WO2008071074A1

WO2008071074A1 - The use of mesenchymal stem cells and the separating and preserving method of stem cells from human tissues

Info

Publication number: WO2008071074A1
Application number: PCT/CN2007/003505
Authority: WO
Inventors: Songling Wang; Songtao Shi; Yan Xu
Original assignee: Songling Wang; Songtao Shi; Yan Xu
Priority date: 2006-12-13
Filing date: 2007-12-10
Publication date: 2008-06-19

Abstract

A method of separating and preserving stem cells from human tissues is illustrated, and mesenchymal stem cells are especially used for functional restoration or regeneration of mammal such as human injured tissues or defected tissues and hairdressing.

Description

Application of mesenchymal stem cells and methods for isolating and preserving stem cells in human tissues.

Field of invention

The present invention relates to the use of mesenchymal stem cells, particularly for functional repair or regeneration of mammalian tissues such as human damaged tissue or defective tissue and beauty. The present invention also relates to a method of preserving stem cells, particularly a method of preserving dental pulp stem cells. Background technique

Mesenchymal stem cells (MSCs) can differentiate into a wide variety of cells, which makes it possible for stem cells to treat tissue regeneration.

Bone marrow mesenchymal stem cells are mesenchymal stem cells with multi-differentiation ability, which can differentiate into various cells such as osteoblasts, chondrocytes, adipocytes, cardiomyocytes, myoblasts and nerve cells. These cells were originally distinguished by a cell population of cells formed in vitro by fibroblast morphology (CFU-F population forming unit-matrix). Each group appears as a cell line in which one cell begins to proliferate. Although bone marrow mesenchymal stem cells have the ability to differentiate into a variety of cells, they usually tend to form bone tissue. For example, bone marrow mesenchymal stem cells can continue to form bone/medullary after being transplanted into the immunodeficient mouse. Organoid structure. Another surprising feature is that in xenogeneic transplantation, bone marrow mesenchymal stem cells activate and support the hematopoietic component during osteogenesis. This manifestation of mesenchymal stem cells with various differentiation potentials strives to establish the microenvironment from which they originate. Mesenchymal stem cells have also been successfully used in fractures, aplastic anemia, and in the treatment of human type IV acute graft-versus-host disease by modulating immune cell responses. In addition, bone marrow mesenchymal stem cells are also used to generate adipose tissue for filling and reconstitution of soft tissues.

Recently, a new mesenchymal stem cell has been isolated from the human periodontal ligament, the periodontal mesenchymal stem cells (PDLSCs). Periodontal disease is the leading cause of tooth loss in adults. Treatment of periodontal disease is a difficult problem in clinical work. Conventional regenerative treatments include guided tissue regeneration (GTR), topical application of enamel derivatives (EMD), or various growth factors that can cause tissue regeneration in the periodontal portion. These treatments have varying effects, mainly related to the missing shape and the amount of healthy normal periodontal ligament. So far, there is no treatment that can achieve functional regeneration of periodontal tissue.

Therefore, the application range of developing mesenchymal stem cells is still very necessary. Summary of the invention

The present inventors have found that mesenchymal stem cells such as bone marrow mesenchymal stem cells or periodontal (ligament) mesenchymal stem cells (PDLSCs) have shown unexpectedly good effects in functionally repairing human damaged tissues or defective tissues and cosmetics. For example, bone marrow mesenchymal stem cells repair facial damage or defects in mammals such as humans, periodontal mesenchyme repair, and the above repairs are not only tissue shape repair, but also recovery or repair of la-texture regeneration and normal function. . The present inventors have further found in the research that the special treatment of stem cells in human tissues can advantageously retain the activity of stem cells in human tissues, thereby providing a possibility for the repair of tissue damage in humans in the future. The present invention has been completed based on the above findings.

Accordingly, a first aspect of the invention relates to the use of mesenchymal stem cells for the preparation of a product for functional repair or regeneration of a mammalian, e.g., human, damaged tissue or defective tissue or cosmetic.

Another aspect of the invention relates to a method of functionally repairing or regenerating a mammalian, such as a human damaged tissue or defective tissue or cosmetic, comprising administering a functionally repairing effective amount of mesenchymal stem cells to a mammal in need of functional repair of damaged or defective tissue. Animals such as humans or humans who need beauty.

The invention also relates to a functional repair or regeneration mammalian such as a human injury group A composition of woven or defective tissue comprising mesenchymal stem cells and hydroxyapatite/calcium triphosphate or gelatin sponge.

The invention further relates to compositions for use in cosmetics comprising mesenchymal stem cells and hydroxyapatite/calcium triphosphate or gelatin sponge.

The invention also relates to a method for isolating and preserving stem cells in human tissues, which comprises chopping human tissue containing stem cells, and then digesting in an enzyme solution consisting of type I collagenase and neutral protease at 37 t; (PH6-9) 10-60 min; single cell suspension was filtered and the resulting single cells were washed with Ca ²⁺ and Mg ^{2+ -free} fluorescent buffer (PBS-) and then at 4. C is mixed with 90% fetal calf serum (FBS) and 10% dimercaptosulfoxide, placed at low temperature, and then refrigerated in liquid nitrogen. Terms and definitions

The term "functional repair or regeneration" means that a damaged tissue or a defective tissue of a mammal such as a human is treated by the mesenchymal stem cells of the present invention, and can not only return or restore or improve to a normal tissue shape but also have normal physiological functions of normal tissues. . If the damaged periodontal tissue is treated by the periodontal mesenchymal stem cells of the present invention, it can be regenerated into normal periodontal tissue, thereby regenerating normal teeth.

The term "mesenchymal stem cells" is exemplified by bone marrow mesenchymal stem cells and periodontal ligament (mesenchymal) stem cells (PDLSCs). The PDLSCs are preferably from humans under the age of 20, and the PDLSCs are freshly removed and frozen and reused. The same function, and there is no limit to the freezing time.

The term "injured or defective tissue" is, for example, a tissue damage or defect caused by disease, genetic or external force such as trauma.

The term "cosmetic" is used, for example, to modify or reshape the contours of a mammal, such as a human face, and to repair or regenerate the aging of a mammal, such as a human skin, such as the removal of facial wrinkles or freckles, and is generally understood by those skilled in the art. Beauty category.

The term "human tissue" generally refers to soft tissue, which generally refers to the removal of bone, teeth. All tissues of the quality and blood components have no hard tissue.

The term "low temperature" generally means below O, for example, -80 °C. DRAWINGS

Figure 1 is: Human bone marrow msc-mediated reconstruction of the mouse face

(A) MSCs grafts with HA/TCP as a carrier can cause significant changes in the appearance of the mouse's face.

(B) Rats without MSC grafts were transplanted as a control group.

(C) H&E staining showed a large amount of active bone tissue (B) and hematopoietic bone marrow (BM) produced by bone marrow MSC grafts. The interface between the regenerated bone and the skull (CB) is complete, as indicated by the yellow dashed line. The black arrow shows the height of the regenerated bone.

(D) In contrast, in the control group, grafts transplanted with HA/TCP particles showed only a limited number of bone gains.

Figure 2 is a description of the MSC regenerated bone in the maxillofacial region

(A, B). After 8 weeks of transplantation, the newly formed bone (^ is combined with HA/TCP particles and combined with the skull (ί3⁄4Α) by the connective tissue (67).

(CF). Immunohistochemical analysis with anti-ALP (C), type III collagen (CIII, D), MEPE (E) antibody revealed that osteogenesis cells (black arrows) or osteoblasts resemble newly formed bone surfaces, Or similar to bone cells in newly formed bone. Immunohistochemistry IgG staining was negative (F)

(G). At 8 weeks post-transplantation, MSC grafts contain well-differentiated bone and bone marrow components, showing typical hematopoietic niche structures, including osteoblasts (empty arrows) and hematopoietic cells (BM). H&E staining

(H). In situ hybridization with the human specific probe alu. showed that the cells produced by the regenerated bone (black arrow) were positive and the bone marrow components were negative.

Figure 3: GFP-positive bone marrow cells homing into the bone marrow component of MSC (A, B) GFP bone marrow was injected into the MSC/HA transplant recipient mice via the tail vein. Immunohistochemical analysis with anti-GFP monoclonal antibody after 2 months of injection showed GFP-positive bone marrow (ΒΑΠ homing into the bone marrow of MSC graft ( )

Immunohistochemistry with IgG showed negative results (B)

(C). Cell flow analysis of the heaviest cells of MSC grafts or untransplanted long bones, GFP-positive cells and hematopoietic cell markers (upper right square) CD45, CD9, CDllb co-expressed in bone marrow from MSC grafts . (lower layer). Bone marrow cells (upper layer) taken from the long bones of the transplanted rat were used as a negative control.

Figure 4: Autologous bone marrow MSCs transplantation can change the contours of the pig's face

(A, B) Facial appearance. Transplantation of bone marrow MSCs-HA/TCP can significantly alter the appearance of pigs after a week (empty arrow) (A).

In the control group, the appearance of pigs without MSC grafts did not change (B)

(C, D) 3D reconstruction showing the skeletal appearance of MSC/HA grafts (empty arrow) (C) and pigs without MSC transplantation (D)

(E, F) Coronal CT plain scan image. The MSC graft (white arrow) is tightly bound to the skull (yellow arrow) (E). There was no bone connection between the graft and the skull in the control group (F).

(G, H) X-ray film. The MSC graft (white arrow) is tightly connected to the skull (yellow triangle) (G). The HA/TCP (empty arrow) and the defect between the skulls (black triangles) show no bone connection between the two (H).

Figure 5 is a description of pig MSC regenerated bone

(A-D) Electron microscopy analysis of MSC/HA and HA/TCP grafts. Numerous micropores (empty arrows) are present on the broken surface, including the inner (A) and outer (B) sides of the HA/TCP. After 8 weeks of MSC transplantation, these spaces became smaller compared to HA/TCP grafts, either inside (C) or outside (D).

(EH). At 8 weeks after transplantation, H&E staining showed that MSC grafts regenerated a large amount of bone tissue (E) at low magnification. In contrast, in the control group, the ligament (CT, mixed with HA/TCP particles (F) was shown. Under the high power microscope, the MSC shift was shown. The inner side (G) and the outer side (H) of the plant. Osteoblasts appear (empty arrows in H) on the regenerated bone surface ( ).

Figure 6 is a view of the reduction of wrinkles caused by human MSCs in rats.

(A, B) When PDLSCs loaded with gelatin sponge (collagen-based gelatin sponge) were implanted into mice for 4 weeks, H&E stained sections showed a large amount of collagen fibers (black arrows in A). The same piece of polarized light shows that these collagen fibers are compressed. (white arrow in B)

(C, D) In contrast, bone marrow MSC/gelatin sponge does not produce collagen fibers, either after 2 weeks of transplantation (C) or after 4 weeks (D). Although some cellular components appeared after two weeks of transplantation, they could not form collagen fibers (C). After 4 weeks of transplantation, only a limited number of cells appeared. (D)

(E-H) In situ hybridization was performed on MSC grafts using human alu. and mouse specific pfl. A large number of human alu. positive (black spots) cells appeared (P) after PDLSCs/gelatin sponges were transferred to plants, and pfl-positive (black spots) cells were detected only outside the graft (G). In situ hybridization with sense mRNA showed negative staining for both alu and pfl (H).

The (IL) single-column PDLSCs act similarly to the mixed colony PDLSCs, both producing compressed collagen fibers, as shown by H&E staining (1, black arrow in K) and polarized light in the same field, U, L White arrow in the middle).

(M, N) PDLSCS was transplanted into the corrugated area subcutaneously with a gelatin sponge as a carrier.

(m, n) Magnifies the area of the drawing in M and N. Compared with the control group (M, m), wrinkles were significantly reduced (Ν, η).

Figure 7 is:

(Α) The alveolar bone in the middle of the animal's maxilla or chin of the first molar was destroyed, and the root ligament was sutured with a 4th thread. The strap was removed after 10 days. The size of the defect is 3mm wide, 7mm long and 5mm deep

(B) 30 days after surgery, the inflammatory response is obvious (C) The gums were red and swollen 120 days after surgery, and large stones were visible.

(D) After 6 months, the infection spread to the surrounding area, and the roots were exposed.

(E) X-ray shows normal alveolar bone, clear cortex

(F) 6 months after surgery showed significant reduction in alveolar bone density

(G) CT three-dimensional reconstruction of the real defect area

(H) Coronal scan showing defect

Figure 8 is:

(A) Histology H&E staining showed that the periodontal ligament consisted of a large number of closely packed collagen

(B) PDLSCs can be extracted to produce similar collagen, but longer.

(C) The quality of collagen produced by PDLSCs was higher than that of dental papilla stem cells in the untreated control group (D) and in the transplanted HA/TCP group (E). After March, only part of the periodontal tissue was regenerated.

(E) In the PDLSCs-HA/TCP experimental group, the proliferating periodontal tissue basically covered the wound

Histology shows regeneration of new bone (C) and periodontal tissue (H), as well as newly formed cementum (F)

CT showed significant bone damage before transplantation (J, L). After three months of transplantation, bone tissue in PDLSCs-HA/TCP group was completely regenerated (K), and there was almost no regeneration in the HA/TCP group (M).

Figure 9 shows: Compared with the other two groups, the PDLSCs experimental group showed significant bone regeneration after 3 months of transplantation.

According to the present invention, the bone marrow mesenchymal stem cells or periodontal ligament stem cells of the present invention are usually used in combination with hydroxyapatite/calcium triphosphate (HA/TCP) or gelatin sponge.

Figure 10 is a view: The cryopreserved pulp mononuclear cells contain dental pulp stem cells (A, B). A single colony, cell colony forming unit (CFU-f) (A), is formed on the medium. In terms of the ability to form (CFU-f), compared with normal pulp mononuclear cells, there was a significant decrease in the number of pulp mononuclear cells after refrigeration (CE). Compared with the PDSC produced by the usual method, the doubling of dental pulp stem cells (DPSCcryo) produced by refrigerated pulp mononuclear cells was reduced (0, which was measured by bromodeoxyuridine binding method for 12-hour proliferation (D), STR0-1 And the expression level of MUC18, the early origin marker (E) of mesenchymal stem cells. However, its number doubling, value-added, and the expression of STR0-1 and MUC18 are still at an acceptable high level (C-E). immunocytochemical staining in frozen pulp produced by monocytes dental pulp stem cells (DPSCcryo) for STR0- 1, MUC18, CD105, and CD73 positive (F.), negative to CD34, I gM and ₁ ₈ 0 1 is Used as a negative control. Figure 11 shows: DPSCcryo has similar differentiation ability to PDSC. (A) Alkaline phosphatase (ALP) staining shows that both have the same level of ALP. (B) L-ascorbic acid-2- Magnesium phosphate, dexamethasone, and inorganic phosphate were stained with alizarin red for four weeks and showed the same level of mineral nodule formation. (C, D) DPSCcryo is similar to normal PDSC, using real-time PCR (C) Detection of bone viscous protein (ON) and bone The nuclease (0CN), measured by the Wes tern b lot method, has RUNX2, ALP, and OCN (D). G3PDH and - used as internal control. (E, F) when cultured containing 0.5 mM phosphodiesterase Inhibitor (IBMX), 0.5 mM hydrocortisone, 60 mM armor. After 5 weeks in a new fat-inducing environment, similar to normal PDSC, a lipid-positive lipid group (E) can be formed. The fat markers PPARg2 and lipoprotein lipase (LPL) (F) were achieved, Figure 12: DPSCcryo can form dentin in vivo. After 8 weeks of transplantation, DPSCcryo can differentiate into dentate cells (empty arrows), the cells It is the cause of the formation of a dentin-like structure on the surface of the hydroxyapatite carrier (HE staining) (A). DPSCcryo produces the same dentin structure (A) and a similar amount of dentin (B) as the DPSC. (0) In immunocytochemical staining, the dentin structures produced by DPSCcryo and DPSC are positive for human-specific mitochondria and DSP antibodies.

The following examples are intended to further illustrate or illustrate the invention, but are not intended to limit the invention in any way.

Experimental materials and experimental procedures

Human periodontal ligament stem cells and human bone marrow mesenchymal stem cells:

The third molars (n = 18) from 16 normal adults ^(18-20 years old). The periodontal ligament was carefully separated from the root surface of the tooth and then placed in 3 mg/ml type I collagenase.

(Worthington Biochemical Company, Freehold, NJ) and 4 mg/ml dispase (Roche Diagnost ic/Boehringer Mannheim Corp., Franklin Lakes, NJ) for digestion. A single cell suspension of PDLSCs ( 1 χ 10 ⁴ ) was seeded in a 10 cm tissue plate (Costar, Cambridge, MA) in a-MEM medium (GIBCO/Invitrogen, Carlsbad, CA) from 15% fetal bovine serum.

(Equitech-Bio Inc, errville, TX), lOOuM L -ascorbic acid-2-phosphate (WAK0, Tokyo, Japan), 2mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin ( Biosource/Invi trogen) provides nutrients and is then incubated with a 37° 5% C0 ₂ environment. Human bone marrow mesenchymal stem cells were purchased from ALLCells LLC, Berkeley, CA. To identify the purchased cells, a single cell suspension of 1 χ 10 ⁶ bone marrow mononuclear cells was seeded in a 15 cm tissue plate for 3 hours, and then the cells that were not attached were removed. Attached cells are placed in the PDLSC tissue environment. In order to further confirm mesenchymal stem cells, monoclonal individuals were examined with STR0-1, a specific stain for mesenchymal stem cells, and showed positive. All primary cells in this experiment were in stages 1-3. Experimental antibody

Rabbit anti-matrixextracellular phosphoglycoprotein (ΜΕΡΕ, LF-155 ), type III collagen (LF-70) and alkaline phosphatase (ALP, LF-47 ) antibody by Dr. Larry Fisher (NIDCR/NIH, Bethesda, MD) provide. Mouse monoclonal antibody ( mAb ) green fluorescent protein (GFP), R-jujube (R-PE)-conjugated murine monoclonal antibody against CD45, CD9, CDllb and IgM molecules, R-PE binding isotype-matched control antibody Becton Dickinson ( Franklin takes, NJ ). Anti-human special mitochondrial antibodies were purchased from Chemicon (Temecula, CA) and anti-STR0-1 antibodies were supplied by Dr. StanGronthos (Institue of Medical and Veterinary Science, Adelaide, Austral.). Negative control antibodies of the same rabbit and mouse type were purchased from Caltag Laboratories (Burlingame, CA). Transplant

About 8. Ο χ 10 ^δ In vitro cultured human bone marrow mesenchymal stem cells were mixed with 80 mg of HA/TCP ceramic particles and then planted in 10-week-old immunodeficient brown rats (NIH-bg-nu/nu-xid, Harlan Sprague Dawley , Indianapolis, IN), above the head. For PDLSC planting, 4 X 10 ⁶ PDLSCs were placed on a lOmm x 10 mm collagen-based gelatin sponge and placed as a carrier under the skin of a rat. These operations are in accordance with animal experiment specifications (NIDCR#04-317 and USC #10874). These plants were taken out 4-8 weeks after planting, soaked in 4% formalin, decalcified with 10% ethylenediaminetetraacetic acid buffer (pH 8.0), and embedded in paraffin. Dewaxed and stained with H&E. Immunohistochemistry

The removed MSC graft was dewaxed and then coated with primary antibody (1:200-1:500 dilution) for one hour. Immunohistochemistry experiments were performed according to the instructions using the Zymed SuperPicTure Polymer Assay Kit (Zymed nvitrogen). Pig model

Six piglets (4-8 weeks, weighing 20-40 kg) were provided by the Chinese Academy of Agricultural Sciences. Animals are placed in a comfortable environment. Piglets were anesthetized with 6 mg/kg chlorinated hexahydrate and 0.6 mg/kg acetonide. Bone marrow mesenchymal stem cells of piglets are taken out and cultured in the same manner as human mesenchymal stem cells. Animals were randomly divided into experimental and control groups. 10 X 10 ⁶ pig bone marrow mesenchymal stem cells were incubated for 90 minutes on a porous HA/TCP blocking platform (diameter 25 mm high 10 mm, containing 60% HA and 40 TCP; pore 200-300 um, manufactured by Sichuan University). Cut the pig's forehead subcutaneously and create a suitable channel under the periosteum. HA/TCP-MSCs were placed in the experimental group of 3 pig periosteum for 8 weeks, and HA/TCP was placed in the body of the control group. Electronic scanning mirror scanning

The MSC graft sample was cut into approximately 1 leg pieces and fixed with 2% polyfurfural and 2.5% glutaraldehyde for 2 hours. Rinse with sodium dicitrate and fix in 1% osmium tetroxide. Dehydrated with graded alcohol and then incubated in isoamyl acetate. After gold plating, the samples were tested under a Toshiba S-520 electron microscope. In situ hybridization

As described above, the digoxigenin-labeled human specific fragment alu and the mouse specific fragment pfl were used as probes for in situ hybridization. The starters include: human alu, sense, 5'-tggctcacgcctgtaatcc-3' (base number: 90-108), ant isense, 5'-TTTTTTGAGACGGAGTCTCGC-3' (BASE NUMBER: 344-364, Genbank accession number: AC004024) ; and mouse pfl, sense, 5'-CCGGGCAGTG GTGGCGCATGCCTTTAAATCCC-3' (base number: 170-201), ant isense, 5'-GTTTGGTTTTTGAGCAGGGTTCTCTGTGTAGC-3' (base number: 275-306, Genbank access ion number: X78319). Probe Prepared by polymerase chain reaction (PCR), including 1 PCR buffer (Perkin Elmer, Fos ter City, CA), 0.1 mM dATP, 0.1 mM dCTP, 0.1 mM dGTP, 0. 065 mM dTTP, 0. 035 mM digoxigenin-l l-dUTP, 10 pmol of a specific initiator, and 100 ng of human chromosomal DNA as a template. Unstained samples were deparaffinized and hybridized with the digoxigenin-labeled a lu or pf 1 probe by mRNA loca tor-Hyb Ki t (Ambion, Inc., Aus t in 。). The presence of alu or pf1 in the tissue can be detected by NBC/BCIP solution (RocheDiagnos tic/Boehringer Mannheim Corp) after an immunological reaction with an anti-digoxigenin ALP binding fragment (RocheDiagnos tic/Boehringer Mannheim Corp). Bone marrow cells homing

Rats transplanted with MSC graft for 8 weeks were intravenously injected with cyclophosphamide (Sigma) diluted in phosphate buffered saline (PBS) at 64 mg/kg once daily for 4 days. Then, the transgenic mouse long bone-expanded GFP (sGFP)-producing bone marrow cells (1.57 ⁷ all nuclear cells/body) were injected into the cyclophosphamide-injected mouse through the tail vein. Eight weeks after the injection, eGFP-positive homing bone marrow cells in the MSC graft can be detected by immunohistochemistry and fluorescence activated cell sorting. Fluorescence activated cell sorting (FACS) analysis

Cells in MSC grafts (1×10 ⁶ ) or long bone cells from transplanted nude mice were placed in PE-conjugated mAbs and incubated for 45 minutes at 4 degrees. PE-conjugated isotype-matched IgG was used as a negative control. After washing with PBS and 0.4% bovine serum albumin, these cells were used for FACS analysis. Example 1

Reconstruction of rat maxillofacial region with bone marrow mesenchymal stem cells

In order to evaluate the bone regeneration induced by bone marrow mesenchymal stem cells in plastic surgery The bone marrow MSCs proliferated in vitro were transplanted to the surface of the frontal bone of the rat by the HA/TCP vector, resulting in a considerable change in the appearance of the maxillofacial region (Figs. 1A and 1B). After 8 weeks of transplantation, bone marrow MSCs differentiated into osteoblasts to form new bone and corresponding hematopoietic components (Fig. 1C). The newly formed bone tissue is attached to the frontal bone to maintain the graft in an appropriate position (Fig. 1C). Moreover, the remodeling of the hematopoietic microenvironment in MSC grafts provides conditions for an important balance between bone marrow MSCs and hematopoietic stem cells, thereby ensuring the formation of this abnormal bone/myeloid organ structure. In contrast, HA/TCP grafts only resulted in a limited amount of induced bone formation between the HA/TCP particles and the calvarial contact surface (Fig. 1D). Further studies revealed that the newly formed bone was connected to the rat skull by connective tissue and directly bound to HA/TCP (Fig. 2B). This also confirmed that osteoblasts in MSC secrete osteogenic factors, including ALP, type III collagen, and MEPE (Fig. 2C-2F). MSCs play a reliable role in the osteogenesis of the maxillofacial region.

This work further reveals that regeneration of bone-like hematopoietic components (Fig. 2G) regenerative bone and associated hematopoietic components have great potential for application in maxillofacial plastic surgery. It was also observed in MSC transplantation that the hematopoietic cord of the recipient aggregated to form a bone/myeloid organ-like structure, which included bone from the graft and a myeloid component derived from the recipient (Fig. 2H). Moreover, osteoblasts differentiated from MSCs interact with primitive hematopoietic cells to form a bone marrow niche microenvironment that is visible in long bones (Fig. 2G). To further demonstrate the difference between MSC-mediated tissue regeneration and other transplantations, immunohistochemical staining revealed that GFP-positive bone marrow cells homing to the bone marrow of MSC grafts two months after tail vein injection of bone marrow from GFP mice. Area 9 Figures 3A and 3B). FACS analysis was used to distinguish cell types that were homing into the medullary region of MSC grafts (Fig. 30. Two months after injection of GFP bone marrow, including CD45, CD9, CD1 lb-positive hematopoietic cells in MSC-mediated niche Long-term homing in the microenvironment, which indicates the regeneration of a functional bone/myeloid organ-like tissue in the maxillofacial region. Bone marrow MSC mediates reconstruction of porcine maxillofacial region

Piglets were used as a transplant model, and homologous bone marrow MSCs were placed on HA/TCP for transplantation into the maxillofacial region. Similar to experiments in rats, bone marrow MSCs were found to alter the appearance of the maxillofacial region (Fig. 4A, 4B). Imaging studies revealed a osseointegration of the MSC graft with the frontal bone of the recipient (Fig. 4C-4H), which provides evidence for the application of this technique to orthopedics. Animals transplanted with autologous bone marrow MSCs showed massive bone regeneration compared to the single-transplant HA/TCP animal model (Fig. 5). According to electron microscopy and histology analysis, pig autologous MSCs caused high-quality bone tissue regeneration on MSC grafts of different surfaces (Fig. 5). Example 2

Human PDLSC-mediated collagen formation for reducing wrinkles in mice

The PDLSCs were transplanted into the skin of the rats to reduce facial wrinkles (Fig. 6). When HA/TCP grafts carrying PDLSCs are placed under the skin, they form collagen fibers and cementum in the body. It was also found in the study that PDLSCs were implanted into a collagen-based gelatin sponge and transplanted into the body to produce a large amount of collagen fibers (Fig. 6A, 6B). Using this feature, the transplanted rat wrinkles are reduced and the appearance is improved (Fig. 6M, 6m, 6N, 6n). Prior to this, collagen gels and stabilized hyaluronic acid have been used for non-surgical cosmetic purposes, and fibroblasts of the dermis are used as a filling for soft tissue. Surprisingly, it was found that grafts of bone marrow MSCs or gingival fibroblasts on the carrier gelatin sponge did not mediate any tissue formation after 8 weeks (Fig. 6, gingival fibroblast data not shown). In contrast, PDLSCs/gelatin sponge grafts can survive for long periods of time with the regeneration of long-acting collagen fibers (Fig. 6A, 6B). Measurement of human specific alu and murine specific pf l by in situ hybridization can be used as evidence for collagen formation in human PDLSCs (Fig. 6E-6H). This test shows that PDLSCs have a unique ability to produce collagen fibers, which is also available in in vivo tests. In order to further evaluate its collagen fiber regeneration ability, a single colony group will be The PDLSCs were transplanted into the mouse subcutaneously using the collagen platform as a vector (Fig. 61-6L). Surprisingly, all single-column PDLSCs showed collagen fiber regenerative capacity, suggesting that collagen regeneration may be a feature common to all PDLSCs. discuss

Human mesenchymal stem cells are thought to be primitive cells with multiple directional differentiation capabilities that can differentiate into osteoblasts, chondrocytes, fat cells, muscles and nerves (3, 9, 20, 30-32). MSCs have now been used to treat severe bone damage that cannot be healed naturally (3, 20, 30, 32-34). Histological analysis showed that MSCs-mediated bone regeneration was similar in the maxillofacial region to the subcutaneous site previously studied (18, 20). Studies on ectopic tissue regeneration from previous MSCs have shown that it has the ability to be used in the reconstruction of maxillofacial bones, accompanied by maintenance of HSC niches. These results show that MSCs will become an important material for maxillofacial tissue reconstruction.

Long-lasting tissue regeneration requires a niche-like hematopoietic microenvironment to maintain the long-term survival of cells, normal function, and the disappearance of tissue-forming tissues. Previous studies have shown that osteoblasts differentiated from MSCs innervate HSC niches via the BMP receptor, PTH, and Tie2/angiopoiet in-l signaling pathways. The evidence here demonstrates that bone marrow MSCs can reconstitute an ectopic HSC niche environment. MSC-mediated bone formation generates bone/myeloid organ-like structures by directing the hematopoietic components of the receptor. In this ectopic organ, bone marrow hematopoietic cells interact with bone-derived cells and play their role in experimental rats. Features up to 18 months. While man-made materials such as heterogeneous implants can also alter the appearance of the face, MSC grafts have the ability to regenerate functional organ-like structures that work in conjunction with the recipient body. The advantage of this tissue regeneration is that it provides a long-term tissue re-establishment by developing a balance between the hematopoietic microenvironment between the graft and the recipient host. Although bone formation was also found in the control group in which HA/TCP particles were transplanted, the number of bone formation was small, and there was no production of hematopoietic components in the control group. These structures all indicate that MSC-mediated osteogenesis is different from bone hyperplasia. Transplanted PDLSCs produce a large amount of collagen fibers that are similar in structure to the original PDL. In contrast, transplanted MSCs and gingival fibroblasts do not produce any tissue, probably because they do not produce the ideal niche to maintain viability. Different carriers have a great influence on the production of collagen fibers. For example, gelatin sponges allow PDLSCs to produce collagen fibers in the body without producing inorganic-containing tissue components, while HA/TCP-PDLSCs grafts produce both collagen fibers and cementum. The mechanism by which different vectors cause PDLSCs to produce different products is still not well understood, and may be due to the ineffective difference in biocompatibility of the vector or the ability to induce differentiation of the MSC. This feature provides a unique opportunity for PDLSCs to improve facial appearance.

In summary, Example 1 demonstrates that MSCs can be used for integers to improve facial appearance. Experimental Example 2 shows that PDLSCs can be used for human beauty, such as wrinkling of the face. Example 3 Method for repairing and regenerating periodontal tissue damage caused by periodontal disease by PDLSCs

Animal

Twelve natural mini-small pigs, large in December, weigh 30-40 kg from China Agricultural University. Animals are fed under normal conditions. All procedures were performed after anesthesia was performed on minipigs with 6 mg/kg chlorinated ketamine and 0.6 mg/kg lanthiophene. Twelve mini-small pigs were made into periodontal defect models, 5 of which were transplanted with PDLSCs, 3 were transplanted only, and the rest were used as negative control.

PDLSCs culture

The canine teeth of the mini-pigs were removed, and the ligaments around the middle third of the roots were separated, and the ligaments were placed in the solution for 37 hours. The digestive juices included 3 mg/ml type I zymogen (Wor thing ton biochemical company) , Freeho ld, NJ ) and 4mg/ml dis pa se (Roche, Mannhe im, Germany), after 70um filter (Fa l con, BD Labware, Frankl in Lakes, NJ, USA) . Filtration to obtain a single cell suspension. PDLSCs from different individuals were cultured separately. To identify stem cells, a single cell suspension was placed in a 10 cm tissue plate (Cos tar, Cambr idge, MA) planted in a-MEM medium (GIBCO/Invitrogen, Carlsbad, CA) from 15% fetal Bovine serum (Equi tech-Bio Inc, Kerrvi l le, TX) l OOuM L-ascorbic acid-2-phosphate (WAK0, Tokyo, Japan), 2 mM L-glutamine, 100 units/ml penicillin and 100 ug/ml chain The mycin (Biosource/Invi trogen) provides nutrients and is then incubated with a 37 ° C 5% CO 2 environment. The efficiency of colony formation was evaluated on day 14. A colony of more than 50 cells was identified as a colony. Stage 3 PDLSCs were placed in the induction medium (0.5 mM 3-isobutyl-1-mercaptopurine (IBMX), 0.5 M hydrocortisone and 60 M armor). Oil red 0 after 4 weeks ( Sigma) is used to detect fat cells. Immunohistochemical staining

PDLSCs were placed in 24-well cell culture slides (2 X 10 ⁴ cells/well, NUNC, Napervi le, IL, USA) for secondary culture. The cells were first fixed in 4% paraformaldehyde for 15 minutes and then blocked and coated with anti-Stro-1 (R&D, 1:200-1:500 dilution) for one hour, according to the manufacturer's instructions. Then put any IgM secondary antibody in the sheep for 45 minutes. Production of animal models of periodontitis

For all 12 animals, the alveolar bone in the middle of the maxillary or mandibular first molar was destroyed, and the i-ligament of the root was sutured with a 4th thread. The strap was removed after 10 days. The size of the defect is 3mm wide, 7 inches long and 5mm deep. A bone valve appears during the procedure and the alveolar bone is removed. A concave cover is placed between the crown of the alveolar bone and the lowermost portion of the damaged portion. The animals after the surgery are trained for 30 days to maintain the periodontal condition and then the next operation can be performed. PDLSCs transplantation

One month after the periodontitis model was manufactured, approximately 2. 0 X 10 ⁷ in vitro cultured phase 3 PDLSCs mixed into hydroxyapatite/calcium triphosphate (HA/TCP) ceramic pellets (Wuhan University of Science and Technology Biomaterials and Engineering Center) . Then, it was transplanted into the subcutaneous skin of the damaged periodontal and placed in a gel film (Nanjing Jinling Medical College) to cover the area. The test animals received antibiotics for 3 days. At the same time, another group of 3 animals was implanted only in HA/TCP, and another group of 3 was used as a control without any operation. Clinical evaluation

Plaque Index (PLI) (Qui gl ey and He in, 1962), sulcus bleeding index (SBI) (Mazza et al., 1981), periodontal probing depth (PD), clinical attachment loss (CAL) All test teeth before and after surgery were evaluated as clinical evaluation indicators. The PD was measured with a periodontal probe (Hu-Fr iedy, Le imen, Germany) and the results were accurate to the millimeter. Histological and imaging evaluation

The histological evaluation of the defect tissue was performed at different time points after operation. The sample was first fixed in 4% formalin, and then a portion of the sample was placed in 10% edetate buffer to remove lime and finally coated with paraffin. The sample was dewaxed and stained with H&E. Another part of the sample was embedded in plastic without lime treatment (Dona th and Breuner, 1982). The unstained portion of the sample was first detected by fluorescence microscopy, and then the sample was stained with indoline blue and observed under a light microscope. Bone mineral density and regeneration at different time periods were analyzed by X-ray scanning and CT (S i emens Company, Germany). Scanning thickness is 0. 75mm. Statistical analysis The mean of the clinical data was statistically tested. The clinical data of the three groups were compared with each other and the pre- and post-operative data were compared by the F test. The p-value was less than 0.05. result

(Starting from Figure 7) Histological sections of H&E staining showed that the periodontal ligament tissue was composed of a large number of parallel-arranged collagen fibers (Fig. 8A). Under light microscopy, the PDSLCs of miniature pigs are in the shape of typical fibroblasts. The ratio of fibroblasts to colonies was 23/105, and about 5.6% of periodontal ligament cells were positive for antibody Stro-1 by flow cytometry. Through the observation of the scanning electron microscope, the cultured phase 3 PDLSCs grew actively, and the intracellular components such as mitochondria, rough endoplasmic reticulum and extracellular substances were abundant. At the same time, by contrast, the collagen produced by PDLSCs is thicker and longer than that produced by dental papilla stem cells (Fig. 8B, 8C). Moreover, in vitro, cultured minipigs PDLSCs have the ability to differentiate into adipocytes.

To determine the stability of the periodontitis model we created, we examined these models with X-ray and C T at 6 months postoperatively. The results showed that inflammatory periodontal tissue was clearly observed at 30 days postoperatively (Fig. 7B), and the P D examination was approximately 10 mm (Table 1). One hundred and twenty days after surgery, the gums were still red and swollen, and large stones were produced. (Fig. 7C). However, the PD examination returned to 5mm and the CAL was checked for 2 legs (Table 1), indicating a partial restoration of the periodontal. After 6 months, the examination revealed that the inflammation developed further, and the root of the first molar was exposed (Fig. 7D). Preoperative imaging showed normal alveolar bone density and clear cortical (Fig. 7E). The alveolar bone density at the affected site was significantly reduced 6 months after surgery. (Fig. 7F). Significant bone damage was observed in both the C T 3D reconstruction and the crown scan (Fig. 7G, H). This shows that surgical removal of part of the alveolar bone while ligating the surrounding portion can produce a stable periodontitis model.

Three months after treatment, the results of inspection PD PDLSCs- HA / TCP group was (3.0 ± 0.5) mm, HA / TCP is a group ⁽⁴ .0 ± 0.8) mm, as a non-treatment group ⁽⁴ · 8 ± 0 · ⁷ ) mm, CAL The results of the check are (0. 2±0. 5) mm, (1. 2+0. 9) mm, (0. 7+0. 4) mm _0. The results are analyzed, only PDLSCs-HA/TCP The group was significantly improved (Table 2). The newly formed alveolar bone height was (3. 65 ± 0.75) mm in the PDLSCs-HA/TCP group and (1.75 ± 0.43) mm in the HA/TCP group, and the untreated control group was ( 0. 5 ±0. 38) mm (Table 2, Figure 8C, 8D, 8E). H&E staining sections showed regeneration of bone and periodontal ligaments in the PDLSCs-HA/TCP group, but not in the other two groups. They were mainly repaired by epithelial cell fusion. CT scans showed that the alveolar bone had substantially recovered to normal height in the PDLSCs-HA/TCP group (Fig. 8J, 8K), while still very low in the other two groups (Fig. 8L, 8M). discuss

Many studies have shown that transplanting cells to the periodontal disease area can regenerate the cementum, periodontal ligament, and alveolar bone. However, the ability to regenerate from different cells is very different. It is not only related to the transplant site, but also related to the active differentiation of the transplanted cells. Previous studies have shown that early transplantation of mesenchymal cells with diseased periodontal can significantly reduce epithelial to root hyperplasia and promote stable structural formation. Although the treatment is different, the goal of periodontal disease treatment is to activate the remaining periodontal stem cells to produce new cementum and bone components. However, in periodontal disease, various inflammatory factors such as IL-1, TNF-a, and MMP destroy periodontal support tissues and alter the function of residual periodontal stem cells. The number of stem cells that can differentiate into bone and cementum surface is reduced. Therefore, direct transfer of stem cells into periodontal tissue should be a good treatment. Previous experiments have shown that stem cells, either from fresh or frozen periodontal ligaments, have the ability to differentiate into cementum/osteoblasts in vitro and form cementum/periodontal ligament-like tissue in vivo. In this study, we successfully isolated PDLSCs from minipigs and demonstrated that these cultured cells have the same characteristics as human cells. In 170 culture from single cells ¹⁰⁵ out of the low density population of single colonies, they behave as mesenchymal stem cell markers STR0-1 positive (5.6% positive). These ones PDLSCs can also produce collagen and differentiate into adipocytes in vitro.

When these PDLSCs are placed back into the periodontal, they exhibit a good ability to form bone, cementum, and periodontal ligaments. In the other two groups, the main manifestations were healing by epithelial cell hyperplasia, which was produced by only a few new substances (P < 0.05). At the same time, in the only transplanted connection organization, the CAL becomes deeper. Therefore, only transplantation of HA/TCP does not activate periodontal stem cells. Instead it prevents the proliferation of apical epithelial cells from delaying healing. When PDLSCs are mixed with HA/TCP, HA/TCP provides PDLSCs with sufficient value-added copy space for PDLSCs, allowing it to quickly cover damaged alveolar bone and roots. HA/TCP is very useful because it allows PDLSCs to quickly cover damaged areas while preventing epithelial coverage.

The maxillofacial region of miniature pigs has many similarities in terms of anatomy, physiology, disease, and disease. Gingivitis is a common disease in small pigs after 6 months. More severe periodontitis can occur in pigs older than 16 months. There are various methods for making periodontal disease models in pigs, such as ligation, bacteria, bone damage, and elastic metal shaping. Our animal model was made with silk thread for 10 days of ligation while bone damage occurred. Observing this method within 6 months after surgery, this method can significantly increase PD and CAL. Within one month after surgery, PD and CAL increased significantly, and repair began one month later, but did not return to normal levels.

This experiment demonstrates that PDLSCs can be successfully isolated from small pigs and have similar functions to human PDLSCs.

Table 1

Clinical examination of periodontal disease animal model

Days after surgery PLI SBI PD (mm) CAL (mm)

30 4 4 10 5

60 3 3 5 2

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Example 3

Isolation and preservation of dental pulp stem cells

Materials and Methods Isolation and culture of dental pulp stem cells (PDSCs)

The specimen was a normal adult (18-27 years old) third molar (n=16) collected from the oral clinic of the University of Southern California (USC). The method of tooth extraction was performed according to the University of Southern California guidelines. The method of separating dental pulp stem cells as previously described (Gronthos et al., 2000) _{0 is} mainly to separate the molars along the cementum enamel junction with a sterile forceps. The pulp tissue is separated from the crown and the root cavity. The sample was minced and digested in a 37 ° C enzyme solution for 30 minutes. The enzyme solution consisted of 3 mg/mL type I collagenase (Worthington Biothech, Freehold, NJ) and 4 mg/mL neutral protein drunk (Roche Diagnotic/Boer inger Mannheim Corp., Indianapol is, IN). Single cell suspensions were obtained by filtration through a 70-μιη filter (Falcon, BD Bioscience, Franklin Lakes, NJ). The resulting monocytes are generally used for pre-culture (Gronthos et al., 2000) and the other half is refrigerated. Briefly, mononuclear cells were washed 3 times with Ca ²⁺ and Mg ²⁺ -free fluorescent buffer (PBS-) and applied at 4 ° C with 90% fetal calf serum (FBS) (Equitech- Bio Inc., Kerrville, TX) mixed with 10% dimethyl sulfoxide (Sigma, St Louis, MO) (operating on ice). The cells were stored at -80 ° C overnight and refrigerated with liquid nitrogen. After storage for 6 months in cold storage, it was at 37. It melts rapidly in the C state and is used for the culture of DPSC. Monocytes were seeded in 100 mm cultures (Coaster, Cambridge, MA) as ΙΟχ ^{3 and} unattached cells were removed 3 hours later. The attached cells were supplied with nutrients from α-ΜΕΜ (Invitorgen Co. Grand Island, NY), including 15% FBS, 100-mM magnesium monophosphate (Wako Purre Chemicals, Osaka Japan), 2 mM glutamine, 100 U/ Ml penicillin I 100 g/ml streptomycin (Biosource, Rockville, MD). The resulting cell colonies are the first stage of PDSCs.

Claims

Rights request

1. Use of mesenchymal stem cells in the preparation of a product for functional repair or regeneration of a mammal, such as a human damaged tissue or defective tissue or cosmetic.

2. A method of functionally repairing or regenerating a mammalian, such as a human damaged tissue or a defective tissue or cosmetic, comprising administering a functionally effective amount of mesenchymal stem cells to a mammal, such as a human or a human, in need of functional repair of damaged or defective tissue. Humans who need beauty.

3. A composition for functionally repairing or regenerating a mammalian, such as a human damaged tissue or a defective tissue, comprising mesenchymal stem cells and hydroxyapatite/calcium triphosphate or gelatin sponge.

4. A composition for cosmetic use comprising mesenchymal stem cells and hydroxyapatite/calcium triphosphate or gelatin sponge.

The use according to claim 1, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells or periodontal ligament (mesenchymal) stem cells.

6. The method of claim 2, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells or periodontal ligament (mesenchymal) stem cells.

The composition according to claim 3 or 4, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells or periodontal ligament (mesenchymal) stem cells.

8. A method for isolating and preserving stem cells in human tissues, comprising: chopping human tissue containing stem cells, and then in an enzyme solution consisting of type I collagenase and neutral protease at 37 ° C (pH 6-9 ) Digestion for 10-60 minutes; single cell suspension was filtered, and the resulting single cells were washed with Ca ²⁺ and Mg ^{2+ -free} fluorescent buffer (PBS-), followed by 90% fetal calf serum (FBS) at 4 °C. Mix with 10% dimercaptosulfoxide, place it at low temperature, and then freeze it in liquid nitrogen.

9. The method of claim 8 wherein said body tissue is human soft tissue.

10. The stem cells of the method of claim 8 or 9 for repairing tissue damage in humans.