CN114099779A

CN114099779A - Waton's gel product capable of promoting bone regeneration

Info

Publication number: CN114099779A
Application number: CN202010885501.8A
Authority: CN
Inventors: 傅毓秀
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-08-28
Filing date: 2020-08-28
Publication date: 2022-03-01

Abstract

The invention relates to a Waton's gel product for promoting bone regeneration, in particular to an umbilical cord Waton's gel product which has the effect of promoting bone regeneration. Wherein the product is prepared by the following two methods: (1) treating the Waton's gel of umbilical cord with an antifreeze agent, freezing and storing, and thawing before use; or (2) directly freeze-drying the Wharton's jelly of umbilical cord, and storing at room temperature under dry condition before use.

Description

Waton's gel product capable of promoting bone regeneration

Technical Field

The invention relates to an umbilical cord Waton's gel product which has the effect of promoting bone regeneration.

Background

Bone is damaged, or removed, in large quantities due to trauma, fractures, infections, and metastases. When the defect range exceeds 2 times of the diameter of the long bone shaft, the defect will not heal naturally even if a bone grafting method or other bone repairing method is adopted, the phenomenon is called critical bone defect, the fracture part is too large, and the risk of subsequent fracture is also increased greatly. Also, regeneration of dense bone defects is relatively difficult. Therefore, how to repair the bone defect of dense bone is a very difficult problem and dilemma in clinical orthopedics nowadays.

At present, the directions of clinical common medical treatment are divided into three types, the first type is autogenous bone, and bone tissues of other parts of a body need to be taken out through an operation to repair the bone at the deficient part. The defects are that the recovery period of the re-operation is long, and the bone tissue with the same area cannot be taken out. The second is allograft bone, which is allograft bone obtained by removing excess bone tissue from other people through surgery and post-processing. Allograft bone grafting has ethical debate beyond the risk of immunological infection. Therefore, researchers in the field wish to develop appropriate medical materials in an attempt to overcome the dilemma of clinical treatment.

Disclosure of Invention

The invention provides an umbilical cord Waton's gel product for promoting bone regeneration, which has the effect of promoting bone regeneration, wherein the product is prepared by the following two methods: (1) treating the Waton's gel of umbilical cord of puerpera with antifreeze agent, freezing for storage, and thawing before use; or (2) directly freeze-drying the Waton's gel of umbilical cord of parturient, and storing at room temperature under dry condition before use.

According to the present invention, the Watton's gel preparation obtained in the process (1) is treated with an antifreeze agent and then stored under refrigeration, and expressed as WJF (Wharton's Jelly Frozen), still retains cell bodies.

According to an embodiment of the invention, wherein the cryoprotectant is a 10% dimethyl sulfoxide (DMSO) solution.

According to an embodiment of the invention, wherein the cryopreservation is performed in liquid nitrogen.

In one embodiment of the invention, the Wawden gel product is first protected with an anti-freeze agent (e.g., a 10% DMSO solution), stored frozen, and thawed prior to use.

The Walton's gel product obtained by the method (2) according to the present invention is prepared by freeze-drying the Walton's gel of umbilical cord of a parturient, and is stored at room temperature and in a dry condition before use, and expressed as WJD (Wharton's Jelly Dried), and does not contain cell forms.

According to an embodiment of the invention, wherein the cryopreservation is at-80 ℃.

In another embodiment of the present invention, the Waton's gel product is prepared by removing fresh Waton's gel, directly freezing and storing at-80 deg.C, freeze-drying, and draining water from tissue.

According to the present invention, the two types of Watton's gel products can increase the expression of alkaline phosphatase in osteoblasts, increase the expression of a bone differentiation gene in osteoblasts, promote differentiation of osteoblasts, enhance the expression of calcium deposition, effectively increase the yield of bone, regenerate bone in a bone defect, enhance the expression of activity of osteoblasts in a bone defect, or promote the development of new bone.

In another aspect, the invention provides the use of the two Watton's gel products for preparing a medicament for promoting bone regeneration.

The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the invention will be apparent from the following detailed description of several embodiments, and from the appended claims.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

In the drawings:

FIG. 1 shows a histological analysis of a freshly frozen, or freeze-dried, Waton's gel preparation of the invention. Wherein: (A) paraffin embedding and sectioning are carried out on a fresh frozen Waton's gel product (WJF) removed from an umbilical cord and a Waton's gel product (WJD) subjected to freeze drying treatment of (B). (A1, B1) shows that the fresh frozen Waton's gel product presents a compact structure by H & E staining, and cell bodies rich in mesenchymal stem cells are arranged in the tissues; the freeze-dried Waton gel product exhibits an evacuated porous structure. (A2, B2) was stained with Sirius Red (Sirius Red) to show that collagen from freshly frozen Waton's gel was densely arranged in bundles and that collagen from freeze-dried Waton's gel was around the periphery of the voids. (A3, B3) shows that polysaccharides are impregnated in the tissue of the Waton's gel by staining with PAS. (C, D) the Watton's gel product (WJD) after freeze-drying is observed under scanning electron microscope to show the tissue pattern with different sizes of porous structure. (E) The Watton's gel after freeze drying shows porous structures with different sizes no matter the Watton's gel is subjected to paraffin embedding and slicing, and then is subjected to HE staining quantification or scanning electron microscope image analysis; furthermore, the diameter distribution of the holes is mostly in the range of 10-70 microns.

FIG. 2 shows that the co-culture of rat osteoblasts and two Watton's gel preparations can increase the expression of the bone differentiation gene in osteoblasts. Wherein: (A) the rat RUNX2 was examined by RT-PCR after culturing rat osteoblasts alone in vitro and co-cultured with freeze-dried watts gel (WJD) or freshly frozen watts gel (WJF) for 14 days, indicating that each group exhibited rat RUNX 2. (B) The expression level of rat RUNX2 in the rat osteoblasts was quantified by real time RT-PCR. The results showed that the expression level of RUNX2 in the white mouse in osteoblasts of ROB/WJD and ROB/WJF groups was significantly increased (p <0.05) compared to ROB group. *: compared with the ROB group, the difference is significant, and p is less than 0.05.

FIG. 3 shows that the quantitative determination of alkaline phosphatase increases the expression level of alkaline phosphatase in rat osteoblasts when cultured together with lyophilized Waston's gel cell preparation (WJD) or freshly frozen Waston's gel preparation (WJF). Wherein: (A) a pattern diagram of co-culture of rat osteoblasts with freeze-dried Waton's gel cell preparation (WJD) or freshly frozen Waton's gel cell preparation (WJF) in a non-direct or direct manner. (B) After 14 days for the culture, alkaline phosphatase staining was performed. (C) To quantify the intracellular concentration of alkaline phosphatase, the expression level of alkaline phosphatase in the osteoblasts of white rats in the ROB/WJD, ROB + WJD, ROB/WJF or ROB + WJF groups was significantly increased (p <0.05) as shown by OD595 absorbance values, as compared with that in the ROB group alone. *: compared with the ROB group, the difference is significant, and p is less than 0.05.

FIG. 4 shows that the white rat osteoblasts co-cultured with two Watton's gel preparations can promote the expression of calcium deposition in osteoblasts. Wherein: (A) the rat osteoblasts were cultured separately in vitro and then co-cultured with freeze-dried Waton's gel cell preparation (WJD) or freshly frozen Waton's gel cell preparation (WJF) for 21 days, stained with alizarin Red (alizarin red S), and the gradually enlarged images showed that the osteoblasts of each group showed calcium deposition after being treated with the bone differentiation medium. (B) To quantify the concentration of calcium ions in each group, the ROB/WJD and ROB/WJF groups showed higher calcium deposition levels (p <0.05) than the ROB and ROB + BMP2 groups, as shown by OD 540 absorbance. *: compared with the ROB group, the difference is significant, and p is less than 0.05.

FIG. 5 shows that the two Waton's gel product treatment groups are implanted respectively by the tomography observation of a microcomputer, and the new bone regeneration of the skull defect of the white rat is effectively promoted. The skull-defect white rat follows the bone healing situation by micro-CT in the first, second, third, fourth and fifth months after operation. Wherein: (A) only a small amount of bone grows along the periphery of the defect in the Injury + Saline group, and free new bone also grows in the defect range except the periphery of the defect in the Injury + WJD or Injury + WJF group. (B) The defect area is marked with a red dashed line and the percentage of area of new bone mass within the defect area is quantified. The results show a significant increase in the percentage of new bone in either the Injury + WJD, or Injury + WJF treated group alone compared to the Injury + salt group (p < 0.05). *: compared with the Injury + Saline group with the same month age, the difference is remarkable, and p is less than 0.05.

FIG. 6 shows that the respective implantation of two Waton's gel preparations treated groups, as observed by H & E staining, effectively increased the area percentage of new bone mass in skull-deficient white rats. Wherein: (A) the horizontal tissue sections of each group of skull are visualized as H & E stained low-power panoramas, and the bone defect range is indicated by white dashed lines. And then gradually enlarging the boundary area (B) of the bone defect or the inner area (C) of the bone defect by low times (scale bar is 1mm and 200 mu m respectively). (D) The area percentage of new bone mass was quantified by H & E staining and the results showed that the area percentage of new bone mass was significantly increased in the Injury + WJD and Injury + WJF groups compared to the Injury + salene groups (p <0.05, n ═ 5). *: compared with the group of Injury + Saline, the gene has significant difference that p is less than 0.05.

FIG. 7 shows that the distribution of mature osteoblasts (bone osteoblasts) of white rats with skull defects is effectively improved by the respective implantation of two types of Watton's gel products, which is observed by ALP staining. Wherein: (A) each group of skull horizontal plane tissue sections were processed by low power panorama stained with ALP and then gradually enlarged from the boundary region (B) of the bone defect or the inner region (C) of the bone defect. (D) To quantify the area percentage of mature osteoblasts (mature osteoblasts) by ALP staining, the results showed that the area percentage of mature osteoblasts (mature osteoblasts) was significantly increased (p <0.05, n 5) compared to the set of Injury + WJD, Injury + WJF, and the set of mature osteoblasts. *: compared with the group of Injury + Saline, the gene has significant difference that p is less than 0.05.

FIG. 8 shows the distribution of immature osteoblasts (osteoblasts) in white rats with skull defects, which are observed by immunohistostaining of anti-osteocalcin (anti-osteopalcin), implanted with two kinds of Watton gel preparations. (A) Each group of skull horizontal plane tissue sections were visualized as a macroscopic panorama stained with anticalcin (anti-osteooccin), followed by macroscopic gradual enlargement of the boundary region (B) of the bone defect, or the inner region (C) of the bone defect. (D) In order to quantify the area percentage of immature osteoblasts (immatureoplasts) by staining with anticalcin (anti-osteopecalin), the results showed that the area percentage of immature osteoblasts (immatureoplasts) was significantly increased (p <0.05, n ═ 5) compared with those of the Injury + WJD group, the Injury + WJF group, and the immatureoplasts (immatureoplasts) were distributed. *: compared with the group of Injury + Saline, the gene has significant difference that p is less than 0.05.

Fig. 9 shows a trend graph of new skull bone development. The method comprises the steps of performing anti-osteocalcin (anti-osteopalcin) immunohistological staining to identify immature osteoblasts, performing ALP histological staining to identify mature osteoblasts, performing H & E histological staining to identify mature bone, summing quantitative data of the three stains to represent bone development at different stages, and comprehensively evaluating the bone neogenesis tendency of the skull defect of the white rat. The results show that the development of new bone mass is significantly higher whether fresh frozen or freeze-dried Waton's gel products are implanted.

Detailed Description

Unless defined otherwise, 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.

As used herein, the singular forms "a", "an" and "the" include plural referents 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 skilled in the art.

The terms "comprises" or "comprising" are generally used in the sense of including/comprising one or more features, elements or components that are permitted to be present. The terms "comprising" or "including" encompass the terms "consisting of or" consisting of.

The term "subject" or "subject" as used herein includes both human and non-human animals, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.), or laboratory animals (e.g., rats, mice, guinea pigs, etc.).

The term "Watton's jelly" as used herein refers to the Watton's jelly in mammalian umbilical cords, which is also known as inter-lamellar jelly (mucus connective tissue mass) found in the umbilical cord, which is a colloid that is composed of a large amount of extracellular matrix (extracellular matrix) into a three-dimensional architecture that is rich in extracellular matrix, collagen, glycoproteins, and hyaluronic acid, among others. The umbilical cord contains two umbilical arteries and one umbilical vein; the connective tissue surrounding the blood vessel is called Walton's gel (Wharton's jelly).

Suitable sources for preparing the products of the invention are "Watton's gel" (from maternal umbilical cord) which can be processed, for example, by dissection, mincing, washing, enzymatic digestion, freezing resistance, freezing, drying, baking, or any combination thereof.

The term "critical bone defect" as used herein refers to the destruction, or removal, of bone caused by trauma, bone fracture, bacterial infection, metastatic erosion of tumors, and the like. When the defect range exceeds 2 times or more the diameter of the long bone shaft, the defect is not healed naturally even by a method of repairing bone such as bone grafting, and this phenomenon is called critical-sized bone defect.

The following examples are provided to illustrate the present invention and are not to be construed as limiting the scope of the invention in any way.

The invention is further illustrated by the following examples, which are intended to be illustrative only and not limiting. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Examples

1. Materials and methods

1.1 preparation of Watton's gel preparation

The umbilical cord after delivery is collected in an aseptic mode and stored in Hank's balanced salt solution (HBSS, Gibco 14185-.

The removed Waton's gel is divided into two treatment modes;

(1) the first group was freshly frozen Watton's gel products, referred to as WJF. Fresh Waton's gel is protected by antifreeze agent (10% DMSO), and then frozen for storage and thawed for use.

(2) The second group is freeze-dried Waton's gel products, known as WJD. Directly storing fresh Waton's gel in a refrigerator at-80 deg.C, freezing, drying with a freeze dryer to remove water, sealing, and storing in a drying oven for direct use.

Both groups of Watton's gel preparations were subjected to in vitro cell experiments and animal experiments. In addition, fresh or freeze-dried Wawden gel preparations were paraffin-embedded and examined for histological morphology by histochemical staining.

1.2 tissue fiber staining

Fresh frozen or freeze-dried Walton's gel products are embedded in paraffin, and after deparaffinization and rehydration of tissue sections, the tissue sections are stained in 0.1% Sirius Red Stain (Sigma 2610-10-8), and then the tissue sections are soaked in secondary water for washing. Then, the tissue slices are soaked in alcohol with gradually increased concentration for dehydration, then soaked in xylene (xylene), and finally, the slices are sealed, observed by an optical microscope and photographed.

1.3 tissue glycoprotein staining

After the Walton's gel tissue pieces were dewaxed and rehydrated, Periodic acid-Schiff staining (PASStain for short) was performed with Periodic acid-Schiff staining kit (Sigma 395B-1 KT). The tissue pieces were stained with periodic acid solution (periodic acid solution) for 5 minutes and washed three times with two minutes of water twice. Subsequently, the tissue piece was stained with Schiff's reagent (Schiff's regent) for 15 minutes, and washed with running water for 5 minutes. After staining was complete, the tissue-specific glycoprotein appeared pink-purple. Then, the tissue slices are soaked in alcohol with gradually increased concentration for dehydration, then soaked in xylene (xylene), and finally the slices are sealed for optical microscope observation and photographing.

1.4 in vitro culture of Osteoblast in rat

In the experiment, a large-sized white rat born one day is adopted, after death is caused by excessive anesthesia, the skull of the white rat is taken down by using a sterilized instrument in an aseptic operating platform, cut into the size of about 1mm, and washed by a sterile HBSS buffer solution to remove red blood cells. Then reacted with 0.1% trypsin (trypsin) and 0.1% collagenase (collagenase) in an oven at 37 ℃ for 2 hours. Thereafter, an appropriate amount of 10% FBS DMEM was added to terminate the effect of trypsin (trypsin), and then centrifuged at 1500rpm for 5 minutes. Taking the lower layer cells, adding 10% FBS DMEM to scatter the cells, planting in a 10cm culture dish, replacing the culture solution once every four days, and carrying out subculture on the cells after about 10-14 days. The experiment was performed using the osteoblasts of generation 2.

1.5 grouping of osteoblasts cultured in vitro

Osteoblasts (osteoplast) in vitro culture were divided into five groups, the first, osteoplast group, i.e. 2 x 10⁴Osteoblasts were cultured in 24-well dishes alone. The second group, the osteoblast/WJD group, 2 x 10⁴Osteoblasts were cultured in the lower layer of a 24-well dish, and lyophilized Waton's gel was cultured in the transfer dish well (transwell) in the upper layer. The third group, the osteoblast + WJD group, 2 x 10⁴Culturing osteoblasts in 24-well dish, and freeze-dryingDry Walton's gel preparation (WJD) was co-cultured. Fourth group, osteoplast/WJF group, 2 x 10⁴The osteoblasts were cultured in the lower layer of a 24-well dish (24well plate), and freshly frozen Waton's gel product (WJF) was cultured in the migration well of the upper layer. Group V, Osteoblast + WJF group, i.e. 2 x 10⁴The osteoblasts were cultured in a 24-well dish and co-cultured with freshly frozen Waton's gel product (WJF). The alkaline phosphatase cell staining experiment was performed after 14 days of culture by changing the DMEM medium containing 10% FBS every three days.

1.6 Transcription-Polymerase Chain Reaction (Reverse Transcription-Polymerase Chain Reaction, RT-PCR for short)

Cells obtained after treatment with 0.05% Trypsin (Trypsin) were reacted with fresh skull tissue to extract total RNA and centrifuged at 12000g for 15 min at 4 ℃ by phenol (Trizol) (Sigma T9424). The supernatant was mixed with isopropanol in equal volume and centrifuged at 12000g for 10 min at 4 ℃. After washing the RNA with ethanol, the RNA was redissolved in DEPC water at 55 ℃. RNA concentration was measured before use. The purified RNA is subjected to reverse transcription reaction to synthesize complementary ribonucleic acid (cDNA). The cDNA was subjected to polymerase chain reaction with Primer and Master Mix. The following primers were used:

Rat-RUNX2

212bp

Forward：5’-GCTTCTCCAACCCACGAATG-3’，

Reverse：5’-GAACTGATAGGACGCTGACGA-3’。

Rat-GAPDH

160bp

Forward：5’-CTCTACCCACGGCAAGTTCAAC-3’，

Reverse：5’-GGTGAAGACGCCAGTAGACTCCA-3’。

after completion of the PCR product, DNA Electrophoresis (Electrophoresis) was performed on a 2% Agarose gel (Agarose gel), and after completion, the PCR product was visualized and photographed by UV lamp.

1.7 alkaline phosphatase cell staining experiments

Washing the cells with physiological test water, and fixing the cells with 2.5% glutaraldehyde (glutaraldehyde) for 15 minutes; A5-Bromo-4-Chloro-3-Indolyl Phosphate/Tetrazolium Blue (5-Bromo-4-Chloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium, BCIP/NBT for short, Sigma B5655) lozenge was dissolved in 10mL of secondary water and added to a 24-well dish. The reaction is carried out in an oven at 37 ℃ in a dark place for 1 hour; then 10% SDS in hydrochloric acid (HCl) (Ocarino et al, 2008) was added and the reaction was carried out in an oven at 37 ℃ for 12 hours in the absence of light. The amount of alkaline phosphatase expressed in each group was quantified by measuring the absorbance at 595 nm. The higher the expression of alkaline phosphatase, the greater the number of osteoblasts differentiated.

1.8 alizarin Red S (alizarin red S) staining

The osteoplast group, osteoplast/WJD group and osteoplast/WJF group were cultured in bone differentiation medium (osteopenic medium, R & D CCM007) for 21 days, and then the medium was changed every three days, and finally, alizarin red S (alizarin red S) staining experiments were performed. Washing the cells with physiological test water, and fixing the cells with 2.5% glutaraldehyde (glutaraldehyde) for 15 minutes; then 2% alizarin red S (ARS for short, Sigma A5533) is added into the 6-well dish. The reaction is carried out in an oven at 37 ℃ in a dark place for 1 hour; then, 10% cetylpyridinium chloride (CPC, Sigma C-0732 for short) (Qiao et al, 2016) was added thereto, and the mixture was dried in an oven at 37 ℃ for 1 hour in the absence of light. The red area of each group was quantified by measuring the absorbance at 540nm, which represents the amount of calcium expressed.

1.9 Experimental animals

The experimental animal source used in the experiment is male Sprague Dawley (S.D.) strain white rat of eight weeks old provided by Yangming university experimental animal center, and the weight of the rat is about 250-280 g. The breeding method is that the breeding is carried out in transparent PC cages of 45x24x20cm, the cages are illuminated for 12 hours every day (from seven am to seven pm), the environment temperature is constant temperature and air-conditioned (22 +/-2 ℃) and the feed (Laboratory Animal Diets MF18) and the drinking water are fully supplied, and the padding is replaced once a week.

1.10 Critical-sized Cranisal bone defect (CRITICAL-SIZED CRANIAL BONE DEFECT) in rats

The rats were anesthetized by intraperitoneal injection of sutent 50(Zoletil 50) and xylazine hydrochloride (Sigma 23076359), and then the heads were fixed with a stereotaxic instrument. The hair is shaved off by using a shaver, the skin of a white rat is disinfected by dipping cotton in iodine solution, the skin is cut by using a scalpel, the subcutaneous tissue is stretched by using surgical scissors, and the external periosteum is separated. Next, a hole was drilled in the rat parietal bone using an 8mm diameter trephine, and the skull and the underlying meninges were separated to prepare an animal model of critical bone defect (critical-sized bone defect). Finally, the subcutaneous tissue and the skin are sutured.

1.11 animal Experimental groups

The white rats are divided into three groups:

the first group was the Injury + Saline group, i.e., after the skull defect, no treatment was given, and only physiological Saline was given

(Saline)。

The second group was the Injury + WJD group, i.e. the treatment group given a freeze-dried watt's gel preparation (WJD) immediately after the skull defect at the defect.

The third group was the Injury + WJF group, i.e., the treatment group given freshly frozen Waston's gel preparation (WJF) immediately after the skull defect at the site of the defect.

1.12 micro-computer Tomography (micro-computer Tomography Scan, micro-CT for short)

The computed tomography image is processed by Amide software (sourcefocus) in a digital geometry mode, and then a three-dimensional radioactive medical image is reconstructed. The experiment used a small animal optical and computed tomography (U-CT) apparatus^UHRMILabs), a live skull scan was performed to track the extent of healing of the defective skull on the first day post-surgery, followed by the first, second, third, fourth, and fifth months, once per month.

1.13 sacrifice, perfusion fixation, decalcification and paraffin section of experimental animal

The white rat is anesthetized by intraperitoneal injection of Sutai 50 (Zolethil 50) and xylazine hydrochloride (xylazine hydrochloride), and fixed by perfusion of 4% paraformaldehyde (paraformaldehyde) and 7.5% picric acid (picric acid) in 0.01M PB. Taking out the skull, putting the skull into a fixing solution for 24 hours, and replacing with 20% EDTA to decalcify; the decalcification solution is replaced by 20% EDTA in 9% NaOH aqueous solution within 9 days of the decalcification time. The decalcified tissue was washed with running water for 8 hours. Next, dehydration was carried out with an alcohol solution of increasing concentration, paraffin embedding was carried out with xylene (xylene) and pure paraffin (pure paraffin), and the skull was sliced into a 7 μm tissue piece in a horizontal section using a paraffin slicer.

1.14 Hematoxylin-Eosin staining (Hematoxylin & Eosin Stain, H & E Stain for short)

After the tissue pieces were dewaxed and rehydrated, they were stained in hematoxylin (martial arts chemical No.3008-1) for 5 minutes and washed with running water for 30 minutes. Then, after 70% alcohol was put in for 3 minutes, the remaining alcohol was left to evaporate in a ventilated environment. Soaking the tissue slice in eosin (Wuteng chemical No.3200-2) for 1 min, washing with flowing water for 15 min, soaking the tissue slice in ethanol with increasing concentration for dehydration, and soaking in xylene twice. And finally, sealing the wafer by using a sealing wafer glue, and carrying out optical microscope observation and photographing. Taking a complete and maximum skull slice, taking the boundary between the primary bone and the new bone as the boundary of the skull defect, and calibrating the skull defect area. Next, the percentage of new bone mass within the defect area was quantified.

1.15 Alkaline Phosphatase staining (alkali Phosphatase Stain, ALP Stain for short)

After the tissue pieces were dewaxed and rehydrated, the tissue pieces were oven-reacted with Alkaline Phosphatase kit (Alkaline Phosphatase kit) (Sigma 85L2-1KT) at 37 ℃ for 1 hour, followed by soaking ddH₂And O2 minutes. And finally, sealing the wafer by using a sealing glue, observing by using an optical microscope, photographing and quantifying. The stained sections were observed to be bluish-purple cells containing alkaline phosphatase, i.e., representative of mature osteoblasts. Taking a complete and maximum skull slice, taking the boundary between the primary bone and the new bone as the boundary of the skull defect, and calibrating the skull defect area. Next, the percentage of the bluish purple area in the defect area was determined as the area ratio of the area where osteoblasts were present.

1.16 anti-osteocalcin antibody (anti-osteopalcin antibody) tissue immunostaining

The tissue sheet was deparaffinized, and then added with a primary antibody, mouse anti-osteocalcin antibody (Abcam ab 13740; 1:200), and reacted at 4 ℃ for 16 to 18 hours. Followed by the secondary antibody, goat anti-mouse IgG-conjugated biotin (goat anti-mouse-IgG-conjugated biotin) (Millipore AP124B, 1:250) was reacted at room temperature for 1 hour. Then use

ABC-conjugated HRP Kit (Vector laboratory PK-4000) was reacted at room temperature for 1 hour. Washed with 0.01M PBS for 5 minutes for a total of three times; finally DAB (5mg DAB, 35% H)₂O₂3.5. mu.L in 10mL Tris-HCl, pH 7.4). After dyeing, the tissue pieces were air-dried at room temperature and dehydrated. Soaking the tissue slices in ethanol with increasing concentration for dehydration, and soaking in xylene (xylene). And finally, sealing the wafer by using a sealing wafer glue, and carrying out optical microscope observation and photographing.

All skull sections were taken and the percentage of Osteocalcin (Osteocalcin) marked as dark brown in the defect area was quantified to identify the active manifestation of ossification.

1.17 statistical analysis

All experimental data are expressed as mean SEM (Standard error of the mean). Comparisons between the means were performed by One-Way ANOVA (One-Way ANOVA) or Two-Way ANOVA (Two-Way ANOVA) and by Fisher's Least Significant Difference test (LSD) test (Fisher's Least Significant Difference). Experimental data all used p <0.05 as the lowest starting criterion with significant differences.

2. Results

2.1 tissue analysis of fresh and lyophilized Waton's gel preparations

The freshly frozen Waton's gel product (FIG. 1A) from the umbilical cord was paraffin-embedded with the freeze-dried Waton's gel product (FIG. 1B) and serially sectioned. The cell and tissue structures of the Waton's gel product were observed by H & E staining. The results showed that there were a large number of nuclei in the freshly frozen Waton's gel preparation, and the structure was also dense and well-aligned (FIG. 1A 1). The Watton's gel product after freeze-drying treatment had not been observed to have any nuclei, the structure had been changed from dense to loose, and large and small pores appeared (FIG. 1B 1). The main components of the Watton's gel product are collagen and glycoprotein. The distribution of collagen and glycoprotein was observed by staining with Sirius red (Sirius red) and PAS, respectively. The results show that the collagen and glycoprotein present in the Waton's gel are not destroyed by freeze-drying, and still serve as a structural scaffold for supporting the Waton's gel (FIGS. 1A 2-A3, B2-B3). The lyophilized Waton's gel product was visualized by scanning electron microscopy (FIG. 1C). Scanning images showed that the freeze-dried Waton's gel product exhibited a high density of pores in cross-section (FIG. 1D). And randomly selecting a plurality of visual fields, selecting holes in a frame, quantifying the sizes of the holes, and calculating the distribution of the average diameters of the holes. Analysis shows that the Watton's gel after freeze drying treatment presents porous structures with different sizes no matter the Watton's gel is subjected to paraffin embedding and slicing, and then is subjected to HE staining quantification or scanning electron microscope image analysis; furthermore, the pore diameter distribution largely falls within the range of 10 to 70 μm (FIG. 1E).

2.2 increasing expression of intracellular bone differentiation Gene in osteoblasts co-cultured with Walton's gel preparation

Separately cultured rat osteoblasts in vitro or cultured together with freeze-dried Waton's gel product or fresh frozen Waton's gel product for 14 days, trypsin-treated cells were removed, and RNA was extracted. RT-PCR was performed using rat Runt-related translation factor 2 (RUNX 2 for short) to investigate the differentiation of rat osteoblast bone. The results show that the ROB group, the ROB/WJD group or the ROB/WJF group has the performance of detecting the white rat GAPDH; furthermore, rat RUNX2 was also expressed (fig. 2A). Further, the difference in gene expression level between the groups was analyzed by a real-time quantitative polymerase chain reaction apparatus. Quantitative results showed that the level of osteoblast expression RUNX2 in the ROB/WJD or ROB/WJF groups was significantly increased compared to the ROB group (FIG. 2B).

2.3 expression of alkaline phosphatase in rat osteoblasts co-cultured with Walton's gel preparation

The rat osteoblasts were cultured in vitro alone or together with freeze-dried Waton's gel or freshly frozen Waton's gel either indirectly or directly, treated with 10% FBS DMEM for 14 days, stained with BICP/NBT, and then measured for absorbance by OD595 to observe the amount of expression of alkaline phosphatase in the rat osteoblasts (FIGS. 3A and 3B). The results showed that the expression level of intracellular alkaline phosphatase was significantly increased in both the white rat osteoblasts and the freeze-dried waton's gel product or the freshly frozen waton's gel product when they were cultured together with each other indirectly or directly (p <0.05, fig. 3C).

2.4 white rat osteoblasts co-cultured with Walton's gel preparation show an increased calcium deposition

The white rat osteoblasts are cultured separately in vitro or together with freeze-dried Watton's gel products and fresh frozen Watton's gel products in vitro indirectly, bone differentiation culture solution is used for treating for 21 days, alizarin red S (alizarin red S) is used for dyeing, and the expression of the white rat osteoblast calcium deposition is marked in deep red. The progressive magnification from the low power image shows that the ROB group has clustered osteoblasts which are more sparse and has only a very small amount of calcium deposition in the visual field. Besides the clustered osteoblasts, the ROB/WJD group also found concentrically grown osteoblasts, and also showed a large amount of calcium deposition in the periphery. The ROB/WJF group can find mature bone cells with tubules, and clustered osteoblasts are surrounded by the bone cells. There was also a distribution of a large amount of calcium crystals in the visual field (fig. 4A). Then, the deep red labeled calcium crystals were redissolved, and the absorbance was measured by OD 540 to analyze the concentration of calcium ions. The results showed that the concentration of calcium ions in the ROB/WJD group, or ROB/WJF group, was significantly increased compared to the ROB group (p <0.05, fig. 4B). The alizarin red S (alizarin red S) staining result conjectures that under indirect co-culture with a freeze-dried Waton's gel product or a fresh frozen Waton's gel product, the differentiation of osteoblasts can be promoted, and the expression of calcium deposition is enhanced.

2.5 the new bone mass at the skull defect position is quantified by the computer tomography, and the Waton's gel product is shown to be given, thereby effectively improving the bone mass yield

And (3) observing the first, second, third, fourth and fifth months of the white rat after the skull defect operation by using micro-computed tomography (micro-CT), and observing the skull healing condition. The new bone of the Injury + Saline group grows inward only to a small extent along the periphery of the bone defect. The Injury + WJD group, or the Injury + WJF treated group, in addition to the ingrown new bone, also found sporadic new bone tissue in the defect area (fig. 5A). The area percentage of new bone mass within the bone defect range was quantified. The results show that 12.5 + -3.4% of the bone neogenesis occurs in the group of Injury + Saline in the first month after operation, the skull healing ascends very slowly with the increase of the time after operation, and only 18.4 + -1.4% of the skull recovery situation occurs in the fifth month after operation. In the treatment groups of Injury + WJD and Injury + WJF, the new bone percentage was 21.2 + -3.9% and 23.0 + -4.1% in the first month after the operation, respectively, which was significantly increased (p <0.05) compared with the treatment group of Injury + Saline, and the new bone percentage was maintained to 30.6 + -3.6% and 35.6 + -3.7% in the bone defect range (p <0.05, FIG. 5B) until the fifth month. By the macroscopic observation of microcomputer tomography, the white rat is found to be subjected to skull defect surgery without any treatment, and the skull healing capacity can only be maintained within the range of 12-18% within five months. Administration of lyophilized, or freshly frozen, Watton's gel preparation promotes new osteogenesis in white rats with skull defects.

2.6H & E staining showed that administration of the Walton gel preparation promoted bone regeneration in the skull defect of white rats

The bone appearance around the bone defect (fig. 6B) and within the bone defect (fig. 6C) was gradually enlarged from low magnification in a low-magnification panorama (fig. 6A) of the H & E staining results. The results show that a small amount of new bone and dense connective tissue exist around the defect of the Injury + Saline group; only connective tissue is present within the bone defect. In addition to the tendency of bone mass to increase at the edge of the bone defect, the treated groups of Injury + WJD and Injury + WJF also observed new bone mass, free and tending to mature, within the bone defect. And quantifying the area percentage of the new bone in the bone defect range in the fifth month after the operation according to the H & E staining result. The content of new bone substances in the defect range of the Injury + Saline group is only 18.8 +/-1.9%. In the Injury + WJD treatment group, the content of new bone substances in the bone defect range respectively reaches 27.1 +/-4.0 percent and 32.7 +/-5.0 percent, and is remarkably increased compared with the Injury + Saline treatment group (p is less than 0.05, and figure 6D). The administration of freeze-dried, or freshly frozen, Watton's gel preparations, as observed by H & E staining, promotes bone regeneration in white rats with skull defects.

2.7 labeling osteoblasts (osteoplast) with alkaline phasphatase staining, showing that administration of Wawden gel preparation increases the expression of activity of osteoblasts in skull defect of rats

The mature osteoblast expression regions around the bone defect (fig. 7B) and within the bone defect (fig. 7C) were gradually enlarged from low magnification in the low-magnification panorama of the ALP staining results (fig. 7A). The results show that a small number of mature osteoblasts exhibit ALP around and within the bone defect in the Injury + Saline group. Both the Injury + WJD and Injury + WJF treatment groups had mature osteoblasts attached to the new bone mass growing inward around the bone defect, and also could observe that the free new bone mass was surrounded by osteoblasts within the bone defect. Quantification of ALP in defect Range by ALP staining results⁺Percentage of area of (c). ALP of the group of Injury + Saline⁺The area is only about 3.7 + -2.1%. Treatment groups Injury + WJD and Injury + WJF, ALP⁺The areas reached 10.0. + -. 5.9% and 8.8. + -. 2.3%, respectively, and there was an increased tendency (p) compared with the Injury + Saline group<0.05, fig. 7D). The lyophilized, or freshly frozen Waton's gel preparation was administered to increase the activity of osteoblasts in skull-deficient white mice, as observed microscopically by ALP staining.

2.8 identification of new bone mass by immunohistostaining with anti-osteocalcin (anti-osteopecalin) antibody, showing that administration of Waton's gel preparation promotes bone neogenesis in the skull defect of rats

Osteocalcin (osteopalcin) is secreted from osteoblasts and released in bone tissue during osteogenesis to form uncalcified bone mass. From the low-power panoramagram (FIG. 8A) of the results of anti-osteocalcin immunohistostaining, the expression of the area of uncalcified bone containing osteopalcin was gradually enlarged from low power (FIG. 8B) around the bone defect to a range of bone defects (FIG. 8C)Amount of the compound (A). The results showed that no osteopecalin expression was observed around the bone defect in the Injury + Saline group; within the bone defect, only a small amount of osteopecalin is released into the tissue. In the treatment group consisting of Injury + WJD and Injury + WJF alone, osteocalcin was found not only in the vicinity of the bone defect or in the range of the bone defect, but also in the presence of a group of osteoblasts, which represented that the osteoblasts were undergoing osteogenesis. Quantification of OCN in bone Defect Range from OCN staining results⁺Percentage of area of (c). Injury + Saline group OCN⁺The area is only 2.4 +/-1.6%. While Injury + WJD, and Injury + WJF treatment groups alone, OCN⁺The area percentage reaches 14.8 +/-4.8 percent and 15.5 +/-1.5 percent respectively, and the area percentage is remarkably increased compared with the Injury + Saline group (p)<0.05, fig. 8D). The freeze-dried and fresh-frozen Waton's gel product treatment group was administered to the microscopic examination of anti-osteocalcin (anti-osteopalcin) tissue immunostaining to further promote osteoprogenitor expression of osteoprogenitor.

2.9 administration of Waton's gel product can significantly promote the development of new bone

Immature (immature) osteoblasts are identified by anticalcin (anti-osteopocalin) immunohistological staining, mature (mature) osteoblasts are identified by ALP histological staining, and mature bone is identified by H & E staining. The quantitative data of the three stains are summed up to evaluate the bone development of the white rat with the skull defect. The results show that the bone development in the Injury + Saline group is about 25%. The study group of Injury + WJD and Injury + WJF had bone development around 52% and 57%, respectively, with higher bone development (fig. 9).

3. Conclusion

The Waton's gel preparation was transplanted into a skull-deficient white rat. As a result, it was found that the bone regeneration was observed by the microcomputer tomography after the treatment of either the freshly frozen or freeze-dried Waton's gel products, and the bone development of the host was also promoted by the evidence of the image showing mature bone through the staining of the section. The experimental results show that the Waton's gel product can be used as a natural medical material for promoting bone regeneration in clinical medicine, and a novel treatment method is provided for patients suffering from bone defects.

It is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent and without further elaboration. Accordingly, the description and claims provided should be understood for illustrative purposes, and are not intended to limit the scope of the present disclosure in any way.

Claims

1. An umbilical cord Waton's gel product with the function of promoting bone regeneration, wherein the product is prepared by the following two methods: (1) treating the Waton's gel of umbilical cord with an antifreeze agent, freezing and storing, and thawing before use; or (2) directly freeze-drying the Wharton's gel of umbilical cord, and storing in dry condition before use.

2. The article of manufacture of claim 1, wherein the umbilical cord is from a human or non-human mammal.

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

4. The article of claim 1, wherein the cryopreservation is in liquid nitrogen.

5. The article of claim 1, 2, 3 or 4 wherein the cryopreserved Watton's gel contains cell bodies.

6. The article of claim 1 wherein the step of freeze drying comprises removing small pieces of fresh waton's gel, storing the fresh waton's gel directly at-80 ℃ for freeze storage, and then using a freeze dryer to remove moisture from the tissue.

7. The article of claim 6, wherein the Watton's gel is free of cell bodies.

8. The product according to claim 1, which is capable of increasing the expression of alkaline phosphatase in osteoblasts, increasing the expression level of a bone differentiation gene in osteoblasts, promoting differentiation of osteoblasts, enhancing the expression of calcium deposition, effectively increasing the yield of bone, regenerating bone at a bone defect, enhancing the expression of activity of osteoblasts at a bone defect, or promoting the development of new bone.

9. Use of a preparation according to claim 1 for the preparation of a medicament for promoting bone neogenesis.

10. The use of claim 9, wherein the preparation increases the expression of alkaline phosphatase in osteoblasts, increases the expression of differentiation genes in osteoblasts, promotes differentiation of osteoblasts, enhances the expression of calcium deposition, is effective in increasing bone mass production, neogenesis of bone mass in a bone defect, increases the expression of activity of osteoblasts in a bone defect, or promotes the development of new bone mass.