阅读说明：本技术 骨移植物成骨潜能的增强 (Enhancement of osteogenic potential of bone grafts ) 是由 G·达姆德赫瑞 J·赫尔姆斯 于 2014-07-16 设计创作，主要内容包括：本发明涉及通过用Wnt多肽如脂质体Wnt多肽的活体外处理增强骨移植物的细胞存活和成骨潜能。特别地,本发明涉及骨移植物用人Wnt3a蛋白,优选脂质体人Wnt3a(LWnt3a)进行的活体外处理。(The present invention relates to enhancing cell survival and osteogenic potential of bone grafts by ex vivo treatment with Wnt polypeptides, such as liposomal Wnt polypeptides. In particular, the invention relates to the ex vivo treatment of bone grafts with human Wnt3a protein, preferably liposomal human Wnt3a (LWnt3 a).)

1. A method of repairing a bone comprising:

(a) obtaining bone graft material from a subject in need thereof;

(b) contacting the bone graft material ex vivo with liposomes comprising a Wnt protein to produce a modified human bone graft material; and

(c) administering the modified bone graft material to a bone of the subject, wherein the subject receives spinal fusion.

2. The method of claim 1, wherein the Wnt protein is Wnt3a protein.

3. The method of claim 1, wherein the bone graft material comprises bone marrow stem cells or bone marrow progenitor cells.

4. The method of claim 1, wherein the bone graft material is an autograft.

5. The method of claim 1, wherein the bone graft material is taken from a site other than the spinal fusion site.

6. The method of claim 1, wherein the bone graft material is obtained from an allogeneic donor.

7. The method of claim 1, wherein contacting comprises incubating the bone graft material with the liposomes comprising a Wnt protein.

8. The method of claim 7, wherein the incubation takes up to 1 hour.

9. The method of claim 7, wherein the incubation takes up to 36 hours.

10. The method of claim 1, wherein contacting is performed at room temperature.

11. The method of claim 1, wherein contacting is performed at about 37 ℃.

12. The method of claim 1, wherein the bone is associated with a bone disease.

13. The method of claim 1, wherein the bone is associated with a metabolic disease.

14. The method of claim 1, wherein the bone is associated with steatosis.

15. The method of claim 1, wherein the subject is a human.

Technical Field

The present invention relates to enhancing the osteogenic potential of bone grafts by ex vivo treatment with Wnt polypeptides, such as liposomal Wnt polypeptides. In particular, the invention relates to the ex vivo treatment of bone grafts with Wnt3a protein, preferably liposomal Wnt3a (L-Wnt3 a).

Background

Orthopaedic and dental implants are used in a variety of joint and tooth replacements and to promote bone repair in humans and animals, particularly hip and knee joints and tooth replacements. Although many individuals experience simple healing and functional recovery, there is also a high complication rate, estimated at 10-20% for total joint replacement. Failures at the implant-bone interface cause most of these failures and necessitate subsequent corrective surgery. In addition, implants used as anchoring devices for orthodontic tooth movement are estimated to have a failure rate of 40% and subsequent placement of additional implants is necessary due to failure at the implant-bone interface.

Orthopedic and dental implants are made of relatively inert materials ("allogenic)" materials), typically a combination of metal and ceramic or plastic. Previous approaches to improve the outcome of orthopedic implant surgery have focused primarily on physical changes in the implant surface designed to increase bone formation. These approaches include the use of implants with porous metal surfaces to promote bone ingrowth and the injection of implants with hydroxyapatite plasma (hydroxyapatite plasma). Approaches to using dental implants also include the use of morphologically enhanced titanium surfaces, wherein surface roughness is imparted by methods such as sandblasting, acid etching, or oxidation.

Attempts have also been made to promote osseointegration, in which the implant surface is greatly altered. For example, short peptides containing the arginine-glycine-aspartic acid (RGD) sequence are attached to the surface of the implant because the RGD sequence is utilized by cells to adhere to the extracellular matrix. Researchers have attempted to reestablish this cellular adhesion to modified implant surfaces, but this strategy resulted in only a slight increase in implant osseointegration and mechanical fixation. Alternatively, in an attempt to stimulate angiogenesis around the implant, the surface of them is coated with a coating comprising the angiogenic growth factor VEGF. Implants immersed in saline solutions have been marketed as a means of increasing the osteointegration of implants, but there is little or no data confirming the claimed effect.

Another strategy for stimulating osseointegration is nano-texturing the implant surface. The rationale for this strategy is that texturing increases surface area and thus prevents "slippage" of the implant relative to the cells in the environment surrounding the implant. However, nano-texturing produces no measurable benefit in clinical trials.

The use of protein-based approaches for stimulating osteointegration of implants has also been extensively studied. Recombinant Bone Morphogenetic Proteins (BMPs) induce robust bone formation in bone fractures and they have also been used in attempts to stimulate direct bone formation around implants. Although in vitro results are encouraging, in vivo data is still not convincing. Recombinant BMP inhibits osteogenic differentiation of cells in the bone marrow cavity and is therefore contraindicated for implant osteointegration. See Sykaras et al (2004) ClinOral Investig 8(4): 196-. The use of BMP is associated with an increased incidence of ectopic ossification and uncontrolled inflammation, and newer metadata analysis also suggests an increased risk of cancer.

Wnt proteins form a family of highly conserved secretory signaling molecules that bind to cell surface receptors encoded by Frizzled and low density lipoprotein receptor-related proteins (LRPs). The WNT gene family consists of structurally related genes that encode secreted signaling proteins. These proteins are involved in tumorigenesis and in several developmental processes, including the regulation of cell fate and pattern formation during embryogenesis. Once bound, the ligand initiates a cascade of intracellular events that ultimately leads to transcription of the target gene by the nuclear activity of the β -catenin and DNA binding protein TCF (Clevels H,2004Wntsignaling: lg-norrin the dogma. curr Biol 14: R436-R437; Nelson WJ, Nusse R2004Convergence of Wnt, beta-catenin, and cadherin pathways. science 303: 1483-1487; Gordon MD, Nusse 2006 sigWnt talking: Multi pathways, Multiple receivers, and Multiple transfer vectors. J. Bioi 281 m: 22429-Chem 22433).

Wnt is also involved in a wide range of cellular decisions associated with the process of osteogenesis. For example, Wnt regulates the expression levels of sox9 and runx2, which affect the commitment of mesenchymal progenitor cells to the fate of chondrogenic or osteogenic cells. Wnt affects the differentiation rate of osteoprogenitor cells. In adult animals, there is a great deal of evidence that Wnt signaling regulates bone mass. For example, gain-of-function mutations in human Wnt co-receptor LRP5 are associated with several high bone mass syndromes, including type I osteopetrosis and endosteal hyperostosis or autosomal dominant osteopetrosis. Loss of Wnt function mutations cause low bone mass diseases including osteoporosis-pseudoglioma. Increased production of the Wnt inhibitor Dkkl is associated with multiple myeloma, a disease with increased bone resorption as one of its distinguishing features. For further details, see s.min et al, Wnt protein bone regeneration. sci.trans.med.2, 29ra30 (2010); ZHao et al, control lingth in vivo activity of Wnt lipomes, Methods Enzymol 465:331-47 (2009); popelut et al, The acquisition of implantation of infection by Liposomal Wnt3a, Biomaterials 319173 e9181(2010) and Morrell NT, Leucht P, ZHao L, Kim J-B, ten BergeD et al (2008) Liposomal Packaging genes Wnt proteins with In Vivo Biologicalcalel Activity PLoS ONE 3(8): e 2930.

It has been demonstrated that combining Wnt proteins with lipid vesicles (liposomes) produces biologically active Wnt formulations (Morrell et al, 2008, supra; and Zhao et al, 2009, supra) (Minear et al, 2010, supra; Popelut et al, 2010, supra). The biological activity of soluble wing-free proteins (wing proteins) is described in van Leeuwen et al (1994) Nature 24: 368(6469): 3424. Biochemical characterization of Wnt-Frizzled interactions using soluble biologically active vertebrate Wnt proteins is described by Hsieh et al (1999) ProcNatl Acad Sci US a 96(7): 3546-51. Bradley et al (1995) Mol Cell Bio i 15(8):4616-22 describe soluble forms of Wnt proteins with mitogenic activity. The use of liposomal Wnt proteins to enhance osteointegration is described in U.S. patent publication No. 20120115788.

Disclosure of Invention

In one aspect, the invention relates to a method of enhancing cell survival in a bone graft comprising subjecting the bone graft to ex vivo treatment with a Wnt polypeptide. In another aspect, the invention relates to a method of enhancing the osteogenic potential of a bone graft comprising subjecting the bone graft to ex vivo treatment with an effective dose of a Wnt polypeptide (including, but not limited to, Wnt 3A). In a further aspect, the invention relates to a method of revitalizing (revitalizing) a bone graft from a subject with reduced healing potential, comprising subjecting the bone graft to in vitro treatment with a Wnt polypeptide. In all aspects, the bone graft may be an autograft or an allograft. In all aspects, the bone graft may comprise a stem cell population, such as, for example, a bone marrow-derived stem cell population, e.g., bone marrow-derived mesenchymal stem cells.

The bone graft is preferably from a human subject. In one embodiment, the human subject is an elderly patient. In certain embodiments, the human subject is at least 50 years old, at least 55 years old, at least 60 years old or at least 65 years old or at least 70 years old or at least 75 years old or at least 80 years old or at least 85 years old. In another embodiment, the human subject has reduced healing potential, e.g., is a smoker, diabetic, or characterized by a nutritional deficiency.

In all aspects and embodiments, the Wnt polypeptide is preferably Wnt3a, more preferably human Wnt3a, most preferably liposomal human Wnt3a (L-Wnt3 a). In a further aspect, the method of the invention further comprises the step of introducing the bone graft into a recipient, such as a human patient. In various embodiments, the bone graft may be used to support dental implants, repair bone fractures. In another embodiment, the bone graft is used to repair or reconstruct diseased bone. In yet another embodiment, the bone graft is used in the hip, knee, or spine of the recipient.

Drawings

1A-1J. bone grafts have osteogenic potential. FIG. 1-A quantification of total DNA in representative aliquots of whole bone marrow harvested from transgenic β actin-enhanced green fluorescent protein (β -actin-eGFP) male mice; each aliquot constitutes a bone graft. Fig. 1-b. bone graft implantation into a critical-size (critical-size) calvarial defect (bounded by circles) of 2-mm diameter, formed in the sagittal suture separating the parietal bones (outlined by vertical white dashed lines). The black dashed line represents the plane of the tissue section. FIG. 1-C. representative tissue sections from the injury site on day 1 post-transplantation; GFP immunostaining identifies transplanted cells from eGFP donors (n ═ 5); the inferior space represents the sagittal sinus. FIG. 1-D. representative tissue sections at day 5 post-implantation; immunostaining with bromodeoxyuridine (BrdU) identified cells in S phase. Figure 1-e. day 7 post-transplant, GFP immunostaining identifies bone grafts (yellow dotted line); the higher magnification image of the boxed area in fig. 1-E (fig. 1-F) shows that most of the cells at the site of injury are derived from GFP-positive grafts. Figure 1-g. day 14 post-implantation, micro-CT reconstruction confirmed that 2-mm calvaria lesions constituted critical-sized non-healing defects (n ═ 6) 40. Fig. 1-h. healing of the same size calvaria injury treated with bone graft (n ═ 6). FIGS. 1-I and 1-J. 7 days post-implantation, aniline blue staining was used to identify new bone-like matrix; no bone-like matrix was formed in the untreated injury site (yellow dotted line). Fig. 1-J show visible bone-like matrix at day 7 post-implantation in representative samples treated with bone graft. Abbreviations: IHC ═ immunohistochemistry. The arrows mark the edges of the intact bone. A scale: 2mm (FIG. 1-B); 200 μm (FIGS. 1-C and 1-D); 100 μm (FIG. 1-E); 40 μm (FIG. 1-F); 2mm (FIG. 1-G) and 200 μm (FIGS. 1-I and 1-J).

Figures 2A-2i. reduction of osteogenic potential in bone grafts from aged animals. On day 7 post-transplantation (d7), aniline blue staining indicated bone-like matrix produced by bone grafts from young donors (fig. 2-a) and elderly donors (fig. 2-B). Fig. 2-c histomorphometric analysis of new bone mass formed from young and old bone grafts. Figure 2-d. at day 7 post-transplant (d7), Green Fluorescent Protein (GFP) immunostaining identified cells derived from bone graft at the time of donor younger, compared to older donors (figure 2-E). Figure 2-f GFP-positivity (GFP) in the injury site when grafts were harvested from young donors (blue bars, n ═ 13) compared to older donors (white bars, n ═ 13)^+ve) The number of cells. On day 5 post-transplantation (d5), bromodeoxyuridine (BrdU) staining identified proliferating cells in bone grafts from young donors (fig. 2-G) and elderly donors (fig. 2-H). Figure 2-i quantitative reverse transcription polymerase chain reaction (qRTPCR) of Proliferating Cell Nuclear Antigen (PCNA) in bone grafts from young and old animals is equivalent. Single asterisk denotes p<0.05. Is marked by an arrowThe edges of the whole skeleton. A scale: 200 μm (FIG. 2-A. the scale in FIG. 2-A also applies to FIG. 2-B]2-D [ the scale in FIG. 2-D also applies to FIG. 2-E]And 2-G [ the scale in FIGS. 2-G also applies to FIGS. 2-H])。

Figure 3 Wnt signaling was reduced in old bone grafts. Figure 3-a quantitative RT-PCR to assess relative expression levels of Wnt ligand and Wnt target gene (figure 3-B) in Bone Marrow (BM) harvested from young donors (blue bars, n ═ 3) and elderly donors (white bars, n ═ 3). The gene expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Asterisks indicate p < 0.05.

Figure 4. liposomal Wnt3a restored osteogenic capacity of aged bone grafts fig. 4-a.l-PBS treated aged bone grafts (n ═ 5) aniline blue staining new aniline blue positive bone-like stroma in fig. 4-B.L-Wnt3a treated bone grafts (n ═ 8) fig. 4-c. determination of histomorphometry quantification of new bone stroma after transplantation, fig. 4-D.L-PBS and L-Wnt3a (fig. 4-E) treated bone grafts aniline blue staining on day 12 (d12) after transplantation, fig. 4-f. activity of β enzymes (β -gal) normalized to total DNA as measured in cell populations from harvested bone marrow (non-adherent floating cells and adherent cells), white bars (n ═ 4) represent reactivity after L-PBS treatment, blue bars (n ═ 4) represent concentration of L-Wnt3 treated (Wnt 0.25 g) effective staining of Wnt derived from Wnt treatment in fig. 8-g 23 g + t β t^LacZ/+Wnt-reactive cells in mouse L-PBS-treated bone grafts were compared to L-Wnt3a treatment (FIG. 4-J). FIG. 4-K Xgal staining on representative tissue sections identified from a young Axin2^LacZ/+Mouse L-PBS treated Wnt-responsive cells in bone grafts. Single asterisk denotes p<0.05; the four asterisks denote p<0.0001. Abbreviations: L-PBS ═ liposomal PBS; L-Wnt3a ═ liposomal Wnt3 a; BM is bone marrow; and DAPI ═ 4', 6-diamidino-2-phenylindoleIndole, dihydrochloride. Arrows mark the edges of intact bones. A scale: 100mm (FIG. 4-A [ the scale in FIG. 4-A also applies to FIG. 4-B)]) (ii) a 200mm (FIG. 4-D [ the scale in FIG. 4-D also applies to FIG. 4-E)]) (ii) a 100mm (FIG. 4-G) and 40mm (FIG. 4-I, [ scale in FIG. 4-I also applies to FIGS. 4-J and 4-K)])。

Figure 5L-Wnt 3a treatment restored osteogenic potential of bone grafts from older animals. Bone marrow from aged rabbit donors was assayed for DNA fragmentation associated with apoptosis. Figure 5-a. terminal deoxynucleotidyl transferase dUTP nick end marker (TUNEL) staining (n-4) demonstrates the degree of apoptosis in L-PBS (10mL) treated aged bone marrow compared to L-Wnt3a treatment (figure 5-B) (effective concentration 0.15 μ g/mL Wnt3 a). Figure 5-measurement of caspase activity in samples of aged bone grafts treated with PBS (white bars) or L-Wnt3a (blue bars). Bone marrow was harvested from aged rabbits, incubated with L-PBS or L-Wnt3a for up to 1h, and then transplanted into critical dimension defects formed in the ulna. Fig. 5-d radiographic evaluation at four weeks post bone grafting. L-PBS treatment was compared to L-Wnt3a treatment (FIG. 5-E). Figure 5-f micro-CT iso-surface reconstruction at eight weeks after bone grafting. L-PBS treatment was compared to L-Wnt3a treatment (FIG. 5-G). Fig. 5-h Bone Volume (BV) and bone volume/total volume (BV/TV) were calculated using the bone analysis tool in GE MicroView software. Single asterisk indicates p < 0.05. Abbreviations: L-PBS liposome PBS and L-Wnt3a liposome Wnt3 a. Arrows mark the edges of intact bones. A scale: 40mm (FIGS. 5-A and 5-B) and 5mm (FIGS. 5-F and 5-G).

Figure 6 histological appearance of regenerated bone from L-Wnt3a treated aged bone graft. Aniline blue staining of the injury sites (boxed regions) of aged bone marrow treatment incubated in L-PBS (FIG. 6-A) or L-Wnt3a (FIG. 6-B). FIG. 6-C Gomori trichrome staining of the aged subject fatty bone marrow cavity and adjacent damaged areas of aged bone graft treated with L-PBS (FIG. 6-D); the fibrous tissue stained emerald blue (turquoise blue). FIG. 6-E Gomori trichrome staining of the aged subject fatty marrow cavity and the adjacent damaged area of aged bone graft treated with L-Wnt3a (FIG. 6-F); mature bone-like matrix stains dark blue-green (dark turquoise) and bone cell nuclei stain red. Fig. 6-g. sirius red staining under polarized light identifies fibrous tissue formed by aged bone grafts treated with L-PBS. Compared to the bone-like matrix formed by aged bone graft treated with L-Wnt3a (FIG. 6-H). Abbreviations: L-PBS vs. liposome PBS and L-Wnt3a vs. liposome Wnt3 a. Arrows mark the edges of intact bones. A scale: 500 μm (FIGS. 6-A and 6-B); 100 μm (FIGS. 6-C to 6-F) and 200 μm (FIGS. 6-G and 6-H).

Fig. 7. the bone graft material comprises stem cell and progenitor cell populations. Gomori staining of BGM harvested from rat femur (a), (B) iliac crest and (C) tibia. (D) Quantitative RTPCR analysis of endogenous osteogenic gene expression in rat BGM freshly harvested from the indicated sources. (E) Schematic of experimental design, in which autologous BGM was transplanted into rat SRC. (F) Representative tissue sections of iliac crest BGM at day 7 post-transplantation, stained to detect BrdU incorporation. The dotted line indicates cancellous bone pieces included in the BGM. (G) Runx2, (H) Sox9 and (I) PPAR γ expression. (J) representative tissue sections of BGM were stained with aniline blue to detect bone-like matrix; asterisks indicate new bone matrix as opposed to old bone fragments (yellow dotted lines). The kidney surface is indicated by white dotted lines in this figure and in G. (K) Safranin O/fast green histology to detect proteoglycan-rich cartilage (red), and (L) Gomori trichrome to detect adipocytes. Abbreviations: BrdU, bromodeoxyuridine; PPAR γ, peroxisome proliferator-activated protein γ. A scale: 50 μm, star number: p < 0.05.

FIG. 8. the bone graft material is Axin2^CreERT2；R26m^TmGWnt-reactive GFP + ve cells, periosteum (a) and endosteum (B) in mice were visualized by immunostaining. (C) GFP in designated microscopic fields^+veQuantification of cells/total cells. (D) GFP in BGM^+veCells were visualized by fluorescence. (E) BGM^{Younger age}(Green bars) and BGM^{Old age}Quantitative absolute RT-PCR results of endogenous Axin2, Lef1, and GAPDH expression in (grey bars). (F) BGM^{Younger age}(Green bars) and BGM^{Old age}Western blot analysis of Wnt3a, total β catenin, Axin2 and β myokinesin (grey bars) scale 50 μm<0.05。

FIG. 9. the osteogenic differentiation potential of BGM decreases with age。(A)BGM^{Younger age}(Green bars) and BGM^{Old age}(grey bars) quantitative RT-PCR analysis of alkaline phosphatase, Osterix and osteocalcin expression. BGM harvested from ACTB-eGFP mice was transplanted into SRC and visualized under bright field (B) and (C) fluorescence to detect GFP signal in BGM. From (D) BGM^{Younger age}(N-5) and (E) BGM^{Old age}Representative tissue sections of (N ═ 5) stained with aniline blue (inset). The dotted lines indicate the surface. (F) Histomorphometric analysis of aniline blue + ve pixels within the total area occupied by BGM on day 7 post-transplantation. From (G) BGM^{Younger age}(N-5) and (H) BGM^{Old age}Representative tissue sections of (N ═ 5) stained to detect ALP activity. (I) ALP in the total area occupied by BGM on day 7 post-transplantation^+veQuantification of pixels. From (J) BGM^{Younger age}(N-5) and (K) BGM^{Old age}Representative tissue sections immunostained for GFP (N ═ 5). (L) GFP in the Total area occupied by BGM on day 7 post-transplantation^+veQuantification of pixels. Abbreviations: ALP, alkaline phosphatase; oc, osteocalcin. A scale: 100 μm. Asterisk p<0.05; double star number: p is a radical of<0.01。

Figure 10 osteogenic differentiation of BGM requires endogenous Wnt signaling. Representative tissue sections stained for ALP activity in BGM treated with (A) a murine IgG2 α Fc fragment (Ad-Fc) or (B) an adenovirus expressing the soluble Wnt antagonist Dkk1 (Ad-Dkk 1). Representative tissue sections immunostained for PPAR γ in BGM treated with (C) Ad-Fc or (D) Ad-Dkk 1. Representative tissue sections immunostained for Dlk1 in BGM treated with (E) Ad-Fc or (F) Ad-Dkk 1. micro-CT remodeling of bone formation in defect sites receiving BGM treated with (G) Ad-Fc or (H) Ad-Dkk1 was examined. The original defect is indicated by a dotted red circle. (I) new bone volume calculated from micro-CT data ± SEM (N ═ 5). Aniline blue staining of representative tissue sections from defect sites receiving BGM treated with (J) Ad-Fc or (K) Ad-Dkk 1. (L) quantification of new bone volume using histomorphometric analysis (see methods). PPAR- γ expression in BM transplants treated with (I) Ad-Fc or (J) Ad-Dkk 1. Single star number: p < 0.05. A scale: A-B,200 μm, CF, J-K,50 μm, G-H,2 mm.

FIG. 11 Wnt3a activates BGM^{Old age}And recoverIts osteogenic differentiation potential. (A) BGM from aged ACTB-eGFP mice were treated with L-PBS or L-WNT3A (0.15. mu.g/ml) for 1h, and then either assayed by qRT-PCR for 24h later for target gene expression or immediately transplanted into SRC for 7 days. (B) BGM treated with L-PBS (grey bars) or L-WNT3A (blue bars)^{Old age}Fold change in Axin2 and Lef1 expression. (C) BGM treated with L-PBS (grey bars) or L-WNT3A (blue bars)^{Old age}Western blot analysis of catenin β, Axin2, and β actin BGM was harvested from SRC on day 4 post-transplantation^{Old age}Representative tissue sections from (D) L-PBS (N ═ 5) and (E) L-WNT3A were then stained for BrdU incorporation (N ═ 5). (F) BrdU in microscopic field of view centered in the middle of bone graft^+veQuantification of pixels. Representative tissue sections from (G) L-PBS (N ═ 5) and (H) L-WNT3A (N ═ 5) treated samples were stained for BrdU incorporation on day 7 post-transplantation. (I) Quantification of BrdU + ve pixels as above. Representative tissue sections from (J) L-PBS (N ═ 5) and (K) L-WNT3A (N ═ 5) treated samples were immunostained for Dlk1 expression on day 7 post-transplantation. (L) quantification of Dlk1+ ve pixels within the total area occupied by BGM on day 7 post-transplant. Representative tissue sections from (M) L-PBS (N ═ 5) and (K) L-WNT3A (N ═ 5) treated samples were immunostained for Oc expression on day 7 post-transplantation. (O) Oc as described for Dlk1^+veQuantification of pixels. Representative tissue sections of bone-like matrix were examined by staining with aniline blue in (P) L-PBS (N ═ 5) and (Q) L-WNT3A (N ═ 5) treated samples. (R) histomorphometric quantification of new bone matrix; see methods for details. Abbreviations as in the previous figure legends. Scale bar 100 μm. Asterisks: p is a radical of<0.05; double asterisks: p is a radical of<0.01。

FIG. 12L-WNT 3A stimulates BGM stem cells and improves spinal fusion. (A) Human MSC cultures were treated with L-PBS or L-WNT3A at 37 ℃ at the indicated time points and qRTPCR for Axin2 expression was used to determine the Wnt-response. (B) Murine SSCs were treated with L-PBS or LWNT3A for 12h at 37 deg.C, and Wnt responses were analyzed by qRT-PCR for Axin2 expression. (C) Quantitative absolute RT-PCR analysis of Axin2 and Lef1 expression in response to 1h incubation with L-PBS (dotted line) or L-WNT3A (0.15. mu.g/mL; blue line) at room temperature. Data are expressed as the ratio of RNA copies/total RNA content over a 24h period. (D) Rat spinous processes were exposed through minimal incisions and standardized volumes of autologous BGM from the iliac crest were treated with L-PBS or L-WNT3A for 1hr, then (E) implanted between the transverse processes of the L4 and L5 vertebrae. At POD2, micro-CT detection (acquisition) was performed on the (pink) pattern treated with (F) L-PBS and (G) L-WNT 3A. At POD49, micro-CT probing was again performed to assess bone growth of grafts treated with (H) L-PBS (grey) and (I) L-WNT3A (blue). (I) Graft growth was plotted as fold volume for each treatment group, comparing the size of each graft at POD2 with its POD49 (indicated by color as described above). Abbreviations: l4, lumbar #4, L5, lumbar #5, AP, apical process, SP, spinous process, TP, transverse process, POD, postoperative day.

Detailed Description

Definition of

Before the present method is described, it is to be understood that this invention is not limited to particular methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that values intermediate between the upper and lower limits of the range (to the tenth unit of the lower limit unless the context clearly dictates otherwise), and any other stated or intermediate values in the stated range, are encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges encompassed within the invention, subject to any specifically excluded limit in the stated range. Unless defined otherwise, 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. Singleton et al, Dictionary of Microbiology and molecular Biology 2nd ed., J.Wiley & Sons (New York, NY 1994) provide one skilled in the art with a general guidance for many of the terms used in this application.

All publications mentioned herein are expressly incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

Wnt proteins. Wnt proteins form a family of highly conserved secretory signal molecules that regulate cell-cell interactions during embryogenesis. The terms "Wnt" or "Wnt gene product" or "Wnt protein" or "Wnt polypeptide" are used interchangeably and include native sequence Wnt polypeptides, Wnt polypeptide variants, Wnt polypeptide fragments, and chimeric Wnt polypeptides. In some embodiments of the invention, the Wnt protein comprises a palmitate covalently bound to a cysteine residue. A "native sequence" polypeptide is a polypeptide having the same amino acid sequence as a Wnt polypeptide of natural origin, regardless of the method used for its production. Such native sequence polypeptides may be isolated from cells that produce the endogenous Wnt protein or may be produced recombinantly or synthetically. Thus, the native sequence polypeptide can have, for example, the amino acid sequence of a naturally occurring human polypeptide, a murine polypeptide, or a polypeptide from any other mammalian species or from a non-mammalian species (e.g., Drosophila, C.elegans, etc.).

The term "native sequence Wnt polypeptides" includes, but is not limited to, human and murine Wnt polypeptides. Human Wnt proteins include the following: wnt1, Genbank reference NP 005421.1; wnt2, Genbank reference NP003382.1, expressed in the brain in the thalamus, in the lungs of fetuses and adults and in the placenta; two isoforms of Wnt2B, Genbank reference NP004176.2 and NP 078613.1. Isoform 1 is expressed in the adult heart, brain, placenta, lung, prostate, testis, ovary, small intestine and colon. In the adult brain, it is found mainly in the caudate nucleus, subthalamic nucleus and thalamus. Also detected in fetal brain, lung and kidney. Isoform 2 is expressed in fetal brain, fetal lung, fetal kidney, caudate nucleus, testis, and cancer cell lines. Wnt3 and Wnt3A exert significant cell-cell signaling during morphogenesis in the developing neural tube and have Genbank reference numbers NP 110380.1 and X56842 (Swiss-Prot P56704), respectively.

The original human Wnt3A amino acid and nucleotide sequences are specifically disclosed as SEQ ID NOs 1 and 2, respectively. Wnt3A was expressed in bone marrow. Wnt 4 has Genbank reference NP 110388.2. Wnt 5A and Wnt 5B have Genbank reference numbers NP003383.1 and AK 013218. Wnt 6 has Genbank reference NP 006513.1; wnt 7A is expressed in placenta, kidney, testis, uterus, fetal lung, and fetal and adult brain, Genbank reference NP 004616.2. Wnt 7B is moderately expressed in fetal brain, weakly expressed in fetal lung and kidney, and weakly expressed in adult brain, lung and prostate, Genbank reference NP 478679.1. Wnt 8A has two alternative transcripts (alternative transcripts), Genbank reference NP114139.1 and NP 490645.1. Wnt 8B is expressed in the forebrain and has Genbank reference NP 003384.1. Wnt10A has Genbank reference NP 079492.2. Wnt 10B was detected in most adult tissues, with the highest levels in heart and skeletal muscle. It has Genbank reference NP 003385.2. Wnt 11 is expressed in fetal lung, kidney, adult heart, liver, skeletal muscle and pancreas and has Genbank reference NP 004617.2. Wnt 14 has Genbank reference NP 003386.1. Wnt 15 is moderately expressed in fetal and adult kidneys and is also found in the brain. It has Genbank reference NP 003387.1. Wnt 16 has two isoforms, Wnt-16a and Wnt-16b, which are produced by alternative splicing. The isoform Wnt-16B is expressed in peripheral lymphoid organs such as the spleen, appendix (apendix) and lymph nodes, in the kidney, but not in the bone marrow. Isoform Wnt-16a is expressed only at significant levels in the pancreas. Genbank references are NP057171.2 and NP 476509.1. All GenBank, SwissProt and other database sequences listed are expressly incorporated herein by reference.

The term "native sequence Wnt protein" or "native sequence Wnt polypeptide" includes native proteins with or without an initial N-terminal methionine (Met) and with or without a native signal sequence. The term specifically includes the 352 amino acid long native human Wnt3a polypeptide, with or without its N-terminal methionine (Met) and with or without the native signal sequence.

A "variant" polypeptide refers to a biologically active polypeptide having less than 100% sequence identity to the original sequence polypeptide, as defined below. Such variants include those in which one or more amino acid residues are added to the N-or C-terminus of the original sequence or within the original sequence; polypeptides in which about one to forty amino acid residues are deleted and optionally substituted with one or more amino acid residues, and derivatives of the above polypeptides, wherein the amino acid residues are covalently modified such that the resulting product has non-naturally occurring amino acids. Typically, the amino acid sequence of a biologically active Wnt variant has at least about 90% amino acid sequence identity with the original sequence Wnt polypeptide, preferably at least about 95%, more preferably at least about 99%.

A "chimeric" Wnt polypeptide is a polypeptide comprising a Wnt polypeptide or portion (e.g., one or more domains) thereof fused or otherwise associated with a heterologous polypeptide. Chimeric Wnt polypeptides generally share at least one common biological property with a native sequence Wnt polypeptide. Examples of chimeric polypeptides include immunoadhesins, polypeptides that bind a portion of a Wnt polypeptide to an immunoglobulin sequence, and epitope-appended polypeptides comprising a Wnt polypeptide or portion thereof fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be raised, and yet is short enough so that it does not interfere with the biological activity of the Wnt polypeptide. Suitable tag polypeptides typically have at least six amino acid residues and typically about 6-60 amino acid residues.

A "functional derivative" of a pristine Wnt polypeptide is a compound that has common qualitative biological properties with pristine Wnt polypeptides. "functional derivatives" include, but are not limited to, fragments of the native sequence and derivatives of the native sequence Wnt polypeptide and fragments thereof, provided that they have the common biological properties of the corresponding native sequence Wnt polypeptide. The term "derivative" encompasses amino acid sequence variants of Wnt polypeptides and covalent modifications thereof.

Biologically active Wnt. The methods of the invention provide Wnt compositions that are active when administered in vivo to an animal (e.g., a lactating animal, such as a human). Specific activity of a Wnt protein can be determined by determining the level of activity after in vivo administration in a functional assay (e.g., an in vitro assay) or in a test model (e.g., accelerated bone regeneration, upregulation of stem cell proliferation, etc.), quantifying the amount of Wnt protein present in a non-functional assay (e.g., immunostaining, ELISA, quantification on coomassie or silver stained gels, etc.), and determining the ratio of biologically active Wnt to total Wnt in vivo.

Lipid structure. As used in the methods of the invention, the lipid structure was found to be important in maintaining the activity of the Wnt protein following in vivo administration. Wnt proteins are not encapsulated in the aqueous phase of these structures, but instead are integrated into the lipid membrane and can insert into the outer layer of the membrane. Such structures are not predicted from conventional methods of formulating proteins, such as in liposomes. Wnt polypeptides having such lipid structures are referred to herein as L-Wnt, e.g., L-Wnt3 a. Methods for tethering Wnt proteins to the outer surface of liposomes or micelles may utilize sequences in order to emphasize the in vitro display of proteins, where crude liposomes are first pre-made; wnt proteins are then added to the crude mixture, which facilitates the addition of extra-liposomal Wnt, followed by various formulation steps, which may include size filtering, dialysis, and the like. Suitable lipids include fatty acids, neutral fats such as triacylglycerols, fatty acid esters and soaps, long chain (fatty) alcohols and waxes, sphingosines (sphingoids) and other long chain bases, glycolipids, sphingolipids, carotenes, polypentenols, sterols, and the like, as well as terpenes and isoprenoids. For example, molecules such as diacetylene phospates may be used. Included are cationic molecules having hydrophobic and hydrophilic moieties, a net positive charge, including lipids, synthetic lipids, and lipid analogs, and which themselves may spontaneously form bilayer vesicles or micelles in water. Liposomes prepared with neutral charges (e.g., DMPC) are preferred. The term also includes any amphipathic molecule that can be stably incorporated into a lipid micelle or bilayer in combination with a phospholipid such that its hydrophobic portion is in contact with the inner hydrophobic region of the micelle or bilayer membrane and its polar head group portion is oriented towards the outer polar surface of the membrane.

The term "cationic amphipathic molecule" is intended to encompass molecules that are positively charged at physiological pH, and more particularly structurally positively charged molecules, including, for example, quaternary ammonium moieties. Cationic amphiphilic molecules are generally composed of a hydrophilic polar head group and a lipophilic fatty chain. Similarly, cholesterol derivatives having a cationic polar head group may also be useful. See, e.g., Farhood et al (1992) Biochim.Biophys.acta 1111: 239-246; vigneron et al (1996) Proc.Natl.Acad.Sci. (USA)93: 9682-. Target cationic amphoteric molecules include, for example, imidazoline salt derivatives (WO 95/14380), guanidine derivatives (WO 95/14381), phosphatidylcholine derivatives (WO 95/35301), and piperazine derivatives (WO 95/14651). Examples of cationic Lipids that can be used in the present invention include DOTIM (also known as BODAI) (Saladin et al, (1995) biochem.34: 13537-.

Although not required for activity, in some embodiments, the lipid structure can include a targeting moiety, such as a targeting moiety covalently or non-covalently bound to a hydrophilic head group. Head groups that may be used to bind the targeting moiety include, for example, biotin, amines, cyano, carboxylic acids, isothiocyanates, thiols, disulfides, α -halocarbonyl (ahalocarbonyl) compounds, a, p-unsaturated carbonyl compounds, alkylhydrazines, and the like. Chemical groups that can be used to attach the targeting moiety to the amphiphilic molecule also include carbamates (amine + carboxylic acid), esters (alcohol + carboxylic acid), thioethers (alkyl halide + thiol; maleimide + thiol), schiff bases (amine + aldehyde), ureas (amine + isocyanate), thioureas (amine + isothiocyanate), sulfonamides (amine + sulfonyl chloride), disulfides, hydrazones (hydrazones), lipids, and the like, as known in the art. For example, targeting molecules can be formed by converting commercially available lipids such as DAGPE, PEG-PDA amines, DOTAP, etc. to isocyanates, followed by treatment with a triethylene glycol diamine spacer to produce amine-terminated thiocarbamate lipids that yield the desired targeted glycolipids by treatment with a targeting moiety of p-isothiocyanatophenyl glycoside. This synthesis provides a water-soluble flexible linker molecule that is spaced between the amphipathic molecule incorporated into the nanoparticle and the ligand that binds to the cell surface receptor, allowing the ligand to readily access the protein receptor on the cell surface. Further information on liposomal Wnt compositions and their uses is found in U.S. patent application publication 20120115788.

The term "bone graft" is used herein in the broadest sense and specifically includes autografts and allografts harvested from a patient's own bone or from individuals other than the individual receiving the graft (including cadavers), respectively. The term "bone graft" also includes autologous or allogeneic pluripotent stem cell populations, such as stem cells harvested from bone marrow, e.g., bone marrow-derived mesenchymal stem cells. Bone grafts may be obtained from donors by a variety of means, including but not limited to, reaming (reamers), perfusion, aspiration methods.

Osteogenic potency is enhanced by incubating cells for bone graft with an effective dose of Wnt protein, e.g., L-Wnt3A, for a period of time sufficient to enhance osteogenic potential.

As used herein, bone graft material refers to a cellular composition obtained from a donor, which may be a living or cadaveric body. Bone graft materials typically comprise a complex population of cells and include stem cells such as mesenchymal stem cells, and may also comprise bone cells and their progenitors. The donor may be allogeneic or autologous with respect to the recipient. The quality of cells used in bone grafts may vary with the donor, recipient, grafting purpose, and the like. The bone graft may comprise up to about 10³At most about 10⁴At most about 10⁵At most about 10⁶At most about 10⁷At most about 10⁸At most about 10⁹At most about 10¹⁰Or more cells.

Bone graft material is obtained from donors, such as from the iliac crest, from the mandibular union (chin area), from the reaming, perfusion and irrigation of the femur and/or tibia, fibula, ribs, anterior mandibular branches, portions of the spinal vertebrae (e.g., those removed during surgery), cadaveric bone, and the like. The graft material may be bone marrow, for example scraped from the endosteal surface of a suitable bone, or may be a block graft comprising bone marrow and small bone pieces. Allograft bone may be harvested from cadavers, bone banks, etc., such as from hip replacement surgery to harvest (sing) the femoral head. The bone graft material may be used fresh or may be cryopreserved as known in the art until needed.

The cells of the bone graft are suspended in a suitable medium in the presence of an effective dose of liposomal Wnt protein (e.g., L-Wnt 3A). Any suitable medium may be used, for example, DMEM, RPMI, PBS, and the like. The cells are typically resuspended at a concentration that maintains viability during incubation, e.g., up to about 10⁴Per ml, up to about 10⁵Per ml, up to about 10⁶Per ml, up to about 10⁷And/ml. The incubation temperature typically does not exceed about 37 ℃ and can be lower, e.g., up to about 32 ℃, up to about 25 ℃, up to about 15 ℃, up to 10 ℃, up to about 4 ℃, but is typically above the freezing temperature unless specifically prepared for cryopreservation.

Effective dosages of Wnt proteins may vary depending on source, purity, method of preparation, and the like. Where the Wnt protein is L-Wnt3A, an effective dose will generally be at least about 0.1. mu.g/ml, at least about 0.5. mu.g/ml, at least about 1. mu.g/ml, at least about 2.5. mu.g/ml, at least about 5. mu.g/ml, at least about 7.5. mu.g/ml, at least about 10. mu.g/ml, at least about 15. mu.g/ml, and may be at least about 25. mu.g/ml, at least about 50. mu.g/ml, at least about 100. mu.g/ml.

The bone graft material is incubated with the Wnt protein for a period of time sufficient to enhance osteogenesis. Enhancement can be measured by various means, for example, by increased Axin2 expression, by increased mitotic activity in the bone graft material (measured from about day 2 to about day 6 of the graft), by increased bone formation after transplantation, by increased Runx2 or osteocalcin expression, by decreased apoptosis after transplantation, or by bone volume produced after transplantation. The increased bone volume can be about 1.5-fold, about 2-fold, about 3-fold, or more relative to the volume obtained in the absence of wnt treatment.

The bone graft material is typically contacted with the Wnt protein for at least about 1 hour, at least about 2 hours, and up to about 36 hours, up to about 24 hours, up to about 18 hours, up to about 15 hours, up to about 12 hours, up to about 8 hours, up to about 6 hours, up to about 4 hours.

After incubation, the bone graft material may be implanted into a recipient according to conventional protocols, e.g., for repairing vertebral bones, fractures, dental supports, and the like.

In particular, the osteogenic capacity of elderly bone grafts was restored by incubation with Wnt proteins. Initially, liposomal Wnt3a treatment reduced cell death in aged bone grafts. Subsequently, after transplantation, the bone grafts treated with liposomal Wnt3a produced significantly more bone (p < 0.05). As is evident from the examples, liposomal Wnt3a treatment enhanced cell survival in the grafts and reconstructed the bone forming ability of the grafts from older animals.

Thus, the present invention provides a safe, effective and clinically applicable regenerative medicine-based strategy for revitalizing bone grafts from elderly patients or from other patients with reduced healing potential, such as smokers, diabetic patients or patients with nutritional deficiencies.

All scientific and patent publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference. Further details of the invention are provided in the following non-limiting examples.

For exemplary purposes only, the present invention includes, but is not limited to, the following technical solutions:

technical solution 1. a method of enhancing cell survival in a bone graft, comprising subjecting the bone graft to ex vivo treatment with an effective dose of a Wnt polypeptide for a period of time sufficient to enhance cell survival of the bone graft upon transplantation.

Technical solution 2. a method of enhancing the osteogenic potential of a bone graft, comprising subjecting the bone graft to ex vivo treatment with an effective dose of a Wnt polypeptide for a time period sufficient to enhance cell survival of the bone graft upon transplantation.

Technical solution 3. a method of revitalizing a bone graft from a subject with reduced healing potential, comprising subjecting the bone graft to ex vivo treatment with an effective dose of a Wnt polypeptide for a period of time sufficient to enhance cell survival of the bone graft upon transplantation.

Scheme 4. the method of any of schemes 1-3, wherein the bone graft is an autograft.

Technical solution 5. the method according to any one of technical solutions 1 to 3, wherein the bone graft is an allograft.

Scheme 6. the method of any of claims 1-3, wherein the bone graft comprises a population of stem cells.

Scheme 7. the method of scheme 6, wherein the bone graft comprises a population of bone marrow-derived stem cells.

Scheme 8. the method of scheme 7, wherein the bone graft comprises bone marrow-derived mesenchymal stem cells.

Technical solution 9. the method according to any one of technical solutions 1 to 3, wherein the bone graft is from a human subject.

Technical solution 10. the method according to technical solution 9, wherein the human subject is an elderly patient.

Technical solution 11 the method of technical solution 10, wherein the human subject is at least 60 years old.

Technical solution 12. the method of technical solution 10, wherein the human subject is at least 65 years old.

Technical solution 13 the method of technical solution 10, wherein the human subject is at least 70 years old.

Technical solution 14 the method of technical solution 10, wherein the human subject is at least 75 years old.

Technical solution 15. the method of technical solution 10, wherein the human subject is at least 80 years old.

Technical solution 16 the method of technical solution 10, wherein the human subject is at least 85 years old.

Technical solution 17. the method according to technical solution 8, wherein the human subject has reduced healing potential.

Technical solution 18. the method of technical solution 16, wherein the human subject is a smoker.

Technical solution 19. the method of technical solution 16, wherein the human subject is diabetic.

Technical solution 20 the method of technical solution 16, wherein the human subject has an auxotrophy.

Technical scheme 21 the method of any one of technical schemes 1 to 19, wherein the Wnt polypeptide is Wnt3 a.

Solution 22 the method of solution 20, wherein the Wnt polypeptide is human Wnt3 a.

Scheme 23 the method of scheme 21, wherein the Wnt polypeptide is liposomal human Wnt3a (LWnt3 a).

Solution 24. the method of any one of solutions 1-23, further comprising the step of introducing the bone graft into a recipient.

Technical solution 25 the method of technical solution 24, wherein the recipient is a human patient.

Solution 26. the method of solution 25, wherein the bone graft is used to support or augment the support of a dental implant.

Solution 27. the method of solution 25, wherein the bone graft is used to repair a bone fracture.

28. The method of claim 25, wherein the bone graft is used to repair or reconstruct a diseased bone.

Solution 29. the method of solution 27 or 28, wherein the bone graft is used in the hip, knee, or spine of the recipient.