3C).

[0081] 2.4 Co-culture of rat osteoblasts and Wharton's jelly increases calcium precipitation levels

[0082] ROBs were cultured in vitro alone or co-cultured indirectly or directly with WJD and WJF, respectively, and treated with osteogenic differentiation medium for 21 days. Then, the cells were stained with Alizarin Red S, and the expression of calcium deposits in ROBs was marked in dark red. Gradually enlarging from low-magnification images, osteoblasts in the ROB group were more sparsely clustered, and there was only a low level of calcium deposits in the field of view. In the ROB / WJD group, in addition to the clusters of osteoblasts, there were osteoblasts growing in a concentric manner, and there was a high level of calcium deposits in the surrounding area. In the ROB / WJF group, mature osteoblasts with tubules were seen, and clusters of osteoblasts surrounded the vicinity. Numerous calcium crystals were distributed under the field of view (Figure 4A). After that, the calcium crystals marked in dark red were dissolved, and the concentration of calcium ions was analyzed by measuring the absorbance at OD540. The results showed that calcium ion concentrations in the ROB / WJD or ROB / WJF groups were significantly increased compared to the ROB group (p<0.05, Fig. 4B). The Alizarin Red S staining results suggested that indirect co-culture with WJD or WJF promoted osteoblast differentiation and enhanced the expression of calcium deposition.

[0083] 2.5 Quantification of bone regeneration in cranial defects by microcomputed tomography shows that Wharton's jelly products effectively increase bone production.

[0084] Skull healing was observed via micro-computed tomography (micro-CT) at 1, 2, 3, 4, and 5 months after skull defect surgery in rats. In the injury + saline group, a small amount of new bone grew inward along the border of the bone defect. In the injury + WJD or injury + WJF groups, in addition to the new bone growing inward, new bone was also found scattered in the defect (Figure 5A). The percentage of the area of ​​new bone within the skull defect area was quantified. The results showed that in the injury + saline group, there was 12.5 ± 3.4% new bone at 1 month after surgery, and the healing of the skull became slower with time after surgery, with only 18.4 ± 1.4% of the skull having recovered at 5 months after surgery. In the injury + WJD group and injury + WJF group, the percentage of new bone at 1 month after surgery was 21.2 ± 3.9% and 23.0 ± 4.1%, respectively, which was significantly increased compared to the injury + saline group (p < 0.05), and the increase was maintained until the 5th month, when the percentage of new bone in the skull defect reached 30.6 ± .6% and 35.6 ± 3.7%, respectively (p < 0.05, Figure 5B). As observed by the naked eye with micro-CT, the skull healing ability of rats that underwent surgery for skull defects was only maintained in the range of 12% to 18% when no treatment was performed for 5 months. Administration of the WJD product or WJF product could promote bone formation in rats with skull defects.

[0085] 2.6 H&E staining showed that Wharton's jelly promoted bone regeneration in rat cranial defects.

[0086] Low-magnification panoramic images of the H&E staining results (Fig. 6A) were used to gradually increase the magnification of bone performance at the border of the bone defect (Fig. 6B) and the internal area of ​​the bone defect (Fig. 6C). The results showed that in the injury + saline group, there was a small amount of new bone and dense connective tissue at the border of the defect, and only connective tissue was present within the bone defect. In the injury + WJD and injury + WJF groups, in addition to a tendency for bone mass to increase at the border of the bone defect, free and mature new bone was also observed within the bone defect. The percentage of new bone within the bone defect was quantified by H&E staining 5 months after surgery. In the injury + saline group, the new bone within the bone defect was only 18.8 ± 1.9%. On the other hand, in the injury + WJD group and the injury + WJF group, the percentage of new bone in the bone defect reached 27.1 ± 4.0% and 32.7 ± 5.0%, respectively, which was significantly increased compared to the injury + saline group (p < 0.05, Figure 6D). As observed microscopically by H&E staining, the WJD product or WJF product promoted new bone formation in rats with cranial defects.

[0087] 2.7 Alkaline phosphate staining for osteoblast marking demonstrated that Wharton's jelly increased activity performance in critical-size cranial defects in rats.

[0088] The results of low-magnification panoramic images of ALP staining (Figure 7A) show the expression areas of mature osteoblasts at the defect border (Figure 7B) and the internal region of the bone defect (Figure 7C), which are gradually enlarged from low magnification. The results showed that in the injury + saline group, a small number of mature osteoblasts at the border and inside of the bone defect showed ALP. In both the injury + WJD and injury + WJF groups, mature osteoblasts were attached to the inwardly grown new bone at the border of the bone defect, and free new bone was surrounded by osteoblasts within the bone defect. ALP within the defect + The area percentage of the lesion was quantified by ALP staining. In the injury + saline group, ALP + In the injury + WJD and injury + WJF groups, the ALP area was only about 3.7 ± 2.1%. +The areas reached 10.0±5.9% and 8.8±2.3%, respectively, and tended to increase compared with the injury + saline group (p<0.05, Figure 7D).As observed microscopically by ALP staining, administration of WJD or WJF products improved the activity of osteoblasts in rats with cranial bone defects.

[0089] 2.8 Marking of new bone by anti-osteocalcin antibody immunohistochemical staining shows that Wharton's jelly products promote the performance of bone regeneration in rat cranial defects.

[0090] Osteocalcin is secreted by osteoblasts and released into bone tissue to form unmineralized bone during bone formation. The low-magnification panoramic (Fig. 8A) results of anti-osteocalcin immunostaining show the expression levels of osteocalcin-containing unmineralized bone at the borders of bone defects (Fig. 8B) and in the areas within the bone defect region (Fig. 8C) from low magnification to gradually enlarged. The results showed that in the injury + saline group, no osteocalcin was observed at the borders of bone defects; only a small amount of osteocalcin was released into the tissues within the bone defects. In the injury + WJD alone treatment group and the injury + WJF alone treatment group, osteocalcin was found not only at the borders of bone defects and within the bone defects, but also in the clusters of osteoblasts, which means that osteoblasts are undergoing bone formation responses. OCN in the bone defects + The percentage of the area of ​​the lesion was quantified by OCN staining. + The area was only 2.4±1.6%. On the other hand, in the injury+WJD and injury+WJF groups, OCN + The area percentage of osteoblasts reached 14.8±4.8% and 15.5±1.5%, respectively, indicating a significant increase compared to the injury + saline group (p<0.05, Figure 8D). As observed microscopically by anti-osteocalcin staining, the WJD or WJF product further promoted osteoblast expression, thereby promoting bone regeneration.

[0091] 2.9 Wharton's Jelly products can significantly promote new bone growth.

[0092] Immature osteoblasts were marked by anti-osteocalcin immunostaining, mature osteoblasts by ALP histological staining, and mature bone by H&E staining. Quantitative data obtained from the three staining methods were summed up to evaluate bone development in rats with cranial defects. The results showed that the injury + saline group had a bone development of about 25%, while the injury + WJD group and the injury + WJF group had a bone development of about 52% and about 57%, respectively, which showed relatively high levels of development (Figure 9).

[0093] 3. Conclusion

[0094] The Wharton's Jelly product was implanted into rats with cranial defects. After treatment with either WJF or WJD, the new bone was observed by micro-CT. Staining of the sections demonstrated that the signals shown in the images were mature bone, and the development of the host bone was also promoted. These experimental results demonstrated that the Wharton's Jelly product could be used in clinical medicine as a natural medicinal material to promote bone regeneration, thereby providing a new treatment method for patients suffering from bone defects.

[0095] It is believed that those skilled in the art to which the present invention pertains can utilize the present invention in the broadest manner based on the description in this specification without further description. Therefore, it should be understood that the description and claims provided are for illustrative purposes only and are not intended to limit the scope of the present invention in any manner.

Claims

1. A pharmaceutical product for promoting bone regeneration, the pharmaceutical product being prepared by one of the following two methods: Method (1): Treating Wharton's jelly collected from the umbilical cord with a cryoprotectant, then storing it frozen and thawing it before use; and Method (2): Freeze-dry Wharton's jelly directly from the umbilical cord and store it under dry conditions before use.

2. 10. The pharmaceutical product of claim 1, wherein the umbilical cord is from a human or non-human mammal.

3. 2. The pharmaceutical product of claim 1, wherein the cryoprotectant is a 10% dimethyl sulfoxide (DMSO) solution.

4. The pharmaceutical product of claim 1, wherein the cryopreservation is performed in liquid nitrogen.

5. 5. The pharmaceutical product of claim 1, 2, 3 or 4, wherein the cryopreserved Wharton's Jelly contains cell bodies.

6. 2. The pharmaceutical product of claim 1, wherein the freeze-drying comprises harvesting fresh Wharton's jelly from the umbilical cord, then directly storing it frozen at -80°C, and then using a freeze-dryer to remove the water from the tissue.

7. 7. The pharmaceutical product of claim 6, wherein the fresh Wharton's jelly does not contain cell bodies.

8. The pharmaceutical product of claim 1, which increases the performance of alkaline phosphatase in osteoblasts, improves the expression level of bone differentiation genes in osteoblasts, promotes osteoblast differentiation, enhances calcium deposition, effectively enhances bone production, enables bone formation in bone defects, enhances the activity of osteoblasts in bone defects, or induces the growth of new bone.

9. 13. Use of the pharmaceutical product of claim 1 in the preparation of a medicament for promoting bone regeneration.

10. The use according to claim 9, wherein the pharmaceutical product increases the performance of alkaline phosphatase in osteoblasts, improves the expression level of bone differentiation genes in osteoblasts, promotes osteoblast differentiation, enhances calcium deposition, effectively enhances bone production, enables bone formation in bone defects, enhances the activity of osteoblasts in bone defects, or induces new bone growth.

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