WO2018162722A1 - Dpp-4 inhibitors for use in treating bone fractures - Google Patents

Abstract

The present disclosure provides DPP-4 inhibitors for use in treating a bone fracture, for use in preventing non-union of a bone fracture or preventing healing complications following bone fracture, for use in reducing the inhibitory effects of marrow adipose tissue (MAT) on fracture healing, or for use in preventing loss of bone mineral density (BMD) in a subject in need thereof. The disclosure also provides methods of preparing cells for transplantation comprising providing a cell culture comprising osteogenic progenitor cells (OPCs) and/or mesenchymal stem cells, and contacting the cells with a DPP-4 inhibitor. The disclosure further provides methods of preparing cells for transplantation comprising providing a cell culture comprising CD45-CD31^- Sca1⁺ CD24⁺ multipotent cells, and treating the cell culture to promote lineage commitment and/or differentiation into osteogenic progenitor cells (OPCs) and/or mature cells of the osteogenic lineage/bone tissues.

Description

DPP-4 INHIBITORS FOR USE IN TREATING BONE FRACTURES

BACKGROUND OF THE INVENTION

An average of 8 million bone breaks occurs in the United States each year. Most bone fractures heal without issues. However, about 3-10% of these breaks are slow to heal or do not heal at all with traditional methods. In particular, about 5-10% of all bone fractures also display non-union and/or delayed unions, which have a substantial economic impact and substantial effects on quality of life. Fracture healing is considered to be a complicated metabolic process that requires the interaction of many factors, including the recruitment of reparative cells and expression of corresponding genes. If these factors are inadequate or interrupted, healing is delayed or impaired, resulting in a nonunion of the bone. The causes of nonunions or delayed healings of fractures are usually unknown.

There is a need for novel treatments for treating bone-related diseases and conditions such as osteoporosis and bone fractures.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a DPP-4 inhibitor for use in treating a bone fracture in a subject in need thereof.

In a second aspect, the present invention relates to a DPP-4 inhibitor for use in preventing non-union of a bone fracture or preventing healing complications following bone fracture in a subject in need thereof.

In a third aspect, the present invention relates to a DPP-4 inhibitor for use in promoting fracture healing in a subject in need thereof.

In a fourth aspect, the present invention relates to a pharmaceutical composition comprising a DPP-4 inhibitor for use in treating a bone fracture in a subject in need thereof, wherein said pharmaceutical composition further comprises a pharmaceutically-acceptable diluent, excipient, or carrier, and wherein the DPP-4 inhibitor is present in an effective amount to treat or prevent bone fractures.

In a fifth aspect, the present invention relates to a pharmaceutical composition comprising a DPP-4 inhibitor for use in preventing non-union of a bone fracture or in preventing healing complications following bone fracture in a subject in need thereof, wherein said pharmaceutical composition further comprises a pharmaceutically-acceptable diluent, excipient, or carrier, and wherein the DPP-4 inhibitor is present in an effective amount to treat or prevent bone fractures.

In an embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the bone fracture is a non-union bone fracture, a compound fracture or a fracture with delayed healing.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the DPP-4 inhibitor is administered systemically.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the DPP-4 inhibitor is administered locally.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the DPP-4 inhibitor is administered to the site of fracture.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the DPP-4 inhibitor is administered in one or more doses over a period of less than 6 months.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the DPP-4 inhibitor is administered in one or more doses over a period of less than 3 months.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the DPP-4 inhibitor is administered in one or more doses over a period of less than 1 month.

In a further embodiment the present invention relates to the DPP-4 inhibitor for use according to the first, second or third aspect, or to the pharmaceutical composition for use according to the fourth or fifth aspect, wherein, prior to diagnosis of the bone fracture, the subject was not receiving a DPP-4 inhibitor.

In a further embodiment the present invention relates to the DPP-4 inhibitor for use according to the first, second or third aspect, or to the pharmaceutical composition for use according to the fourth or fifth aspect, wherein the subject in need thereof was not, prior to the bone fracture, diagnosed with or treated for Type II diabetes. In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the subject in need thereof is over age 65.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the subject in need thereof is under age 40.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the subject in need thereof has a BMI less than 25.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the subject in need thereof has a BMI of greater than or equal to 25 and less than 30.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the subject in need thereof has a BMI of equal to or greater than 30.

In a further embodiment the present invention relates to the DPP-4 inhibitor for use according to the first, second or third aspect, or to the pharmaceutical composition for use according to the fourth or fifth aspect, wherein the DPP-4 inhibitor is administered in one or more doses, and wherein at least one of the doses is administered locally during surgery to set the fracture.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, wherein the subject in need thereof is a human subject.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the healing complications are osteoporosis related healing complications to bone fracture.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the DPP-4 inhibitor is selected from one or more of alogliptin, linagliptin, saxagliptin, sitagliptin, vildapliptin, gemigliptin, or teneligliptin.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the use further comprises administering metformin, in the same or a different formulation as the DPP-4 inhibitor.

In a further embodiment of the DPP-4 inhibitor for use according to the first, second or third aspect, or of the pharmaceutical composition for use according to the fourth or fifth aspect, the use further comprises administering one or more other therapeutic agent.

In a sixth aspect, the present invention relates to a DPP-4 inhibitor for use in reducing the inhibitory effects of marrow adipose tissue (MAT) on fracture healing in a subject in need thereof.

In a seventh aspect, the present invention relates to a method of preparing cells for transplantation, comprising

providing a cell culture comprising osteogenic progenitor cells (OPCs) and/or mesenchymal stem cells,

contacting the cells with a DPP-4 inhibitor in an amount effective to increase osteogenic gene expression, or osteo-/ chondrogenic cell differentiation, or osteogenic lineage commitment, thereby generating a culture comprising DPP-4 treated cells.

In an eighth aspect, the present invention relates to a method of preparing cells for transplantation, comprising

providing a cell culture comprising CD45^~CD31^~Scal⁺CD24⁺ multipotent cells, treating the cell culture to promote lineage commitment and/or differentiation into osteogenic progenitor cells (OPCs) and/or mature cells of the osteogenic lineage/bone tissues, and sorting the cells to isolate or enrich for OPCs.

In a ninth aspect, the present invention relates to DPP-4 treated cells prepared by the method of the seventh or eights aspect for use in transplantation to a fracture in a subject in need thereof.

In a tenth aspect, the present invention relates to a DPP-4 inhibitor for use in preventing loss of bone mineral density (BMD) in an astronaut or other individuals exposed to an altered gravity environment.

In an embodiment of the DPP-4 inhibitor for use of the tenth aspect, the astronaut is exposed to an altered gravity environment for greater than one week, and the DPP-4 inhibitor is administered to the astronaut prior to and/or during and/or after the exposure.

In an eleventh aspect, the present invention relates to a DPP-4 inhibitor for use in preventing loss of bone mineral density (BMD) in a subject in need thereof. FIGURES

Figure 1A shows flow cytometric separation of CD45 CD31^" (upper and lower left quadrants in dot plot) cells by Seal -selection.

Figure IB shows Oil Red-0 (Adipogenesis), Alizarin Red S (Osteogenesis) and Alician Blue (Chondrogenesis) staining of Scal⁺ and Seal^" cells differentiated under corresponding conditions.

Figure 1C shows FACS analysis plot of CD45^"CD31^"Scal⁺ cells separated by CD24 expression.

Figure ID shows Oil Red-0 (Adipogenesis), Alizarin Red S (Osteogenesis) and Alician Blue (Chondrogenesis) staining of CD45^"CD31^"Scal⁺CD24⁺ and CD45 CD31^" Scal⁺CD24^" cells differentiated under corresponding conditions.

Figure 2A shows FACS-analysis of viable cells from 2-month old male -eGFP reporter mice for expression of GFP followed by Seal and CD45/CD31 expression analysis within GFP⁺ cells.

Figure 2B shows adipogenic (Oil Red O) and osteogenic (Alizarin Red S) differentiation assays of CD45^"CD31^"Scal⁺Pa⁺ and CD45^"CD31^"Scal^"Pa⁺ populations.

Figure 3 shows an analysis of the colony forming potentials (CFU-F) (n=6/cell population) of the indicated bone populations. All results are shown as mean ± SEM.

Figure 4 shows an analysis of the CFU-F total recovery rate (n=3/cell population) of the indicated bone populations. All results are shown as mean ± SEM.

Figure 5 shows flow cytometric dot plot analyses of bone resident CD45^"CD31^"Scal^" Pa⁺ cells (left panel) and bone resident CD45^"CD31^"Scal^"Pa^" cells separated into CD24^" and CD24⁺ cells by FACS.

Figure 6 shows FACS-analysis of CD45 CD31^" cells from 2-month old male Zfp423- eGFP reporter mice for expression of Seal and GFP.

Figure 7A shows analysis in individual cells (n=72) after immunofluorescence co- staining for Seal and Zfp423-eGFP in the CD45 CD3 l^"Scal⁺ population at day 5 of adipogenic differentiation.

Figure 7B shows significant correlation of average fluorescence intensities (Avlnt) in individual cells (n=72) after immunofluorescence co-staining for Seal and Zfp423-eGFP in the CD45^"CD31^"Scal⁺ population at day 5 of adipogenic differentiation. Figure 8 shows FACS-analysis of cultured Scal⁺ cells at day 3 (d3), 5 (d5), and 8 (d8) of adipogenesis showing frequencies of Scal^~Zfp423⁺, Scal⁺Zfp423⁺, and Scal^~Zfp423^~ cell populations (n=3). All results are shown as mean ± SEM.

Figure 9 upper panel shows quantification of osteogenic Scal^"Pa+, multipotent Scal⁺Pa⁺CD24⁺ and adipogenic Scal⁺Pa⁺CD24^" cells in metaphysis or diaphysis of bones derived from -eGFP mice. The lower panel shows quantification of osteogenic Scal^"Pa+, multipotent Scal⁺Pa⁺CD24⁺ and adipogenic Scal⁺Pa⁺CD24^" cells localizing at the endosteum, or to areas <40 μιη from the endosteum, and to areas >40 μm from the endosteal layer of bones of -eGFP reporter mice. All results are shown as mean ± SEM (n= 14-24 bone marrow sections were analyzed from n=4 mice per reporter strain; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 10 shows quantification of bone marrow- localized Pa-GFP+ cells associated to blood vessels with diameters smaller or larger than 10 μιη (upper panel). The lower panel shows quantification of Zfp423⁺ preAd distribution in bones from the Zfp423-eGFV reporter mouse strain in metaphysis or diaphysis. All results are shown as mean ± SEM (n= 14-24 bone marrow sections were analyzed from n=4 mice per reporter strain; ****p<0.0001).

Figure 11 shows frequency analyses of the four investigated cell populations in different bone compartments by FACS. All results are shown as mean ± SEM (n=3; p<0.05: a vs. long bones; b vs. sternum; c vs. thoracic spine; d vs. caudal spine; e vs. calvarium).

Figure 12A shows transgene alleles of the rep^AdlLuc reporter mouse strain: The Zfp423- eGFP reporter mouse strain was crossed to a strain expressing Cre-recombinase under control of the Adiponectin promoter (Adipoq-Cre) and a constitutive Luciferase (Luc)-reporter where the Luc-encoding cDNA is suppressed by a loxP-flanked Stop-signal. Thus, in this reporter strain only mature adipocytes expressing the adipogenic marker Adiponectin undergo Cre- mediated recombination. This leads to the excision of the loxP-flanked stop-cassette, activating the expression of Luciferase that can be detected by in vivo imaging techniques. Figure 12B shows transgene alleles of the rep^tdTom reporter mouse strain: The Zfp423-eGFV reporter was crossed to an mTmG-reporter mouse strain without presence of a Cre-transgene. Thus, the cells maintained constitutive red fluorescence and can be detected by immunofluorescence for tdTomato or by in vivo imaging.

Figure 13 shows the FACS-gating strategy for the isolation of the four investigated cell populations (PreAd, APC, CD45 CD31 Scal⁺CD24⁺, OPC) from both the rep^AdiLuc reporter mouse strain and the rep^tdTom reporter mouse strain for subsequent in vivo transplantation assays. Figure 14 shows in vivo luciferase imaging (top panels) and macroscopic identification (arrows; middle panels) of transplants 8 weeks after sternal s.c. -injection of the indicated cell populations isolated from repAdiLuc mice. The lower panels show microscopy of corresponding Movat-Pentachrome stains (yellow: mineralized structure; blue: cartilage; purple: nuclei). Scale bars, 30μιη.

Figure 15 shows FACS-analysis of transplants of initially CD45 CD31 Scal⁺CD24⁺ cells identified by tdTomato-expression giving rise to the CD45^~CD31^~Scal⁺CD24^~ population within the transplant.

Figure 16 shows a summary of sternal transplantation experiments of the four investigated cell populations including numbers of transplanted animals and respective differentiation fates as determined by histological analysis and engraftment efficiency.

Figure 17 shows a summary of the four investigated cell populations with phenotypic marker expression and differentiation potential performances during in vitro and in vivo experiments. In the column for immunophenotypes/ marker phenotypes, markers that are required to define and isolate the respective populations by flow cytometry are labeled in bold.

Figure 18 shows m NA levels of general adipogenic markers Pparg and Cebpa as well as brown adipogenic markers Ucpl and Cidea after adipogenic differentiation. Results of two independent experiments with 3 replicates each are shown as mean ± SEM (n=6; p<0.05: a vs. Bone, b vs. iWAT, c vs. BAT, d vs. Muscle).

Figure 19 shows a western blot analysis of UCP1 protein with β-Actin as a loading control measured in differentiated CD45^"CD31^"Scal⁺ populations isolated from inguinal white adipose tissue (iWAT), brown adipose tissue (BAT), bone or muscle after adipogenic differentiation in the presence of the browning agent rosiglitazone (Rosi).

Figure 20 shows gene expression analysis of Sp7 (Osx) and Pparg in whole-bones (femur and tibia) from young (2 -months) and old (25-months) male C57BL/6J mice (n=4). Results are shown as mean ± SEM (*p<0.05; **p<0.01).

Figure 21 shows gene expression analyses of Pparg, Cebpa, Fabp4 and Leptin (Lep) in Scal⁺ cells from young (2-months) and old (25-months) after adipogenic differentiation. Results are shown as mean ± SEM of two independent experiments (n=6).

Figure 22 shows hematoxylin and eosin (H&E) stains of femora from 2-month old or

25 -month old male mice maintained on standard diet (SD), or high fat diet for 24 h (ldHFD) or 10 days (lOdHFD). Figure 23A shows Relative quantifications of CD45 CD31 Scal⁺CD24⁺, APC (CD45^" CD31 Scal⁺CD24 ) and OPC (CD45 CD31 Scal-CD24-Pa⁺) cell populations in 2-month and 25-month old mice on SD (white bars) compared to ldHFD (grey bars, applies to all panels) (n=9).

Figure 23B shows quantification of multipotent CD45^"CD31^"Scal⁺CD24⁺, adipogenic

CD45 CD31 Scal⁺CD24- (APC), and osteogenic CD45 CD31 Scal-CD24-Pa⁺ (OPC) subpopulations in 2-month old and 25 -month old male mice fed SD (white bars) or high fat diet for 10 days (lOdHFD) (black bars) (n=9). Results are shown as mean ± SEM (****p<0.0001).

Figure 24 shows BrdU incorporation into multipotent CD45^"CD31^"Scal⁺CD24⁺ and APCs (CD45 CD3 l Scal⁺CD24 ) from 2-month old and 15-month old mice on SD or ldHFD (n=7-9).

Figure 25 shows quantification of BrdU incorporation into bone-resident OPC cells in 2-month old and 15 -month old mice after 1 day high fat diet compared to mice fed a Standard diet (n=7-8). All graphs show cumulative data from at least three independent experiments. Results are shown as mean ± SEM.

Figure 26 shows quantification of BrdU incorporation into multipotent CD45 CD31^" Scal⁺CD24⁺, APC, and OPC subpopulations in 2-month old and 25 -month old mice after 10- days high fat diet (lOdHFD) compared to mice fed a standard diet (SD) (n=3).

Figure 27 shows quantification of GFP+ cells in 2-month old and 15 -month old male Zfp423-eGFP reporter mice on standard diet (SD) or 1 day high fat diet (ldHFD) (n=6). Graphs show cumulative data from at least two independent experiments. Mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 28 shows red fluorescence in tibiae (top panels) and μCT images (lower panels) of fracture calluses 14 days after fracture and intratibial injection of the indicated cell populations.

Figure 29 shows flow cytometric analysis of fracture calluses two weeks after surgery either injected with bone-derived Scal⁺Pa⁺ cells or Scal^"Pa⁺cells isolated from animals constitutively expressing GFP. Shown are viable cells previously gated for CD45^"CD31^" to show retention of cells after transplantation.

Figure 30 shows total bone mineral density (BMD) by μCT following transplantation of the indicated cell populations into the fracture (n=6). Bars are from left to right in each panel: No cells, OPC, CD45-CD31-Scal+CD24+, APC, PreAd. Mean ± SEM; p<0.05: a vs. no cells; b vs. OPC; c vs. CD45 CD31-Scal+CD24+; d vs. APC. Figure 31A shows histomorphometry of mineralized bone volume (BV) and non- mineralized callus as fibrous tissue (FV) and cartilage tissue (CV) volumes normalized to total callus volume (TV) (n=8). Mean ± SEM; p<0.05: a vs. no cells; b vs. OPC; c vs. CD45 CD31 Scal⁺CD24⁺; d vs. APC. Bars are from left to right in each panel: No cells, OPC, CD45-CD31- Scal+CD24+, APC, PreAd.

Figure 3 IB provides immuno florescence showing the contribution of transplanted multipotent CD45 CD31 Scal⁺CD24⁺ (upper panels) and osteogenic CD45 CD31 Scal Pa⁺ (OPC, lower panels) cell populations to osteochondrogenic structures in the fractured tibiae that were not observed in adipogenic cell transplants (Red: tdTomato; Blue: DAPI; right panels indicate merge of immuno florescence and light microscopic images). Scale bar, 20 μιη.

Figure 31C provides immuno florescence showing the contribution of transplanted multipotent CD45^"CD31^"Scal⁺CD24⁺ (upper panels) and osteogenic CD45 CD3 rScal Pa⁺ (OPC, lower panels) cell populations to endosteal bone linings in the fractured tibiae (Red: tdTomato; Blue: DAPI; dotted lines indicate areas of compact bone as seen in right-side panels of merged immunoflorescence and light microscopic images). Scale bar, 10 μιη.

Figure 3 ID provides immunoflorescence co-staining of tdTomato⁺ cells (red fluorescence) with Osteocalcin to show an osteogenic differentiation fate of transplanted multipotent CD45^"CD31^"Scal⁺CD24⁺ (upper panels) osteogenic CD31^"CD45^"Scal Pa⁺ (lower panels) cell populations. No co-staining detected in adipogenesis-committed populations, e.g. APCs and preAds (not shown). Scale bar, 10 um.

Figure 3 IE provides immunoflorescence co-staining of tdTomato+ cells (red fluorescence) with Aggrecan to show a chondrogenic differentiation fate of transplanted multipotent CD45 CD31 Scal⁺CD24⁺ (upper panels) osteogenic CD45 CD31 Scal Pa⁺ (lower panels) cell populations. No co-staining detected in adipogenesis-committed populations, e.g. APCs and preAds (not shown). Scale bar, 10 um.

Figure 32 shows characterization results of RNA-Seq samples with read counts (left panel) and the fraction of reads mapped to exons (right panel).

Figure 33A shows the Principal Component Analysis (PCA) of the RNA-seq samples. Figure 33B shows the correlation scores of top 10 genes driving PCI and PC2 in Figure 33 A. Figure 33C shows hierarchical clustering analyses of RNA-Seq data from all four cell populations. Figure 34 shows a heat map of selected differentially expressed (DE) genes, divided by candidates reported in the literature (known, asterisks indicate no significant DE between individual groups) and novel markers, enriched in CD45^~CD31^~Scal⁺CD24⁺ cell populations.

Figure 35 shows a heat map of selected differentially expressed (DE) genes, divided by candidates reported in the literature (known, asterisks indicate no significant DE between individual groups) and novel markers, enriched in OPC cell populations.

Figure 36 shows a heat map of selected differentially expressed (DE) genes, divided by candidates reported in the literature (known, asterisks indicate no significant DE between individual groups) and novel markers, enriched in APC cell populations.

Figure 37 shows a heat map of selected differentially expressed (DE) genes, divided by candidates reported in the literature (known, asterisks indicate no significant DE between individual groups) and novel markers, enriched in preAd cell populations.

Figure 38 shows gene expression intensities of Dpp4 from RNA-Seq analysis. Mean ± SEM; *p<0.05, **p<0.01, ****p<0.0001.

Figure 39 shows FACS analysis of DPP4/CD26 surface marker expression in

CD31⁺CD45⁺ populations and CD31 CD45 Scal⁺CD24⁺, OPC, APC and preAd cell populations from 2-month old male mice.

Figure 40 shows DPP4 release into the culture medium by OPCs, multipotent CD45^" CD31^"Scal⁺CD24⁺, and APC cell populations that underwent in vitro adipogenic differentiation (n=3). Results are shown as mean ± SEM; *p<0.05.

Figure 41 shows Dpp4-mRNA expression (as a percentage versus that in young mice) in whole proximal and distal tibiae of 2-month (young) and 15-month (old) male mice (n=5) (left panel). The right panel shows DPP4-secretion by whole tibia explants from young and old mice (n=4). Mean ± SEM; *p<0.05.

Figure 42A shows mRNA expression (as a percentage of control) of Runx2 and Osterix

(Osx/Sp7) in CD45 CD31 Scal⁺CD24⁺ and OPCs either treated with PBS (control, white bars) or Sitagliptin (100 μΜ, black bars) during osteogenic differentiation (n=3). Mean ± SEM; *p<0.05, **p<0.01. Mean ± SEM; *p<0.05, **p<0.01.

Figure 42B shows Alizarin Red S staining (four left panels) and quantification of staining (right panel) of CD45 CD3 l^"Scal⁺CD24⁺ and OPCs either treated with PBS (control, white bars) or Sitagliptin (100 μΜ, black bars) during osteogenic differentiation (n=3). Mean ± SEM; *p<0.05, **p<0.01. Figure 43A shows Oil Red-0 staining of CD45 CD31 Scal⁺CD24⁺ and APCs either treated with PBS or Sitagliptin (100 μΜ) during adipogenic differentiation.

Figure 43B shows mRNA expression (as a percentage of control) of Pparg and Lep in multipotent CD45 CD31 Scal⁺CD24⁺ and APCs either treated with PBS (control; white bars) or Sitagliptin (100 μΜ, black bars) during adipogenic differentiation (n=6 from two independent experiments). Results are shown as mean ± SEM.

Figure 44A shows Alizarin Red S staining and quantification of staining in multipotent CD31 CD45 Scal⁺CD24⁺ and OPCs (CD3 rCD45 Scal Pa⁺) either treated with PBS (control) or recombinant mouse rDPP4 (250 ng/ml) during osteogenic differentiation (n=3). Mean ± SEM; *p<0.05.

Figure 44B shows mRNA expression (as percentage of control) of Osx and Runx2 in multipotent CD31^"CD45^"Scal⁺CD24⁺ and OPCs (CD31^"CD45^"Scal Pa⁺) either treated with PBS (control; white bars) or recombinant mouse rDPP4 (250 ng/ml; blue bars) during osteogenic differentiation (n=3). Results are shown as mean ± SEM.

Figure 44C shows Oil Red-0 staining and mRNA expression levels of Pparg and Lep in multipotent CD31 CD45 Scal⁺CD24⁺ and APCs (CD3 rCD45 Scal⁺CD24 ) cells either treated with PBS (control; white bars) or rDPP4 (250 ng/ml; blue bars) during adipogenic differentiation (n=3). Results are shown as mean ± SEM.

Figure 45A shows Histomorphometric Movat Pentachrome stains (top panels) analysis and corresponding μCT images of the fracture callus (bottom panels) of mice either treated with PBS (control), Diprotin A, or Sitagliptin for 9 days (n=6-7).

Figure 45B shows quantification of mineralized (BV/TV) and fibrotic areas (FV/TV) of mice either treated with PBS (control), Diprotin A, or Sitagliptin for 9 days (n=6-7) (right panel). Results are shown as mean ± SEM (*p<0.05; **p<0.01).

Figure 46 shows FACS analysis of OPC, CD45 CD31 Scal⁺CD24⁺ and APC cell frequencies in non-fractured tibiae from male mice either i.p. -injected with PBS (white bars) or Sitagliptin for 9 days (black bars; n=9-10). Mean ± SEM; *p<0.05, **p<0.01, ****p<0.0001.

Figure 47A shows representative Movat Pentachrome stains of fracture calluses from control PBS-treated mice that received osteogenic (PBS/OPC) or adipogenic (PBS/APC) intratibial transplants and animals treated with Sitaglitpin for 1 week after fracture and receiving the same transplants of osteogenic (Sita/OPC) or adipogenic (Sita/APC) cells.

Figure 47B shows quantification from histomorphometric analysis of fracture calluses from control PBS-treated mice that received osteogenic (PBS/OPC) or adipogenic (PBS/APC) intratibial transplants and animals treated with Sitaglitpin for 1 week after fracture and receiving the same transplants of osteogenic (Sita/OPC) or adipogenic (Sita/APC) cells. Fracture callus total volumes (TV) were analyzed for mineralized callus volume (BV/TV) and fibrous tissue volumes (FV/TV) (n=5-7). p<0.05: a vs. PBS /OPC; b vs. PBS /APC; c vs. Sitagliptin /OPC; d vs. Sitagliptin /APC, Mean ± SEM.

Figure 48 shows FACS analysis of bone-resident MSCs, APCs, and OPCs from animals treated with Sitagliptin (Sita) or control (Ctrl) for 3 days at a dose of 10 mg/kg body weight, either by intraperitoneal injections (i.p.) or by oral gavage (per oral - p.o.). Results are shown as mean ± SEM.

DETAILED DESCRIPTION OF THE DISCLOSURE

The disclosure provides dipeptidyl peptidase-4 (DPP-4) inhibitor agents (which may be referred to herein simply as "agents" or "DPP-4 inhibitors" or "gliptins") for use in any of the methods or compositions described herein. In certain embodiments, the agents are for use in treating a subject having a bone fracture. In some embodiments, the agent is a DPP-4 inhibitor. In some embodiments, the agent is for use in promoting fracture healing in a subject in need thereof (e.g., a subject having a fracture; a subject diagnosed with a fracture). In some embodiments, the agent is for use in decreasing or preventing complications following fracture, such as non-union. In some embodiments, the agent is for use in reducing inhibitory effects of marrow adipose tissue (MAT) on fracture healing in a subject in need thereof. In some embodiments, the agent is for use in preventing loss of bone density. In some embodiments, the disclosure provides methods of preparing cells for transplantation into a subject, wherein the cells have been treated with the agent in an amount effective to increase osteogenic gene expression and differentiation, e.g. osteogenic (or chondrogenic) bone cell formation. Such cells are suitable for transplantation into a subject having a bone fracture, such as to promote fracture healing or to prevent or decrease complications from bone fracture. Other uses and methods are described in further detail herein. Also contemplated are compositions comprising a DPP-4 inhibitor, and any such compositions may be used in any of the methods described herein. Also contemplated are formulations of a DPP-4 inhibitor to be applied systemically or topically/locally in or onto the fracture and any such compositions, such as to promote fracture healing or to prevent or decrease complications from bone fracture.

A. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods and examples are illustrative only, and are not intended to be limiting. All publications, patents and other documents mentioned herein are incorporated by reference in their entirety.

As used herein, the singular forms "a," "an," and "the" include the plural referents unless the context clearly indicates otherwise.

By the terms "has the ability" or "is capable of is meant that the recited agent, proteins or polypeptides will carry out the stated bioactivity under suitable conditions (e.g., physiological conditions or standard laboratory conditions). In some embodiments, the term "can" may be used to describe this ability (e.g., "can bind" or "binds" to a given sequence).

The terms "inhibit", "inhibitor", "antagonist" and "antagonize," when used to refer to any of the agents disclosed herein, mean that the agent is capable of blocking, reducing, attenuating and/or reversing activation of the protein targeted by the agent (e.g., DPP-4). For example, in particular embodiments, a DPP-4 inhibitor or antagonist is an agent that is capable of blocking, reducing, attenuating and or reversing DPP-4 serine exopeptidase activity.

B. DPP-4 Inhibitors

In some embodiments, the agent for use in any of the methods disclosed herein is a DPP-

4 inhibitor. In some embodiments, the DPP-4 inhibitor is a small organic molecule. In some embodiments, the DPP-4 inhibitor is a polypeptide or peptide. In some embodiments, the DPP- 4 inhibitor is an antibody (e.g., an antibody that binds to and inhibits the activity of DPP-4). In some embodiments, the DPP-4 inhibitor is a polynucleotide.

In some embodiments, the agent is isolated and/or purified. Any of the agents described herein, including those provided in an isolated or purified form, may be provided as a composition, such as a composition comprising an agent formulated with one or more pharmaceutical and/or physiological acceptable carriers and/or excipients. Any of the agents described herein, including compositions (e.g., pharmaceutical compositions) may be used in any of the methods described herein. Examples of particular pharmaceutical compositions formulated for preferred routes of delivery are provided herein.

In some embodiments, the agents inhibit a biological activity of DPP-4. In some embodiments, the agent binds to DPP-4 and inhibits a biological activity of DPP-4. In some embodiments, the agent binds to another protein or agent and indirectly inhibits a biological activity of DPP-4. In some embodiments, the agent binds to a substrate of DPP-4 and prevents DPP-4 from interacting with that substrate. In some embodiments, the agent is an antibody or antigen-binding fragment that binds to DPP-4 or a DPP-4 substrate in a manner that prevents DPP-4 or the substrate from interacting with each other. In some embodiments, the agent is capable of binding to a DPP-4 protein, or fragment thereof, having an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 97% or 100% identical to the amino acid sequence of SEQ ID NO: 1 or 3, or a fragment thereof. In some embodiments, the agent is capable of binding to the caveolin-1 binding domain, the fibronectin binding domain and/or the ADA binding domain of DPP-4. In some embodiments, the agent is capable of binding to the amino acid residues corresponding to residues 201-211 and/or 603 of SEQ ID NO: 1. In some embodiments, the agent is capable of binding to residues GWSYG of SEQ ID NO: 1. In some embodiments, the agent is capable of binding to residues corresponding to Ser630, Asp708 and/or His740 of SEQ ID NO: 1. In some embodiments, the agent inhibits or prevents homodimerization of DPP-4. In some embodiments, the agent inhibits secretion of DPP-4, such as from cells expressing DPP-4 on their surface in or near a fracture site.

In some embodiments, the agent inhibits the expression of DPP-4. In some embodiments, the agent inhibits transcription of the DPP4 gene. In some embodiments, the agent inhibits translation of the DPP4 mRNA transcript. In some embodiments, the agent is capable of binding to a polynucleotide having a nucleotide sequence that is at least 80%>, 85%, 90%, 92%, 95%, 97% or 100% identical to the nucleotide sequence of SEQ ID NO: 2 or 4, or a portion or complement thereof. In some embodiments, the agent is an antisense molecule, an RNAi molecule, an siRNA or a CRISPR-based therapeutic agent (e.g., a CRISPR/Cas9 complex) that inhibits DPP4 expression. In some embodiments, the agent inhibits the expression or activity of DPP-4 in a cell by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to DPP-4 expression or activity in the same or substantially the same cell in the same or substantially the same conditions in the absence of the agent.

By the terms "biological activity", "bioactivity", "bioactive" or "functional", when used in the context of DPP-4, is meant the ability of the DPP-4 polypeptide to carry out one or more functions associated with wildtype DPP-4 polypeptides {e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 1), for example, serine exopeptidase activity. The terms "biological activity", "bioactivity", and "functional" are used interchangeably herein. In some embodiments, any of the DPP-4 inhibitors disclosed herein is capable of inhibiting activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the DPP-4 activity as compared to DPP-4 activity under the same or substantially the same physiological conditions in the absence of the agent. In some embodiments, the DPP-4 inhibitor preserves the action of DPP-4 substrate molecules, e.g., glucagon- like peptide- 1, gastric inhibitory polypeptide, peptide histidine methionine, substance P, neuropeptide Y, CXCL12, and other molecules typically containing alanine or proline residues in the second aminoterminal position. In some embodiments, treatment with DPP-4 inhibitors prolongs the duration of action of DPP-4 peptide substrates and increases levels of their intact, undegraded forms.

In some embodiments, any of the agents disclosed herein decreases the half-life (tm) of a DPP-4 polypeptide. In some embodiments, the half-life of the DPP-4 polypeptide is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to the half-life of the DPP-4 polypeptide in the absence of the agent. In some embodiments, the protein half- life is determined in vitro, such as in a buffered saline solution or in serum. In other embodiments, the protein half-life is an in vivo half life, such as the half-life of the DPP-4 in the serum or other bodily fluid of an animal.

In the present context "a DPP-4 inhibitor" is also intended to comprise active metabolites and prodrugs thereof, such as active metabolites and prodrugs of DPP-4 inhibitors. A "metabolite" is an active derivative of a DPP-4 inhibitor produced when the DPP-4 inhibitor is metabolized. A "prodrug" is a compound that is either metabolized to a DPP-4 inhibitor or is metabolized to the same metabolite(s) as a DPP-4 inhibitor. In the present context the term "a DPP-4 inhibitor" is also intended to comprise pharmaceutical salts thereof.

Representative DPP-4 inhibitors are known in the art. For example, representative DPP- 4 inhibitors are disclosed in WO 98/19998, DE19616 486 Al, WO 00/34241, WO 95/15309, WO 01/72290, WO01/52825, WO03/002553, WO 9310127, WO 99/61431, WO 9925719, WO 9938501, WO 9946272, WO 9967278, WO 9967279, WO 02053548, WO 02067918, WO 02066627, WO 02/068420, W0 02083128, WO 2004/037181, WO 0168603, EP1258480, WO 0181337, WO 02083109, WO 030003250, WO 03035067, WO 03/035057, US2003216450, WO 99/46272, WO 0197808, WO 03002553, WO 01/34594, WO 02051836, EP1245568, EP1258476, US 2003087950, WO 02/076450, WO 03000180, WO 03000181, WO 03004498, WO 0302942, U.S. Pat. No. 6,482,844, WO 0155105, WO 0202560, WO 03004496, WO 03/024965, WO 0303727, WO 0368757, WO 03074500, WO 02038541, WO 02062764, WO 02308090, US 2003225102, WO 0214271, US 2003096857, WO99/38501, W099/46272, U.S. Pat. Nos. 6,124,305; 6,107,317, WO 9819998, WO 9515309 and WO 9818763. Each of the foregoing, as well as each of the DPP-4 inhibitors disclosed therein, is incorporated herein in its entirety.

In some embodiments, the agent (e.g., the DPP-4 inhibitor) is or comprises alogliptin, sitagliptin, vildagliptin, saxagliptin, gemigliptin, anagliptin, teneligliptin, trelagliptin, omarigliptin, evogliptin, dutogliptin and/or linagliptin or derivatives or pharmaceutically acceptable salts thereof. In some embodiments, the agent is or comprises diprotin A, berberine and/or lupeol, or a derivative or pharmaceutically acceptable salt thereof. In some embodiments, the agent is or comprises any natural plant extract or bioactive compounds which inhibit DPP-4 activity, or a derivative or pharmaceutically acceptable salt thereof. Such agents may be provided as pharmaceutical compositions and, as noted above, as prodrugs. In some embodiments, the agent is combined (e.g., in the same or different formulation) with metformin. In other words, in certain embodiments, the methods of the present disclosure include administering a DPP-4 inhibitor and metformin, either as a co-formulation or in separate formulations. When administered as separate formulations, the two may be administered at the same or different times and via the same or different routes of administration. In other embodiments, metformin is not used (e.g., the methods of the disclosure do not include administering metformin).

In some aspects, the present disclosure contemplates any number of combinations of the foregoing agents. For example, any of the methods disclosed herein may include treatment with a single DPP-4 inhibitor or with more than one DPP-4 inhibitor, such as two DPP-4 inhibitors that act via different mechanisms of action. When more than one DPP-4 inhibitor is used, the two agents may be administered at the same or different times and via the same or different routes of administration. Moreover, in certain embodiments, the methods further include metformin.

In certain aspects, any of the agents disclosed herein is conjugated to a heterologous agent. In some embodiments, the heterologous agents include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, and an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), which are particularly useful for isolation of the agents by affinity chromatography. In some embodiments, the agent is conjugated to a detectable moiety. In some embodiments, the moiety is a fluorescently labeled or radiolabeled detectable moiety. C. Methods of Administration

Various delivery systems are known and can be used to administer any of the agents of the disclosure, such as any of the DPP-4 inhibitors of the disclosure, e.g., various formulations, encapsulation in liposomes, nanoparticles, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction can be enteral or parenteral, including but not limited to, intraosseous, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary, intranasal, intrathecal, intraocular, epidural, and oral routes. In particular embodiments, parenteral introduction includes intramuscular, subcutaneous, intravenous, intravascular, and intrapericardial administration. In some embodiments, the agents are administered locally to the subject. In some embodiments, the agents are administered systemically to the subject. In some embodiments, the agents are administered directly to a bone fracture site, e.g., during a surgical procedure or when setting a compound fracture. In some embodiments, the agents are administered as a topical formulation (e.g., as a gel formulation) to the site of the bone fracture. In some embodiments, routes of administration are combined. For example, a DPP4-inhibitor can be administered locally, such as during a surgical procedure to set a bone. Subsequent doses of agent over subsequent days, such as over 1-4 weeks, may be by systemic administration (e.g., oral, intravenous, subcutaneous, or intraperitoneal). Alternatively, the same route of administration may be used throughout a multi-dose regimen, such as systemic administration over 1-4 weeks.

In some embodiments, the agents are administered by means of a device implanted within the subject. In some embodiments, the implantable device is coated by a composite surface coating comprising any of the DPP-4 inhibitor agents disclosed herein. In some embodiments, the implantable device delivers drug to the site of bone fracture. In some embodiments, the implantable device releases DPP-4 inhibitor in a controlled fashion (e.g., a controlled release device).

In certain embodiments, the agents of the disclosure are administered in one or more doses over a period of less than 1 year, less than or equal to 9 months, less than or equal to 6 months, less than or equal to 3 months, less than or equal to 1 month, less than or equal to 3 weeks, less than or equal to 2 weeks, or less than or equal to 1 week. In some embodiments, regardless of the total treatment period (e.g., 1, 3, 6 months, etc.) the subject is administered the agent on a dosing schedule. For example, the schedule may be daily, every other day, twice weekly, weekly, twice monthly or monthly. Regardless of the infusion period, the disclosure contemplates that each infusion is part of an overall treatment plan where a composition of the disclosure is administered according to a regular schedule (e.g., weekly, monthly, etc.).

In some embodiments, the agents of the disclosure are prepared in a formulation/composition appropriate for a specific route of administration. The composition and route of administration is chosen depending on the particular use of the technology. For example, a different composition and/or route of administration may be appropriate when using the compositions of the disclosure for research purposes, such as in vitro or in an animal model, versus when using for diagnostic or therapeutic purposes in human patients. One of skill in the art can select the appropriate route of administration depending on the particular application of the technology.

The amount of the compositions of the disclosure for use in the methods of the present disclosure can be determined by standard clinical techniques and may vary depending on the particular indication or use. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Dosages may be determined by techniques known to those of skill in the art or as taught herein. Toxicity and therapeutic efficacy of any of the agents disclosed herein may be determined by standard pharmaceutical procedures in experimental animals.

D. Pharmaceutical Compositions

In certain embodiments, agents of the disclosure (any one or more of which is referred to as "compositions of the disclosure") are formulated with a pharmaceutically acceptable carrier. For example, the disclosure provides a composition comprising an agent of the disclosure formulated with one or more pharmaceutically acceptable carriers and/or excipients. Such pharmaceutical compositions include, where applicable, pharmaceutically acceptable salts of a DPP-4 inhibitor.

In some embodiments, the disclosure provides for a pharmaceutical composition for use in treating a bone fracture in a subject in need thereof, comprising a DPP-4 inhibitor in admixture with a pharmaceutically-acceptable diluent, excipient, or carrier, wherein the DPP-4 inhibitor is present in an effective amount to treat or prevent bone fractures. In some embodiments, the disclosure provides for a pharmaceutical composition for use in preventing non-union of a bone fracture or for use in preventing healing complications following bone fracture in a subject in need thereof, comprising a DPP-4 inhibitor in admixture with a pharmaceutically-acceptable diluent, excipient, or carrier, wherein the DPP-4 inhibitor is present in an effective amount to treat or prevent bone fractures. Any of the compositions of the disclosure can be administered alone or as a component of a pharmaceutical formulation (composition). The compositions of the disclosure may be formulated for administration in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Formulations of the compositions of the disclosure include those suitable for oral, nasal, topical, parenteral, rectal, and/or intravaginal administration. In some embodiments, the disclosure provides for a composition for administration directly to a bone fracture, e.g., by a liquid formulation applied directly to the site of bone fracture {e.g., by a spray). In some embodiments, the disclosure provides for a gel-based formulation for direct or topical administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

In certain embodiments, methods of preparing these formulations or compositions include combining the therapeutic agent and a carrier and, optionally, one or more accessory ingredients. In general, the formulations can be prepared with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Pharmaceutical compositions suitable for parenteral administration may comprise one or more compositions of the disclosure in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers {e.g., HEPES buffer), bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

In certain embodiments of the present disclosure, the compositions of the disclosure are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

In certain embodiments, compositions of the disclosure, including pharmaceutical preparations, are non-pyrogenic. In other words, in certain embodiments, the compositions are substantially pyrogen free. In one embodiment the formulations of the disclosure are pyrogen- free formulations which are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined inside a microorganism and are released only when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, even low amounts of endotoxins must be removed from intravenously administered pharmaceutical drug solutions. The Food & Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one hour period for intravenous drug applications (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins are administered in relatively large dosages and/or over an extended period of time (e.g., such as for the patient's entire life), even small amounts of harmful and dangerous endotoxin could be dangerous. In certain specific embodiments, the endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.

The foregoing applies to any of the compositions, methods and uses described herein. The disclosure specifically contemplates any combination of the features of compositions of the present disclosure (alone or in combination) with the features described for the various pharmaceutical compositions and route of administration described in this section.

E. Therapeutic Uses

For any of the methods and uses described herein, the disclosure contemplates the use of any of the compositions of the disclosure (whether alone or in combination with any of the additional therapeutic treatments disclosed herein). Compositions of the disclosure may be described based on any combination of structural and/or functional features provided herein. In addition, for any of the methods and uses described herein, the disclosure contemplates the combination of any step or steps of one method or use with any step or steps from another method or use. These methods and uses involve administering to an individual in need thereof an effective amount of a compound of the disclosure (including as a pharmaceutical composition) appropriate for the particular disease or condition (e.g., a bone fracture). In specific embodiments, these methods and uses involve delivering any of the agents disclosed herein to the cells of a subject in need thereof.

In certain embodiments, the disclosure provides uses in treating a bone fracture in a subject in need thereof. In some embodiments, the disclosure provides a use in preventing nonunion of a bone fracture in a subject in need thereof. In some embodiments, the disclosure provides a use in preventing healing complications following bone fracture in a subject in need thereof. In some embodiments, the disclosure provides a use in promoting fracture healing in a subject in need thereof. In some embodiments, the disclosure provides a use in reducing the inhibitory effects of marrow adipose tissue (MAT) on fracture healing in a subject in need thereof. In some embodiments, the disclosure provides a use in preventing loss of bone mineral density (BMD) in a subject (e.g. , an astronaut) exposed to an altered gravity environment. In some embodiments, any of the methods and uses disclosed herein comprises administering to the subject an effective amount of any of the DPP-4 inhibitors disclosed herein. In some embodiments, any of the methods and uses disclosed herein comprises administering an effective amount of any of the agents disclosed herein (alone or in combination with any of the additional therapeutic treatments disclosed herein), to a subject in need thereof according to a dosing regimen (e.g., a dose and dosing schedule) and/or dosing schedule. For example, a method or use may comprise administering a DPP-4 inhibitor to a subject in need thereof, such as a subject having a fracture, in one or more doses over a period of time (e.g., a single administration or multiple administrations over a period of less than or equal to 6, 3, 2, 1 month).

The terms "treatment", "treating", "alleviation" and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect, and may also be used to refer to improving, alleviating, and/or decreasing the severity of one or more symptoms of a condition being treated. The effect may be prophylactic in terms of completely or partially delaying the onset or recurrence of a disease, condition, or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for a disease or condition and/or adverse effect attributable to the disease or condition. "Treatment" as used herein covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing complications from a fracture or other bone related disease or condition, such as non-union; (b) preventing further complications of an existing disease or condition (e.g. preventing formation of non-union fractures in osteoporosis patients or preventing healing complications in osteoporosis patients having a fracture); (c) inhibiting the disease or condition (e.g., arresting its development or further progression); (d) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms); promoting healing of a tissue (e.g. , bone) that was damaged or compromised as a result of the disease or condition (e.g. , a bone fracture) ; or (e) acceleration of the healing process or decrease of processes that inhibit the healing process .

In certain embodiments of any of the foregoing, the disclosure provides a use in treating a bone-related disease (e.g. , osteoporosis) or bone-related condition (e.g., a bone fracture) or preventing or decreasing complications associated with a bone fracture with any of the agents described herein (alone or in combination with any of the additional therapeutic treatments disclosed herein). In some embodiments, the subject is a human.

In certain embodiments of any of the foregoing, the disclosure provides a use in treating a therapeutically induced bone loss or bone-related disease such as those induced by medication (e.g. corticosteroid-induced bone loss) or a medical procedure (e.g. patients who have undergone a hysterectomy) or preventing or decreasing complications associated with a bone fracture with any of the agents described herein (alone or in combination with any of the additional therapeutic treatments disclosed herein). In some embodiments, the subject is human. Treating a bone-related disease (e.g., osteoporosis) or bone-related condition (e.g. , a bone fracture) in a subject refers to improving (e.g. , healing a bone fracture), alleviating, delaying or slowing progression or onset, decreasing the severity of one or more symptoms associated with the disease or condition. For example, treating a bone -related disease (e.g., osteoporosis) or bone-related condition (e.g. , a bone fracture) or secondary complications arising after bone injury (e.g., a non-union) includes any one or more of: improved or accelerated healing of a bone fracture, reduction in pain associated with a bone fracture (e.g., back pain), preventing height loss, improving posture, reducing frequency of bone fractures, or increasing bone mineral density (BMD) to prevent fractures and to decrease general proneness to bone fractures in subjects (for instance in patients with metabolic diseases such as diabetes and obesity, or in subjects affected by osteoporosis, or in subjects of advanced age alone, or subjects primarily affected by a combination of these disorders and advanced age).

In some embodiments, the effects of administration of any of the agents disclosed herein (whether alone or in combination with any of the additional therapies described herein) may be determined by assessing the treated subject before and after treatment, and determining whether the treatment has any effect on the bone-related disease (e.g., osteoporosis) or bone-related condition (e.g., a bone fracture). The above parameters for assessing successful treatment and improvement in the condtion are readily measurable by routine procedures familiar to a physician. Efficacy can be measured, for example, by: the ability of a bone to heal from a fracture; a reduction in pain experienced by the subject due to the bone fracture; assessing the time for a bone to heal from a fracture or the time for pain associated with a bone fracture to be alleviated; an improvement in posture or height loss. This can be assessed by means of instruments such as X-rays and/or bone scans, or by asking the patient about their degree of pain associated with the site of injury.

A "therapeutically effective amount" or "effective amount" of a composition is a predetermined amount calculated to achieve the desired result (e.g. , effective in promoting bone fracture healing). The activity contemplated by the present methods and uses includes both medical therapeutic and/or prophylactic treatment, as appropriate. The specific dose of an agent administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the agent administered, the route of administration, and the condition being treated. A therapeutically effective amount of agent of this invention is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient. Therapeutically effective amounts may be administered according to a dosing schedule such that the amount of each dose is effective such that, in the aggregate, the combined doses achieve an end result. Each dose is still considered effective even if it adding to an overall therapeutic effect.

A "patient," "subject" or "individual" are used interchangeably and refer to either a human or non-human animal. The term includes mammals such as humans.

"Mammal" for purposes of the treatment of, alleviating the symptoms of or diagnosis of a bone-related disease (e.g. , osteoporosis) or bone-related condition (e.g. , a bone fracture) refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, ferrets, etc. In some embodiments, the mammal is human. In some embodiments, the mammal is postnatal. In some embodiments, the mammal is pediatric. In some embodiments, the subject is at a life stage that is associated with a greater risk of developing a bone-related condition such as osteoporosis. In some embodiments, the mammal is adult. In some embodiments, the subject is a human that is at least 50 years in age, at least 60 years in age, at least 65 years in age, at least 70 years in age, at least 80 years of age, at least 90 years of age, at least 100 years of age, at least 1 10 years of age or at least 120 years of age. In other embodiments, the subject is a human subject under 40 years of age, under 35 years of age, under 30 years of age, or under 20 years of age. In some embodiments, the subject is a human subject that is still growing (e.g., whose growth plates have not yet fused).

In some embodiments, the subject treated with any of the DPP-4 inhibitors disclosed herein is not a diabetic patient. In some embodiments, the subject in need thereof has Type I or Type II diabetes. In some embodiments, the subject was not, prior to the bone fracture, diagosed with or treated for Type II diabetes. In some embodiments, the subject was not receiving a DPP-4 inhibitor prior to the diagnosis with a bone fracture. In some embodiments, the subject has not previously received a treatment for diabetes. In some embodiments, the subject has been administered metformin. In some embodiments, the subject has not been and/or is not being administered metformin. In some embodiments, the subject has a body mass index (BMI) less than 50, less than 45, less than 40, less than 39, less than 38, less than 37, less than 36, less than 35, less than 34, less than 33, less than 32, less than 31 , less than 30, less than 29, less than 28, less than 27, less than 26 or less than 25. In some embodiments, the subject has a BMI of greater than or equal to 25 and less than 30. In some embodiments, the subject has a BMI of equal to or greater than 30. In some embodiments, the subject has a BMI of 18.5- 24. In some embodiments, the subject has a BMI of less than 18.5. In some embodiments, the subject to be treated with any of the DPP-4 inhibitors disclosed herein is a subject having increased marrow adipose tissue (MAT) as compared to a healthy control subject.

In some embodiments, the subject in need thereof has, at the time of treatment of or immediately prior to the fracture, an Ale (hemoglobin Ale) level below 5.7%. In some embodiments, the subject in need thereof has, at the time of treatment of or immediately prior to the fracture, an Ale level of 5.7-6.4%. In some embodiments, the subject in need thereof has, at the time of treatment of or immediately prior to the fracture, an Ale level greater than 6.5%.

In some embodiments, the subject treated with any of the DPP-4 inhibitors disclosed herein is a subject to be exposed to, currently exposed to, or previously exposed to altered gravity environment. In some embodiments, the subject is exposed to an altered gravity environment for at least 3 days, one week, two weeks, one month, two months, three months, four months, five months, six months, nine months, and/or one year. In some embodiments, the subject is administered the DPP-4 inhibitor prior to and/or during the exposure. In some embodiments, the subject is an astronaut.

In some embodiments, the agents of the present disclosure may be used to treat a bone- related disease. In some embodiments, the bone-related disease is selected from the group consisting of osteoporosis, osteogenesis imperfect, primary bone cancer, cancer that has metastasized to bone, rickets, osteomalacia, renal osteodystrophy, and/or Paget's Disease. In some embodiments, the osteoporosis is primary osteoporosis. In some embodiments, the primary osteoporosis is idiopathic primary osteoporosis or age-related osteoporosis. In some embodiments, the osteoporosis is secondary osteoporosis. In some embodiments, the osteoporosis is caused by idiopathic hyper-calcinuria, cystic fibrosis, glucocorticoid treatment, cyclosporine A treatment, and/or tacromilus treatment. In some embodiments, the bone related disease occurs in post-meopausal women, patients who have undergone hysterectomy, patients who are undergoing or have undergone long-term administration of corticosteroids, patients suffereing from Cushing's syndrome, patients who display one or more symptoms of the frailty syndrome, patients with cachexia, patients with conditions associated with wasting of body tissues, or patients who have gonadal dysgenesis. In some embodiments, the disclosure provides any of the foregoing methods or uses comprising administering any of the agents of the disclosure. In some embodiments, the subject is suffering from more than one of the diseases disclosed herein. In some embodiments, the subject is suffering from a bone -related condition, such as a fracture of a bone. In some embodiments, the subject is suffering from a fracture in more than one bone. In some embodiments, the bone is a flat, long, short, irregular or sesamoid bone. In some embodiments, the bone is a long bone (e.g., a femur, humerus, tibia, metacarpal, metatarsal and/or phalange). In some embodiments, the bone is a short bone (e.g., a carpal or tarsal). In some embodiments, the bone is a flat bone (e.g., a scapula, sternum, cranium, os coxae, pelvis and/or rib). In some embodiments, the bone is an irregular bone (e.g., vertebrae, sacrum and/or mandible). In some embodiments, the bone is a sesamoid bone (e.g., knee cap and/or pisiform). In some embodiments, the fracture is a compound fracture.

In some embodiments, the disclosure provides for a use in treating a bone fracture. In some embodiments, the fracture is a non-union fracture, a compound fracture, a fracture with delayed healing, a stable fracture, a displaced fracture, a non-displaced fracture, an open fracture, a closed fracture, a Greenstick fracture, a complete fracture, an incomplete fracture, a transverse fracture, an oblique fracture, a comminuted fracture, a buckled fracture, a pathologic fracture, and/or a stress fracture. In preferred embodiments, the bone fracture is a non-union bone fracture, a compound fracture or a fracture with delayed healing. As used herein, the term "non-union bone fracture" is meant to relate to a permanent failure of healing following a broken bone unless intervention (such as surgery) is performed. A "fracture with delayed healing" is meant to relate to a failure to reach bony union by 6 months post-injury. This also includes fractures that are taking longer than expected to heal (ie. distal radial fractures). In some embodiments, the fracture is the result of a disease or condition such as osteoporosis. In some embodiments, the fracture is the result of a trauma to the fractured bone. In some embodiments, the fracture is the result of overuse of the bone (e.g. , as the result of repetitive motion, such as in an athlete).

In some embodiments, the disclosure provides for a use in inducing differentiation of a multipotent stem cell or a progenitor cell into a cell of the bone lineage (e.g., into an osteoblast or an osteocyte) in a subject in need thereof. In some embodiments, the disclosure provides for a use in inducing differentiation of a multipotent stem cell or a progenitor cell into a cell of the cartilage lineage (e.g., into a chondrocyte) in a subject in need thereof. In some embodiments, the multipotent stem cell or its progeny, the adipogenic progenitor cell (APC) is a cell that expresses high levels of genes associated with adipocytic lineage (e.g., CD34, EBF2, VIM, PPARA and/or DPP4). In some embodiments, the multipotent stem cell or its progeny, the osteochondrogenic progenitor cell (OPC) is a cell that expresses high levels of genes associated with osteogenic lineage (e.g., AlpI, Dmpl, Collal/2) or is a cell that expresses high levels of genes associated with chondrogenic lineage (e.g., Acan, Col2al, Sox9). In some embodiments, the multipotent stem cell expresses elevated levels of GREM1 or other markers such as Cxcll2, Kitl/Scf, Vcam-1, Lepr. In some embodiments, the multipotent stem cell is a CD45 CD31^" Scal⁺CD24⁺ multipotent stem cell. In some embodiments, the adipogenic progenitor cell (APC) is a CD45^~CD31^~Scal⁺CD24^~ cell. In some embodiments, the osteogenic progenitor cell (OPC) is a CD45^~CD31^~Scal^~Pa⁺ cell. In some embodiments, treatment of the multipotent stem cell with any of the DPP-4 inhibitors disclosed herein enhances osteogenic gene expression. In some embodiments, treatment of the multipotent stem cell with any of the DPP-4 inhibitors disclosed herein enhances chondrogenic gene expression. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

In some embodiments, the disclosure provides for a method of preparing cells for transplantation into a subject having a bone-related disease {e.g., osteoporosis) or bone-related condition {e.g., a bone fracture). In some embodiments, the method comprises providing a cell culture comprising osteogenic progenitor cells and/or mesenchymal stem cells, contacting the cells with any of the DPP-4 inhibitors disclosed herein in an amount effective to increase osteogenic or chondrogenic gene expression thereby generating a culture comprising DPP-4 treated cells. In an embodiment, the thus generated DPP-4 treated cells from the culture are transplanted to a fracture in a subject in need thereof. In some embodiments, the cells for transplantation are multipotent stem cells. In some embodiments, the multipotent stem cell is a cell that expresses high levels of genes associated with adipocytic lineage {e.g., CD34, EBF2, VIM, PPARA and/or DPP 4). In some embodiments, the multipotent stem cell is a CD45-CD31 - Scal+CD24+ multipotent stem cell. In some embodiments, the multipotent stem cell or its progeny, the osteochondrogenic progenitor cell (OPC) is a cell that expresses high levels of genes associated with osteogenic lineage {e.g., AlpI, Dmpl, Collal/2) or is a cell that expresses high levels of genes associated with chondrogenic lineage {e.g., Acan, Col2al, Sox9). In some embodiements, the OPC is a cell whose surface marker configuration is CD45^"CD31^"Scal^"Pa⁺. In some embodiments, treatment of the multipotent stem cell with any of the DPP-4 inhibitors disclosed herein enhances osteogenic gene expression and/or osteogenic lineage commitment of the initially multipotent cell.

In some embodiments, any of the agents described herein may be administered in combination with any of the additional therapeutic treatments described herein. In some embodiments, the additional therapeutic treatment is reduction {e.g. , closed reduction) and or the use of medical devices (e.g., casts, pins, plates, screws, rods or glue) to hold the fracture in place. In some embodiments, the additional therapeutic treatments may include treatment with one or more compounds selected from the group consisting of: an anti-infective agent, a pain and/or inflammation reliever (e.g., acetominophen, ibuprofen), a growth factor (e.g., bone morphogenic proteins (BMPs), TGF-beta, insulin-like growth factor (IGF), fibroblast growth factor (FGF), FGF-2, platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF)), a hormone (e.g., parathyroid hormone (PTH) or growth hormone (GH)) and a soluble receptor (e.g., an ActRIIA receptor). In some embodiments, the BMP is selected from the group consisting of: OP-1 , OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP- 10, BMP-1 1 , BMP- 15, BMP- 16, DPP, Vgl , Vgr-1 , 60A protein, GDF-1 , GDF- 2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-1 1 , GDF-12, NODAL, UNIVIN, SCREW, ADMP, NEURAL, and amino acid sequence variants thereof. In some embodiments, the additional therapeutic treatment is a compound for treating osteoporosis, such as a bisphosphonate (e.g., Alendronate (Fosamax), Risedronate (Actonel), Ibandronate (Boniva) and Zoledronic acid (Reclast)), a hormone (e.g., raloxifene (Evista)), a RANKL inhibitor (e.g., Denosumab (Prolia)) and/or a synthetic hormone (e.g., a synthetic parathyroid hormone such as Teriparatide (Forteo)).

Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. In some embodiments, any of the agents described herein are administered to a subject in combination with an additional therapeutic treatment, wherein the subject has a bone-related disease (e.g., osteoporosis) or bone-related condition (e.g., a bone fracture). In some embodiments, the additional therapeutic treatment is a physical therapy, massage therapy, electrical and electromagnetic stimulation, ultrasound, extracorporeal shock waves and/or rest.

In some embodiments, the combined administration includes coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. In some embodiments, the additional therapeutic treatment and the agent are administered consecutively. In some embodiments, the additional therapeutic treatment is administered concurrently with the agent. In some embodiments, the additional therapeutic treatment is administered prior to the administration of the agent. In some embodiments, combined therapy results in a cumulative therapeutic effect. In some embodiments, combined therapy results in a synergistic therapeutic effect. In some embodiments, the therapeutically effective amount of each of: a) the DPP-4 inhibitor, and/or b) the additional therapeutic treatment is less than that required to achieve a therapeutic effect when one or both agents is administered as a monotherapy.

F. Other Uses

The compositions of the disclosure have numerous uses. For example, the DPP-4 inhibitor agents disclosed herein are useful for studying effects on different cells and tissues in vitro and/or in vivo. Similarly, the agents are useful as imaging agents, such as for ex vivo or in vivo diagnostic applications. For example, agents conjugated to a radioactive moiety are useful for ex vivo or in vivo imaging studies.

In some embodiments, the disclosure provides for a method of inducing differentiation of a multipotent stem cell into a cell of the bone lineage (e.g., into an osteoblast). In some embodiments, the multipotent stem cell is a cell that expresses high levels of genes associated with adipocytic lineage (e.g. , CD34, EBF2, VIM, PPARA and/or DPP4). In some embodiments, the multipotent stem cell is a CD45-CD31-Scal+CD24+ multipotent stem cell. In some embodiments, treatment of the multipotent stem cell with any of the DPP-4 inhibitors disclosed herein enhances osteogenic gene expression and/or osteogenic cell differentiation and/or osteogenic lineage commitment of the multipotent cell. In some embodiments, treatment of the multipotent stem cell with any of the DPP-4 inhibitors disclosed herein enhances chondrogenic gene expression and chondrogenic cell differentiation. In some embodiments, the multipotent cell is in vitro. In some embodiments, the multipotent cell is in a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

When used in vitro or in vivo, the agents of the disclosure are suitable for identifying binding partners and/or tissue distribution for the agents delivered and for evaluating localization and trafficking. Similarly, effects of the agent on gene expression or DPP-4 half- life may be studied in vivo or in vitro.

G. Model Systems

Numerous animal models of bone-related diseases or conditions are known in the art. Suitable animals for studying the effects of any the DPP-4 inhibitors disclosed herein include mice, rats, dogs, and primates. Representative animal models are described in Histing et al, 2011, Bone, 49(4):591-9. An example of a model is also discussed in the Example section below. In this model, anesthetized mice are injected with 1.5 x 10⁴ cells in a 50 % matrigel suspension through the proximal articular surface of the tibia. A steel pin (diameter 0.35 mm) is inserted into the medullary cavity for stabilization and a fracture was induced with scissors 0.5 cm distal from the knee. The effects of any of the DPP-4 inhibitors disclosed herein on the healing of the bone-related diseases or conditions may be assessed in these models.

EXAMPLES The disclosure now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the disclosure.

Example 1. Identification of the Bone Adipocytic Lineage

Flow cytometry was used to delineate the heterogeneous composition of the mesenchymal lineage of the bone. Distinct populations of non-hematopoietic, non-endothelial (CD45 CD31 ) cells were isolated that could be subdivided by expression of Stem cell antigen (Sca)l into two subsets of culture-adherent fibroblasts also expressing the surface receptor Platelet-derived growth factor-a (Pa) (Figures 1 A, 2 A and 2B). During in vitro differentiation, CD45 CD3 l^"Scal⁺ cells exhibited a highly adipogenic but limited osteochondrogenic potential. CD45^"CD31^"Scar cells in turn were non-adipogenic but markedly osteochondrogenic (Figure IB). Consistent with white adipose tissue, a population of unilaterally committed adipogenic progenitors was isolated that was CD45 CD3 l^"Scal⁺CD24^" and a population that displayed tri- lineage differentiation potentials and was CD45^"CD31^"Scal⁺CD24⁺ (Figures 1C and ID). Colony forming potentials (CFU-F) and in vitro recovery rates were highest in fibroblastic cells expressing Seal and/or Pa and were highest in the CD45 CD3 l^"Scal⁺Pa⁺CD24⁺ subset (Figures 3 and 4). CFU-F potential was enriched in the osteogenic CD45 CD3 rScal^"Pa⁺ population but absent in CD45^"CD31^"Scal^"Pa^" cells. Separation of the osteogenic CD45^"CD31^"Scal^"Pa⁺ population by CD24-expression was also possible but these subsets showed identical differentiation capacities. Similarly, the CD45^"CD31^"Pa^"Scal^" cells were mostly CD24⁺ but were non-CFU-F (Figures 3 and 4). Importantly, two separate clonal analyses of the tri-potent CD45^"CD31^"Scal⁺CD24⁺ population revealed a marked homogeneity of a multipotent phenotype, where 64 out of 68 (94%) and 45 out of 54 (83%) clones were able to differentiate into all three lineages when cultivated in the presence or absence of supporting feeder cells, respectively (See Table 1). Table 1.

The transcription factor zinc finger protein (Zfp)-423 labels adipogenic cells in white adipose tissue (WAT). Surprisingly, but consistent with the initial report using a Zfp423-driven enhanced-GFP reporter strain (Zfp423-eGFV), Zfp423-eGFV⁺ (Zfp423⁺) cells occurred as a small subpopulation of less than 1% within the CD45^"CD31^"Sca population while all adipogenic CD45 CD3 l^"Scal⁺ cells were GFP^" (Figure 6). This small subset of adipogenic cells was likely not initially detected due to a dilution effect within the strongly osteochondrogenic cell fraction. In culture, CD45 CD3 Scal^~Zfp423⁺ cells maintained GFP-expression before and after differentiation into adipocytes. In contrast, all CD45 CD3 l^"Scal⁺ cells uniformly acquired GFP-expression only during differentiation, a process that correlated with a concomitant loss of Seal -expression (Figures 7A, 7B, and 8).

In -eGFP reporter mice, multipotent cells and osteogenic progenitors were more abundant in the metaphysis compared to the diaphysis whereas adipogenic progenitors were evenly distributed. Further analyses of Pa, Seal and CD24 expression localized osteogenic progenitors to the endosteum. The majority of multipotent and adipogenic progenitors cells resided in a non-endosteal localization within 40 μιη of the bone surface and revealed a perivascular association of all non-endosteal Pa⁺ cells to blood vessels of less than 10 μιη in diameter (Figures 9 and 10). These findings suggest different micro-anatomical localizations of these functionally distinct cell populations and a preferential association of perivascular multipotent and adipogenic cells to L type vessels, while the distribution of endosteal osteogenic cells resembles more closely localizations of H type endothelium. Consistent with the preferential occurrence of adipocytes in the bone tips of young mice, Zfp423⁺ cells mainly localized to metaphyseal regions of the bone, and labeled a subset of blood vessel-associated progenitors alongside all mature adipocytes of the marrow cavity (Figure 10).

Together, these findings indicate the presence of at least four distinct cell populations in defined micro-anatomies within the bone: A tri-potent, perivascular population with stem cell-like characteristics (CD45^~CD31^~Scal⁺CD24⁺), two functionally and anatomically distinct progenitor populations that are fate-committed towards either the osteochondrogenic (CD45^" CD31 Scal Pa⁺; also referred to as OPC) or adipogenic (CD45 CD31 Scal⁺CD24-; also referred to as APC) lineages, and a more mature CD45 CD3 rScal^"Zfp423⁺ adipocyte precursor stage (also referred to as preAd) cell population. All four populations were present in different bone compartments (Figure 11) and all CD45^"CD31^"Scal⁺ cells were adipogenic. No correlations between MAT occurrence and frequencies were evident. Without wishing to be bound by theory, it is possible that regulatory signals such as leptin may determine the progression into mature adipocytes. Without wishing to be bound by theory, it is possible that signals preventing adipogenic maturation could be differentially distributed within the bones of young mice, since Scal⁺ adipogenic progenitors are also more evenly distributed compared to mature Zfp423⁺ cells (Figures 9 and 10).

To test in vivo differentiation potentials, a triple transgenic mouse strain was generated carrying alleles of the Zfp423-eGFF reporter, Adiponectin (Adipoq)-Cre, and a luciferase reporter within the Rosa26-locus that is only expressed after Cre -mediated recombination, e.g. in mature adipocytes (rep^AdlLuc, Figure 12 A) and a second strain with constitutive red fluorescence (mTmG reporter allele) crossed to the Zfp423-eGFF reporter (rep^tdTom, Figure 12B). Fresh cells of all four populations isolated from _rep^AdlLuc or rep^tdTom by FACS (Figure 13) were transplanted into the sternal region of B6/ Albino mice. After eight weeks, in vivo imaging of luciferase and Perilipin immunofluorescence showed that transplants of the CD45^" CD31^"Scal⁺CD24- and CD45^"CD3 rScal Zfp423⁺ adipogenic populations consistently gave rise to bona fide mature adipocytes, while in vz^'tro-osteogenic CD45^~CD31^~Scal^~Pa⁺ cells did not (Figure 14). Interestingly, CD45^~CD31^~Scal⁺CD24⁺ cells gave rise to Luciferase-positive and negative transplants (Figure 14; top row). Movat-Pentachrome staining of Luciferase- negative tissues of CD45 CD31 Scal⁺CD24⁺ and CD45 CD3 rScal Pa⁺ cells revealed bonelike osteochondrogenic/mineralized structures but never adipocytes (Figures 14; bottom row). Consistent with this observation, intratibial injections of the four cell populations showed that only CD45^"CD31-Scal⁺CD24⁺, CD45-CD31^"Scal⁺CD24-, and CD45-CD3 rScarZfp423⁺ cells, but never CD45^"CD3 Scal^"Pa⁺, gave rise to Zfp423⁺ adipogenic cells in their endogenous microenvironment. Moreover, only CD45^~CD31^~Scal⁺CD24⁺ transplants were able to give rise to adipogenic CD45^~CD31^~Scal⁺CD24^~ cells (Figure 15). In summary, these findings reveal a multi-lineage potential of the CD45^~CD31^~Scal⁺CD24⁺ cell population that can give rise to a lineage of fate-committed adipogenic progenitor cells (APCs: CD45 CD31^" Scal⁺CD24^") that in turn gives rise to a more mature pre-adipocyte (preAd: CD45 CD3 TScal^" Zfp423⁺), and that in parallel could yield a population of unilaterally committed osteochondrogenic progenitor cells (OPCs: CD45^"CD3 rScal^"Pa⁺) under in vitro and in vivo conditions (Figures 16 and 17). Example 2. Marrow Adipocytic Lineage is Closely Related to White Adipocytes.

Gene expression patterns of in vitro differentiated progenitors from bone resembled most closely those derived from inguinal white adipose tissue (iWAT), with similar adipogenic differentiation capacity and expression of general adipogenic genes Peroxisome proliferator- activated receptor-γ (Pparg) and CCAAT/enhancer-binding protein-a (Cebpa) and absent or low expression of brown adipose tissue (BAT) markers Uncoupling protein- 1 (Ucpl) and Cell Death-Inducing DFFA-Like Effector A (Cidea) (Figures 18 and 19), altogether indicating that the marrow adipocytic lineage is more closely related to white rather than brown adipocytes.

Example 3. The Adipocytic Lineage Responds to Diet and Aging

Gene expression was next examined in femora and tibiae from young (2 months) and old (25 months) mice. Consistent with previous reports, expression of adipogenic marker Pparg was increased in old bones. However, adipogenic potential of CD45-CD31-Scal+ progenitors isolated from old bones was unchanged. Conversely, osteogenic marker Osterix (Osx/Sp7) expression was significantly reduced, as was osteogenic capacity of CD45-CD31-Scal- progenitors (Figures 20 and 21). Next, mice of both ages were fed a high fat diet for either 24 hours (ldHFD) or 10 days (lOdHFD). Accumulation of MAT was more pronounced in old animals after lOdHFD, while loss of trabecular bone was observed in aged animals independent of diet (Figure 22). FACS-analysis of young bones revealed a significant induction of CD45- CD31-Scal+CD24+ and APC frequencies after ldHFD that was no longer apparent in lOdHFD mice, suggesting that the rapid induction of adipocytic progenitor proliferation is a mechanism of short-term adaptation to diet (Figures 23A and 23B). In mice aged 25 months, the same ldHFD stimulus significantly increased the multipotent CD45-CD31-Scal+CD24+ and APC populations by approximately 3- and 2-fold, respectively (Figure 23A). BrdU-incorporation was tested in young and 15 -month old Zfp423-eGFP mice and was significantly induced in multipotent CD45-CD31-Scal+CD24+ cells and APCs, an effect that was more pronounced in APCs from old mice (Figure 24). Conversely, the OPC population was not affected by 1 dHFD (Figure 23A and Figure 25) and even showed a reduction in frequency and proliferation rates upon lOdHFD, an effect that was restricted to young animals (Figures 23B and 26). In a cohort of aged, 15-month old Zfp423-eGFP mice, cell frequencies of the Zfp423+ preAds were significantly enhanced after ldHFD, an effect that was significantly less pronounced in young animals (Figure 27). Example 5. The Adipocytic Lineage Inhibits Bone Regeneration

To determine the pathophysiological role of the adipocytic lineage during bone healing, all four populations isolated from reptdTom mice were transplanted into the vicinity of a stabilized tibia fracture and analyzed after 14 days (Figure 28 and Figure 29). μCT- quantification showed a significant decrease of total bone mineral density (BMD) at the fracture site following transplantation of adipogenic populations when compared to the no-cell control group (Figure 28, lower panels, and Figure 30). Histomorphometric analysis of facture/callus sites indicated reduced areas of mineralized tissue and increased amounts of cartilaginous tissues following transplantation of the adipogenic populations, e.g. APCs and preAds, compared to all other groups (Figure 31 A). Due to the lineage-restrictions shown in the cell culture and sternal transplants, these observations likely indicate delayed healing and thus that the cartilaginous structures are entirely derived from the host in the adipogenic transplant groups. Aside from adopting an adipogenic fate after intratibial injection, multipotent CD45- CD31-Scal+CD24+ cells and the two adipogenic populations produced some fibrous tissue, whereas only multipotent CD45-CD31-Scal+CD24+ cells and OPCs contributed to chondrogenic and osteogenic tissue structures and cell types (Figure 3 IB, 31C, 3 ID, and 3 ID). Without wishing to be bound by theory, these observations suggest a negative regulatory role of adipocytic cells during facture healing, further suggesting the detrimental role of MAT in bone homeostasis. Example 6. DPP4 released from MAT inhibits bone healing

To identify potential negative regulators of regeneration processes, RNA-Sequencing (RNA-Seq) was used to further characterize the molecular identity of all four populations (Figure 32). Principal component and hierarchical clustering analyses clearly supported the distinct nature of each population, providing a second line of evidence for the lineage restriction of adipogenic commitment of the closely related APC and pre Ad populations (Figure 33 A, Figure 33B, and Figure 33C). Differential expression (DE) analysis produced several sets of known and potential new candidate genes to define each population (Figures 34-37). For instance, canonical stem cell markers (e.g. Nog, Illrn, Myc) were enriched in the CD45-CD31- Scal+CD24+ multipotent stem cell population (Figure 34). Moreover, signals known to regulate HSC quiescence and maintenance (e.g. Cxcll2, Kitl/Scf, Vcam-1), showed highest expression in this population, along with the highest, but not exclusive, expression level of LepR that was also expressed in the other cells types. The OPC population expressed the classical osteogenic (e.g. AlpI, Dmpl, Collal/2) and chondrogenic markers (e.g. Acan, Col2al, Sox9), as well as previously described skeletal stem cell markers (Itga5, CD200) at elevated levels (Figure 35). The adipogenic populations expressed high levels of markers that have been linked to the adipocytic lineage (i.e. Cd34, Ebf2, and Dpp4) or adipocyte differentiation (i.e. Vim, Ppara; Figures 36 and 37). As expected, expression of Zfp423 was highest in Zfp423+ preAds (Figure 36). Thus, our RNA-Seq analysis confirmed the cellular characteristics of the four populations and establishes the CD45-CD31-Scal+CD24+ multipotent stem cell population as a population expressing elevated levels of Cxcll2 and Lepr that are important regulators of HSCs and osteogenesis.

To identify signals that could mediate the negative effects of adipogenic cells on bone healing, the dataset was screened for secreted factors that were significantly enriched in the adipogenic populations. Among the most significantly regulated secreted factors was the gene encoding for Dipeptidyl peptidase-4 (Dpp4), a protease shed from the plasma membrane that is an important target of clinical diabetes treatments (Figure 38). Consistent with the RNA-Seq data, CD26 (the membrane bound form of DPP4), was enriched on the surface of adipogenic cell populations, and only CD45-CD31-Scal+CD24+ and APCs, but not OPCs, released DPP4 into the medium after adipogenic differentiation (Figures 39 and 40). Expression of Dpp4 was increased in distal tibiae of old mice that contain most ectopic adipocytes, and explant cultures of old tibiae released greater amounts of DPP4 (Figure 41). While treatment of CD45-CD31- Scal+CD24+ and APCs with the DPP4 inhibitor Sitagliptin had no effect on adipogenesis, it significantly enhanced osteogenic gene expression and mineralization of multipotent CD45- CD31-Scal+CD24+ and OPCs during osteogenic differentiation (Figures 42A, 42B, 43A and 43B). While no positive effect was found in untreated OPC transplants (Figure 28, 30, and 31), the improved OPC function following Sitagliptin may serve to promote bone healing. Exposure to recombinant DPP4 slightly impaired osteogenic, but did not alter adipogenic differentiation (Figure 44A, 44B, and 44C). Treatment of mice with two DPP4-inhibitors, Diprotin A and Sitagliptin, significantly accelerated tibia fracture healing (Figure 45) and i.p. injections of Sitagliptin for 9 days significantly increased the frequency of osteogenic progenitors while decreasing the frequency of APCs in non-fractured tibiae (Figure 46). Administration of Sitagliptin was sufficient to abolish the negative effects of transplanted adipogenic cells on bone healing while surprisingly promoting bone healing after OPC transplants (Figure 47A and 47B), suggesting that under such conditions DPP4-inhibtiors may enhance the bone healing capacity of OPCs.

Example 7. Comparison of application methods of 3-day Sitagliptin treatment

To compare the effects of intraperitoneal vs. per oral application of Sitagliptin on the distribution of bone -resident cell populations (MSCs, APCs and OPCs), animals were treated daily with Sitagliptin for three days before analysis of the indicated cell populations. Therefore, Sitagliptin (Sita) or control (Ctrl) were administered to the animals daily at a dose of 10 mg/kg body weight, either by intraperitoneal injections (i.p.) or by oral gavage (per oral - p.o.). For each condition, 3 animals were used. Flow cytrometric analysis of bone-resident mesenchymal stromal cells (MSCs), APCs, and OPCs was conducted as described below. Per oral administration and intraperitoneal injection of Sitagliptin showed equal effects on the distribution of these cell poulations (Figure 48).

Materials and Methods

The Materials and Methods section provided below correspond with the experiments described in Examples 1-7 set forth above.

Experimental Model and Subject Details: All procedures were approved by the ethics committee for animal welfare of the State Office of Environment, Health, and Consumer Protection (State of Brandenburg, Germany). Animals were housed in a controlled environment (20±2 °C, 12 hour/12 hour light/dark cycle), maintained on a standard diet (SD) (Ssniff, Soest, Germany), or fed a high fat diet (HFD) (45% energy from fat, D12451, Research Diets, New Brunswick, NJ,USA) for 1 or 10 days. Male mice were used for all experiments at the indicated ages, where applicable. All following mouse strains were obtained from The Jackson Laboratory: C57BL/6J, B6(Cg)-Tyr^{c 2J}/J (B6-albino), B6.Cg-Tg(Gt(ROSA)26Sor- EGFP)IlAble/J, B6.129S4-Pdgfra^{tml l(EGFp)So}7J (Ρα-eGFP reporter), B6;FVB-Tg(Zfp423- EGFP)7Brsp/J (Zfp423-eGFP reporter), B6.129(Cg)-Gt^O&4 2^or^im^^^cra-^irfromflto -^£G ^"°/J (mTmG-reporter), FVBA29S6(B6)-Gt(ROSA)26So}^Jml(Luc)Kael/J (Rosa26-Luciferase reporter), B6;FVB-Tg(Adipoq-cre)lEvdr/J. Mouse strains expressing Cre-recombinase under promoter control of the mature adipocyte (AdipoQ) lineage markers were intercrossed with the mTmG- reporter mouse strain that constitutively expresses the membrane-bound red fluorescent protein tdTomato (from a loxP-fianked cDNA). Cre-mediated recombination leads to excision of the tdTomato-cassette and activates expression of green fluorescent protein instead. For transplantation experiments, Zfp423-eGFP reporter mice were either intercrossed with mTmG- reporter mice (rep^tdTom), or to AdipoQ-Cre mice and a lox-Stop-lox reporter strain expressing luciferase after Cre-mediated removal of the fioxed Stop-cassette from the Rosa26-locus (rep^AdlLuc). Freshly sorted primary murine cells were used throughout this study and isolated by FACS and cultured as previously described (Schulz et al, 2011, Proc. Natl. Acad. Sci. U. S. A. 108, 143-148 and Steenhuis et al, 2008, Calcif. Tissue Int. 82, 44-56). For cultivation, a complex medium of 60% DMEM low glucose (Invitrogen) and 40% MCDB201 (Sigma) was supplemented with 100 U/ml penicillin and 1,000 U/mL streptomycin (Invitrogen). 2% FBS, l x insulin-transferrin-selenium (ITS) mix, l x linoleic acid conjugated to BSA, 1 nM dexamethasone, and 0.1 mM L-ascorbic acid 2-phosphate (all from Sigma) were added. Before use, growth factors were added to the medium: 10 ng/ml epidermal growth factor (PeproTech), 10 ng/ml leukemia inhibitory factor (MerckMillipore), 10 ng/ml platelet-derived growth factor BB (PeproTech), and 5 ng/ml basic fibroblast growth factor (bFGF; Sigma- Aldrich). The bFGF was added daily throughout the culture period except where stated otherwise. For adipogenic differentiation, cells were induced for 48 hours after three days of expansion, followed by a differentiation period of 5 days. For adipogenic differentiation, induction medium (growth medium without growth factors) containing 5 μg/mL human insulin (Roche Applied Science), 50 μΜ indomethacin, 1 μΜ dexamethasone, 0.5 μΜ isobutylmethylxanthine, 1 nM 3,3',5- triiodo-L-thyronine (T3) (all from Sigma-Aldrich) was added for 48 hours, followed by further differentiation in growth medium without growth factors and the addition of T3 and insulin only. Oil Red O staining was performed by fixing cells with 4% Histofix for 15 minutes at room temperature. For the preparation of Oil Red O working solution, a 0.5% stock solution in isopropanol was diluted with distilled water at a ratio of 3:2. The working solution was filtered and applied to fixed cells for at least one hour at room temperature. Cells were washed four times with tap water before evaluation. For quantification, Oil Red O was extracted by adding a defined volume of isopropanol and absorbance was read in a micro-plate reader (Synergy HI , BioTek) at 510 nm. To induce osteogenic differentiation, pre-confluent cells were supplemented with osteogenic medium (DMEM low glucose (Invitrogen)) with 10%> FBS, 100 nM Dexamethasone, 0.2 mM L-ascorbic acid 2-phosphate, lOmM β-glycerophosphate, and 50 ng/ml L-thyroxine) for 14 days. Cells were then formalin- fixed and stained with 2% Alizarin Red S (Roth) in distilled water. Wells were washed twice with PBS and once with distilled water. De-staining was conducted to quantitatively determine mineralization by adding a 10% cetylpyridinium chloride solution. Absorbance was measured in a micro-plate reader (Synergy HI, BioTek) at 570 nm.

A micromass culture was used for the chondrogenesis assay. A 5 μΐ droplet of cell suspension (appr. 1.5 x 10⁷ cells/ml) was pipetted in the center of a well (48-well plate). After cultivating the micromass culture for 2 hours in the incubator, warm chondrogenic media (DMEMhigh (Invitrogen)) with 10% FBS, 100 nM Dexamethasone, 1 μΜ L-ascorbic acid-2- phosphate, lOx ITS mix, and 10 ng/ml Transforming growth factor βΐ) was added. Cell media was changed every other day. After 21 days, cells were fixed and stained with 1% Alcian-Blue staining (Sigma) for 30 minutes at room temperature. Cells were rinsed three times with 0.1 M HC1. To neutralize acidity, a washing step with dH20 was conducted before microscopic analysis.

For DPP4 in vitro experiments, cell populations were differentiated with adipogenic or osteogenic assays as described above. Mouse recombinant DPP4 (250 ng/ml; R&D Systems) or the DPP-4 inhibitor Sitagliptin (100 μΜ; biomol) were added to differentiation cocktails from day 3 (adipogenic induction) during adipogenesis or day 0 during osteogenesis until the end of differentiation experiments. DPP4 secretion into cell culture media was determined by ELISA (ThermoFisher). Either supernatant of freshly isolated tibia explants maintained in culture media for 24 hours or supernatant from cell populations following 10 days of adipogenic differentiation were used.

CFU-F assay was conducted as follows: Freshly isolated cell populations were seeded in expansion media at 500 cells per 6-well plate. Medium was changed every other day. At day 10, cells were fixed and stained with Crystal Violet (Sigma). Colonies consisting of more than 20 cells were counted as CFU. At least 6 independent assays were performed per cell population. For total recovery rate experiments, cell populations were seeded as described for the CFU-F assay. Analysis of fixed and crystal violet stained cell populations was conducted on day 7, 11, and 15 by quantification of total cell invasion area of well-plate surface using ImageJ software.

Flow cytometry & cell sorting: Flow cytometry and cell sorting were performed on a FACS Aria III cell sorter (BD Biosciences) and analyzed using Flow Jo software (Tree Star). Soft-tissue free bones (tibia/femur) were crushed with bone scissors and incubated for 1 hour in a shaking water bath at 37 °C in 10 ml of 20% FBS/PBS containing 0.5% type-2 collagenase (CellSystems). The suspension was filtered through a 70 μιη mesh to remove bone fragments and centrifuged at 1200 rpm for 5 minutes at 4 °C. The pellet was re-suspended in ACK (Ammonium-Chloride -Potassium) lysing buffer to eliminate red blood cells and centrifuged again at 1200 rpm for 5 minutes at 4 °C. The pellet was re-suspended in 100 μΐ sorting buffer (2% FBS/PBS) and stained with antibodies for at least 30 minutes at 4 °C. The applied FACS antibodies can be found in the Key Resources Table. Living cells were gated for lack of PI (propidium iodide; 1 : 1,000 diluted stock solution: 1 μg/mL in water) fluorescence and accumulation of Calcein (1 : 1,000 dilution; stock of 1 mg in 215

DMSO). Compensation, fluorescence-minus-one control based gating, and FACS-isolation was conducted as described previously (Schulz et al, 2011, Proc. Natl. Acad. Sci. U. S. A. 108, 143-148).

Single-cell clonal assays: For the co-culture approach, a feeder layer of CD45 CD31^" Pa⁺ cells was isolated from long bones of 8-week old male C57BL/6J mice and seeded in 100 μΐ of expansion medium at 750 cells per well of a 96-well plate. On the next day, a single CD45^" CD3 Scal⁺CD24⁺tdTomato⁺ cell freshly isolated from 8-week old male Rosa26-mTmG mice was FACS-sorted into each well. Cells were expanded for 10 days to sub-confiuency with media changes every other day. After 10 days, clonal expansion of a single cell was verified by fluorescence microscopy. Wells containing a readily detectable single colony of tdTomato⁺ cells were trypsinized, washed, and collected in 100 μΐ sorting medium. Five to ten cells (per condition) of each clone were directly FACS-sorted onto freshly prepared 96-well plate feeder layers of expanded CD45^"CD31^"Pa⁺ cells for adipogenic and osteogenic differentiation protocols, or onto a micromass culture for chondrogenic differentiation. At the end of the differentiation assays clones were analyzed for their differentiation capacity by immunocytochemistry. A tdTomato positive clone was considered adipogenic if it co-stained with Perilipin, osteogenic if it co-stained with Osteocalcin, and chondrogenic if it co-stained with Aggrecan. Alternatively, in a feeder cell-free assay, a single CD45 CD31^" Scal⁺CD24⁺tdTomato⁺ cell, freshly isolated from 8-weeks old male C57BL/6J mice, was FACS-sorted into a well of a 96-well plate without feeder cells. Single cells were expanded for 10 days with media changes every other day. After 10 days, clones giving rise to colonies were re-seeded in a new 96-well plate and expanded until sub-confluency. Clones were then used for tri-differentiation assays. At the end of differentiation Oil Red O staining was conducted for Adipogenesis and immunocytochemistry for osteogenesis (Osteocalcin) and chondrogenesis (Aggrecan). Images were acquired with a Keyence BZ-9000 (Biorevo) fluorescence microscope.

Histology: Isolated bones were cleaned from surrounding tissue and fixed/decalcified in Richard-Allan Scientific Cal-Rite fixative (Thermo Scientific), followed by paraffin embedding. Sections (3 μιη) were used for immunohistological staining or H&E overview staining. For immunohistochemistry, sections were de-paraffinized and re-hydrated in xylene and decreasing ethanol concentrations. Heat-mediated antigen retrieval was conducted by placing sections in blocking buffer (40 mM Tris and 1.2 mM EDTA in distilled water) in a microwave for 5 minutes at 330 W. Slides were left for cooling and rinsed with water. Nearly dried samples were circled with a PAP pen (Kisker) and incubated with blocking solution (1% BSA/PBS) for 60 minutes at room temperature. Primary antibodies, diluted in 1% BSA/PBS, were added and samples were incubated in a humidified chamber at 4 °C overnight. Sections were washed with PBS three times. Secondary antibody and DAPI nucleus staining were applied for 10 minutes at room temperature in the dark. Samples were washed twice with PBS. To reduce auto-fluorescence 0.3% Sudan Black solution (in 70% EtOH) was applied for 20 minutes. Sections were washed three times and mounted with Fluoromount G (eBioscience, GER). Samples were stored at 4 °C in the dark before evaluation via fluorescence microscopy. For the quantification of the different bone-resident populations, bone marrow regions of 0.05 mm² from bone sections were selected on fluorescence images. For immunocytochemistry, fixated cells in well plates were permeabilized with 0.1% Triton X-100 solution and blocked with 3%) BSA in PBS. Antibodies were used as listed in the resources table. For nuclear staining, specimens were treated with DAPI. Sections and cells were analyzed using a Keyence BZ-9000 (Biorevo) fluorescence microscope (for up to two fluorescences) or a Zeiss confocal laser scanning microscope (LSM) 700 (for three fluorescences). Sternal transplantation: Sorted cell populations from luciferase-expressing rep or tdTomato-expressing _rep^tdTom mice were subcutaneously injected at 1.5 x 10⁴ cells in a 50% matrigel suspension into the sternal area of B6-albino mice. Eight weeks after transplantation, engrafted tissues were excised, fixed, and histologically analyzed. Mice injected with cells from rep^Luc animals were additionally subjected to Luciferase imaging with an IVIS imaging system (Perkin Elmer) before sacrifice. To this end, animals were intraperitoneally injected with luciferin (150 mg/kg) and subsequently anesthetized. After 12 to 18 minutes, the animals were imaged. Image analysis was performed with Living Image 4.4 software (Xenogen).

BrdU cell proliferation in vivo assay: For 24 hour experiments, mice were i.p. -injected with a single dose of 100 mg BrdU/kg (Sigma Aldrich) diluted in sterile PBS. Mice receiving a SD or HFD for ten days were given BrdU via drinking water at a concentration of 0.5 mg/ml. Drinking water was refreshed every other day. For single-cell immunostaining was approximately 2 x 10³ cells/mouse of each population of interest were double-sorted on glass cover slips pre-coated with a 5 μΐ drop of DMEM(low). Coverslips were incubated for 30 minutes, allowing cells to attach. Cells were fixed by gently adding 4% Histofix for 10 minutes and washed three times for 3 minutes with PBS. Permeabilization solution (0.2 % saponin/PBS) was applied for 6 minutes. Washing solution (0.02 % saponin/PBS) for 5 minutes was followed by administering DNA-denaturation solution (2 M HC1 in 0.02 % saponin/PBS) for 20 minutes at 37 °C. Cells were washed for 5 minutes and blocked for 30 minutes with 2% BSA in washing solution. Incubation with anti-BrdU antibody (Cedarlane) in blocking solution was done overnight at 4 °C. On the next day, three times washing for 10 minutes and incubation with Alexa Fluor 488 donkey anti-rat (FisherScientific) and DAPI staining in blocking solution for 30 minutes at room temperature in the dark was performed. Cells were washed with washing solution three times and PBS once. Coverslips were mounted with Fluoromount G (eBioscience). Samples were stored at 4 °C in the dark before evaluation via fluorescence microscopy (Keyence). The percentage of BrdU-positive cells within each population was calculated as compared to total numbers of D API-positive cells.

Fracture model: Mice were given an analgetic (MediGel, ClearFbO) starting two days prior to surgery. Anesthetized mice were injected with 1.5 x 10⁴ cells in a 50% matrigel suspension through the proximal articular surface of the tibia. A steel pin (diameter 0.35 mm) was inserted into the medullary cavity for stabilization and a fracture was induced with scissors 0.5 cm distal from the knee. At the indicated time point after fracture induction, tibiae were harvested for analyses. After removal of the pin from extracted tibiae, μCT analysis was conducted with LaTheta LCT-200 (Hitachi-Aloka) using manufacturer's pre-defined parameters for isolated bone measurements. Alternatively, tibiae were fixed and decalcified followed by paraffin embedding and sectioning at 3 μιη per slice. Samples were stained using SafraninO/Fast green and Movat Pentachrome. ImageJ software was used for computer-assisted histomorphometric analysis of fracture calluses. Six representative sections of each callus were analyzed for bone, fibrous, and cartilaginous tissue areas in a blinded manner.

For DPP4 in vivo experiments mice received a daily dose of PBS, Diprotin A (5 mg/kg body weight; Sigma) or Sitagliptin (10 mg/kg body weight; biomol) i.p. for 9 consecutive days. For fracture healing experiments, application started two days before induction of injury/cell injection. Fracture healing was assessed one day after the last DPP4-inhibitor administration.

Capture, Library Preparation, and Sequencing of cell population: RNA extraction, reverse transcription and cDNA pre-amplification, Nextera XT libraries and R A-sequencing of the cell populations was done as previously described. A total of 17,000 CD45^"CD31^"Sca Zfp423⁺, 50,000 CD45 CD31 Scal⁺CD24-, 5,000 CD45 CD31 Scal⁺CD24⁺ and 30,000 CD45^" CD3 l^"Scal^"Pa⁺ cells were FACS-sorted from bones of 4 mice (the 3 biological replicates were done on 3 different days), collected in a 1.5 ml Eppendorf tube containing 50 μΐ RLT Plus Buffer (Qiagen) supplemented with 1% 2-Mercaptoethanol, immediately frozen in dry-ice and kept at -80 °C. The time elapsed between mouse euthanasia and the termination of the FACS procedure was -400 minutes. Briefly, RNA was extracted using the RNeasy Plus Micro Kit (Qiagen), together with genomic DNA eliminator (Qiagen), according to the manufacturer's instructions. Reverse transcription and cDNA pre-amplification were performed using the SMARTer PCR cDNA Synthesis kit (Clontech) and the Advantage 2 PCR kit (Clontech). cDNA was harvested and quantified with the Bioanalyzer DNA High-Sensitivity kit (Agilent Technologies). Libraries were prepared using the Nextera XT DNA Sample Preparation Kit and the Nextera Index Kit (Illumina). Multiplexed libraries were pooled, and paired-end 100- bp sequencing was performed on one flow-cell (two lanes) of an Illumina HiSeq 2500.

RNA-seq data processing and analysis: Sequencing data were aligned to the Mus musculus genome (Ensembl version 38.82) using GSNAP (version 2014-10-07) with default parameters. HTseq-count was used to count the number of reads mapped to each gene (default options). Almost all libraries showed good quality, with sizes ranging between 2-3.5xl0⁷ read counts and a fraction of reads mapped to exons greater than 75%. One library yielded less than 300 reads and was excluded from downstream analysis. The data was normalized for sequencing depth using size factors. The union of the top 1,000 genes expressed in each library was selected, which resulted in a list of 2,120 genes. Principal component analysis was carried out on the standardized loglO-transformed normalized counts (after adding a pseudo-count of 1 to avoid infinities). Hierarchical clustering analysis was performed using Euclidean distances with Ward's method on the same dataset. Differentially expressed genes between groups of libraries were identified by using the bioconductor R-package DESeq2 library at a FDR of 0.1. Genes that were not detected in any library were removed prior to the analysis and possibly confounding factors were taken into account (i.e., the animal each sample was taken from). RNA-seq data was statistically analyzed using the R-statistical package and Paleontological Statistics (PAST, version 3.10, http://folk.uio.no/ohammer/past/, accessed December 2015). For DE analyses, gene expression was compared between all investigated cell populations. A p-value of < 0.05 was used as a cut-off for differentially expressed genes. Heat-maps contain representative top-regulated genes, which were further divided by known cell type specific functions as previously described in the literature and unknown novel marker genes.

Gene expression analysis: Total RNA isolation and gene expression analysis was conducted using standard methods as described before (Schulz et al, 2011, Proc. Natl. Acad. Sci. U. S. A. 108, 143-148) using column-based RNA-isolation, reserve transcription for cDNA synthesis, and SYBR green-based detection during quantified real-time PCR. Primer sequences were used as noted in the Key Resources Table.

Quanitification and Statistical Analyses: All data are presented as mean ± standard error of the mean (SEM). The sample size for each experiment and the replicate number of experiments are included in the figure legends. Statistical significance was defined as p < 0.05. Statistical analyses were performed using unpaired, two-tailed Student's t test or Mann- Whitney-U-test where applicable for comparison between two groups, and an AN OVA test was used for experiments involving more than two groups (GraphPad Prism; version 6.04).

Data Resources: All RNA-seq data generated in this study was deposited at the European Nucleotide Archive (http://www.ebi.ac.uk/ena) under secondary sample accession number (ID code) ERP013883.

Key Resources Table

Αηΐί-β-Αοΐίη Peroxidase-conjugated Sigma Cat#: A3854

Anti-human/mouse UCP1 R&D Systems Cat#: MAB6158

Peroxidase goat anti-mouse Abeam Cat#: ab97023

Chemicals, Peptides, and Recombinant Proteins

Calcein eBioscience Cat#: 65-0855-39

Propidium Iodide (PI) Sigma Cat#: P4170

Recombinant Mouse DPPIV/CD26 Protein R&D Systems Cat#: 954-SE

Diprotin A (Ile-Pro-Ile) Sigma Cat#: 19759

Sitagliptin biomol Cat#: Cay- 13252-250

Cal-Rite Fixative ThermoFisher Cat#: 10599428

Roti-Histofix 4 % Carl Roth Cat#: P087.3

Sudan Black B Sigma Cat#: 199664

Fluoromount-G eBioscience Cat#: 00-4958-02

Oil Red O Sigma Cat#: O0625

Alizarin Red S Carl Roth Cat#: A5533-25G

Alcian Blue 8GX Sigma Cat#: A3157

Crystal Violet Sigma Cat#: C0775

BrdU Sigma Cat#: B5002

D-Luciferin - K+ Salt Bioluminescent Substrate Perkin Elmer Cat#: 122796

MCDB201 Media Sigma Cat#: M6770

Dexamethasone Sigma Cat#: D-4902

L- Ascorbic acid 2-phosphate Sigma Cat#: A8960

Insulin-transferrin-selenium (ITS) mix Sigma Cat#: 13146

Linoleic acid- Albumin Sigma Cat#: L9530

Epidermal growth factor PeproTech Cat#: 315-09

Leukemia inhibitory factor MerckMillipore Cat#: ESG1107

Platelet-derived growth factor BB PeproTech Cat#: 315-18

Basic fibroblast growth factor Sigma Cat#: F0291

Indomethacin Sigma Cat#: 17378

Recombinant Human Insulin Roche Cat#: 11376497001

Isobutylmethylxanthine Sigma Cat#: 15879

3,3',5-triiodo-L-thyronine (T3) Sigma Cat#: T6397 β-glycerophosphate Sigma Cat#: G9891

L-thyroxine Sigma Cat#: T0397

Transforming growth factor βΐ PeproTech Cat#: 100-21

Critical Commercial Assays

DPP4 ELISA ThermoFisher Cat#: EMDPP4

RNeasy Plus Micro Kit Qiagen Cat#: 74034

SMARTer PCR cDNA Synthesis kit Clontech Cat#: 634925

Advantage 2 PCR kit Clontech Cat#: 639207

Bioanalyzer DNA High- Sensitivity kit Agilent Cat#: 5067

Technologies

Nextera XT DNA Sample Preparation Kit Illumina Cat#: FC-131

Nextera Index Kit Illumina Cat#: FC-131

Deposited Data

RNAseq Data European ERP013883

Nucleotide

Archive (ENA),

http://www.ebi.ac.

uk/ena

Experimental Models: Organisms/Strains Mouse: R6/2: C57BL/6J The Jackson JAX: 000664

Laboratory

Mouse: R6/2: B6(Cg)-Tyr^c-^2J/J The Jackson JAX: 000058

Laboratory

Mouse: R6/2: B6.Cg-Tg(Gt(ROSA)26Sor- The Jackson JAX: 007897

EGFP)IlAble/J Laboratory

Mouse: R6/2: B6.129S4-Pdgfra^{tml l(EGFP}>^So7J The Jackson JAX: 007669

Laboratory

Mouse: R6/2: B6;FVB-Tg(Zfp423-EGFP)7Brsp/J The Jackson JAX: 019381

Laboratory

Mouse: R6/2: 6.\29{Cg)-Gt(ROSA)26Sor ⁴<^ACTB- The Jackson JAX: 007676

tdTomato,-EGFP)Luo Laboratory

Mouse: R6/2: FVB.129S6(B6)- The Jackson JAX: 005125

Gt(ROSA)26Soi^Jml(Luc>^Kael/J Laboratory

Mouse: R6/2: B6;FVB-Tg(Adipoq-cre)lEvdr/J The Jackson JAX: 010803

Laboratory

Sequence-Based Reagents

qPCR Primer sets: please see Table 2

Table 2

5 SEQUENCE INFORMATION

SEQ ID NO: 1 - Human Dipeptidyl Peptidase 4 Amino Acid Sequence (GenBank Accession No. NP_001926.2) MKTPWKVLLGLLGAAALVTIITVPVVLLNKGTDDATADSR TYTLTDYLK TYRLK LYSLRWISDHEYLYKQENNILVFNAEYGNSSVFLENSTFDEFGHSINDYSISPDGQFILL EYNYVKQWRHSYTASYDIYDLNKRQLITEERIP NTQWVTWSPVGHKLAYVW NDI YVKIEPNLPSYRITWTGKEDIIYNGITDWVYEEEVFSAYSALWWSPNGTFLAYAQFND TEVPLIEYSFYSDESLQYPKTVRVPYPKAGAVNPTVKFFVVNTDSLSSVTNATSIQITA PASMLIGDHYLCDVTWATQERISLQWLRRIQNYSVMDICDYDESSGRWNCLVARQHI EMSTTGWVGRFRPSEPHFTLDGNSFYKIISNEEGYRHICYFQIDKKDCTFITKGTWEVI GIEALTSDYLYYISNEYKGMPGGRNLYKIQLSDYTKVTCLSCELNPERCQYYSVSFSK EAKYYQLRCSGPGLPLYTLHSSVNDKGLRVLEDNSALDKMLQNVQMPSKKLDFIILN ETKFWYQMILPPHFDKSK YPLLLDVYAGPCSQKADTVFRLNWATYLASTENIIVAS FDGRGSGYQGDKIMHAINRRLGTFEVEDQIEAARQFSKMGFVDNKRIAIWGWSYGG YVTSMVLGSGSGVFKCGIAVAPVSRWEYYDSVYTERYMGLPTPEDNLDHYRNSTVM SRAENFKQVEYLLIHGTADDNVHFQQSAQISKALVDVGVDFQAMWYTDEDHGIASS TAHQHIYTHMSHFIKQCFSLP

SEQ ID NO: 2 - Human DPP4 Nucleotide Acid Sequence (GenBank Accession No.

NM_001935.3)

CTTTCACTGGCAAGAGACGGAGTCCTGGGTTTCAGTTCCAGTTGCCTGCGGTGGG CTGTGTGAGTTTGCCAAAGTCCCCTGCCCTCTCTGGGTCTCGGTTCCCTCGCCTGT CCACGTGAGGTTGGAGGAGCTGAACGCCGACGTCATTTTTAGCTAAGAGGGAGC AGGGTCCCCGAGTCGCCGGCCCAGGGTCTGCGCATCCGAGGCCGCGCGCCCTTTC CCCTCCCCCACGGCTCCTCCGGGCCCCGCACTCTGCGCCCCGGCTGCCGCCCAGC GCCCTACACCGCCCTCAGGGGGCCCTCGCGGGCTCCCCCCGGCCGGGATGCCAGT GCCCCGCGCCACGCGCGCCTGCTCCCGCGCCGCCTGCCCTGCAGCCTGCCCGCGG CGCCTTTATACCCAGCGGGCTCGGCGCTCACTAATGTTTAACTCGGGGCCGAAAC TTGCCAGCGGCGAGTGACTCCACCGCCCGGAGCAGCGGTGCAGGACGCGCGTCT CCGCCGCCCGCGGTGACTTCTGCCTGCGCTCCTTCTCTGAACGCTCACTTCCGAGG AGACGCCGACGATGAAGACACCGTGGAAGGTTCTTCTGGGACTGCTGGGTGCTGC TGCGCTTGTCACCATCATCACCGTGCCCGTGGTTCTGCTGAACAAAGGCACAGAT GATGCTACAGCTGACAGTCGCAAAACTTACACTCTAACTGATTACTTAAAAAATA CTTATAGACTGAAGTTATACTCCTTAAGATGGATTTCAGATCATGAATATCTCTAC AAACAAGAAAATAATATCTTGGTATTCAATGCTGAATATGGAAACAGCTCAGTTT TCTTGGAGAACAGTACATTTGATGAGTTTGGACATTCTATCAATGATTATTCAATA TCTCCTGATGGGCAGTTTATTCTCTTAGAATACAACTACGTGAAGCAATGGAGGC ATTCCTACACAGCTTCATATGACATTTATGATTTAAATAAAAGGCAGCTGATTAC AGAAGAGAGGATTCCAAACAACACACAGTGGGTCACATGGTCACCAGTGGGTCA TAAATTGGCATATGTTTGGAACAATGACATTTATGTTAAAATTGAACCAAATTTA CCAAGTTACAGAATCACATGGACGGGGAAAGAAGATATAATATATAATGGAATA ACTGACTGGGTTTATGAAGAGGAAGTCTTCAGTGCCTACTCTGCTCTGTGGTGGT CTC C AAAC GGC ACTTTTTT AGC AT ATGC CC AATTT AAC G AC AC AG AAGTC CC ACT TATTGAATACTCCTTCTACTCTGATGAGTCACTGC AGTACCC AAAGACTGTACGG GTTCCATATCCAAAGGCAGGAGCTGTGAATCCAACTGTAAAGTTCTTTGTTGTAA ATACAGACTCTCTCAGCTCAGTCACCAATGCAACTTCCATACAAATCACTGCTCCT GCTTCTATGTTGATAGGGGATCACTACTTGTGTGATGTGACATGGGCAACACAAG AAAGAATTTCTTTGCAGTGGCTCAGGAGGATTCAGAACTATTCGGTCATGGATAT TTGTGACTATGATGAATCCAGTGGAAGATGGAACTGCTTAGTGGCACGGCAACAC ATTGAAATGAGTACTACTGGCTGGGTTGGAAGATTTAGGCCTTCAGAACCTCATT TTACCCTTGATGGTAATAGCTTCTACAAGATCATCAGCAATGAAGAAGGTTACAG ACACATTTGCTATTTCCAAATAGATAAAAAAGACTGCACATTTATTACAAAAGGC ACCTGGGAAGTCATCGGGATAGAAGCTCTAACCAGTGATTATCTATACTACATTA GTAATGAATATAAAGGAATGCCAGGAGGAAGGAATCTTTATAAAATCCAACTTA GTGACTATACAAAAGTGACATGCCTCAGTTGTGAGCTGAATCCGGAAAGGTGTCA GTACTATTCTGTGTCATTCAGTAAAGAGGCGAAGTATTATCAGCTGAGATGTTCC GGTCCTGGTCTGCCCCTCTATACTCTACACAGCAGCGTGAATGATAAAGGGCTGA GAGTCCTGGAAGACAATTCAGCTTTGGATAAAATGCTGCAGAATGTCCAGATGCC CTCCAAAAAACTGGACTTCATTATTTTGAATGAAACAAAATTTTGGTATCAGATG ATCTTGCCTCCTCATTTTGATAAATCCAAGAAATATCCTCTACTATTAGATGTGTA TGCAGGCCCATGTAGTCAAAAAGCAGACACTGTCTTCAGACTGAACTGGGCCACT TACCTTGCAAGCACAGAAAACATTATAGTAGCTAGCTTTGATGGCAGAGGAAGTG GTTACCAAGGAGATAAGATCATGCATGCAATCAACAGAAGACTGGGAACATTTG AAGTTGAAGATCAAATTGAAGCAGCCAGACAATTTTCAAAAATGGGATTTGTGG ACAACAAACGAATTGCAATTTGGGGCTGGTCATATGGAGGGTACGTAACCTCAAT GGTCCTGGGATCGGGAAGTGGCGTGTTCAAGTGTGGAATAGCCGTGGCGCCTGTA TCCCGGTGGGAGTACTATGACTCAGTGTACACAGAACGTTACATGGGTCTCCCAA CTCCAGAAGACAACCTTGACCATTACAGAAATTCAACAGTCATGAGCAGAGCTG AAAATTTTAAACAAGTTGAGTACCTCCTTATTCATGGAACAGCAGATGATAACGT TCACTTTCAGCAGTCAGCTCAGATCTCCAAAGCCCTGGTCGATGTTGGAGTGGAT TTCCAGGCAATGTGGTATACTGATGAAGACCATGGAATAGCTAGCAGCACAGCA CACCAACATATATATACCCACATGAGCCACTTCATAAAACAATGTTTCTCTTTACC TTAGCACCTCAAAATACCATGCCATTTAAAGCTTATTAAAACTCATTTTTGTTTTC ATTATCTCAAAACTGCACTGTCAAGATGATGATGATCTTTAAAATACACACTCAA ATCAAGAAACTTAAGGTTACCTTTGTTCCCAAATTTCATACCTATCATCTTAAGTA GGGACTTCTGTCTTCACAACAGATTATTACCTTACAGAAGTTTGAATTATCCGGTC GGGTTTTATTGTTTAAAATC ATTTCTGC ATCAGCTGCTGAAACAAC AAATAGGAA TTGTTTTTATGGAGGCTTTGCATAGATTCCCTGAGCAGGATTTTAATCTTTTTCTA ACTGGACTGGTTCAAATGTTGTTCTCTTCTTTAAAGGGATGGCAAGATGTGGGCA GTGATGTCACTAGGGCAGGGACAGGATAAGAGGGATTAGGGAGAGAAGATAGC AGGGCATGGCTGGGAACCCAAGTCCAAGCATACCAACACGAGCAGGCTACTGTC AGCTCCCCTCGGAGAAGAGCTGTTCAC AGCC AGACTGGCAC AGTTTTCTGAGAAA GACTATTCAAACAGTCTCAGGAAATCAAATATGCAAAGCACTGACTTCTAAGTAA AACCACAGCAGTTGAAAAGACTCCAAAGAAATGTAAGGGAAACTGCCAGCAACG CAGGCCCCCAGGTGCCAGTTATGGCTATAGGTGCTACAAAAACACAGCAAGGGT GATGGGAAAGCATTGTAAATGTGCTTTTAAAAAAAAATACTGATGTTCCTAGTGA AAGAGGCAGCTTGAAACTGAGATGTGAACACATCAGCTTGCCCTGTTAAAAGAT GAAAATATTTGTATCACAAATCTTAACTTGAAGGAGTCCTTGCATCAATTTTTCTT ATTTCATTTCTTTGAGTGTCTTAATTAAAAGAATATTTTAACTTCCTTGGACTCATT TTAAAAAATGGAACATAAAATACAATGTTATGTATTATTATTCCCATTCTACATAC TATGGAATTTCTCCCAGTCATTTAATAAATGTGCCTTCATTTTTTCAGAAAAAAAA AAAAAAA

SEQ ID NO: 3 - Murine Dipeptidyl Peptidase 4 Amino Acid Sequence (GenBank Accession No. NP_034204.1)

MKTPWKVLLGLLGVAALVTIITVPIVLLSKDEAAADSRRTYSLADYLKSTFRVKSYSL WWVSDFEYLYKQENNILLLNAEHGNSSIFLENSTFESFGYHSVSPDRLFVLLEYNYVK QWRHSYTASYNIYDVNKRQLITEEKIPNNTQWITWSPEGHKLAYVWKNDIYVKVEP HLPSHRITSTGEENVIYNGITDWVYEEEVFGAYSALWWSPNNTFLAYAQFNDTGVPLI EYSFYSDESLQYPKTVWIPYPKAGAVNPTVKFFIVNIDSLSSSSSAAPIQIPAPASVARG DHYLCDVVWATEERISLQWLRRIQNYSVMAICDYDKINLTWNCPSEQQHVEMSTTG WVGRFRPAEPHFTSDGSSFYKIISDKDGYKHICHFPKDKKDCTFITKGAWEVISIEALT SDYLYYISNQYKEMPGGRNLYKIQLTDHTNVKCLSCDLNPERCQYYAVSFSKEAKY YQLGCWGPGLPLYTLHRSTDHKELRVLEDNSALDRMLQDVQMPSKKLDFIVLNETR FWYQMILPPHFDKSK YPLLLDVYAGPCSQKADASFRLNWATYLASTENIIVASFDG RGSGYQGDKIMHAINRRLGTLEVEDQIEAARQFVKMGFVDSKRVAIWGWSYGGYVT SMVLGSGSGVFKCGIAVAPVSRWEYYDSVYTERYMGLPIPEDNLDHYRNSTVMSRA EHFKQVEYLLIHGTADDNVHFQQSAQISKALVDAGVDFQAMWYTDEDHGIASSTAH QHIYSHMSHFLQQCFSLH

SEQ ID NO: 4 - Murine DPP4 Nucleotide Acid Sequence (GenBank Accession No.

NM_010074.3)

GACTGACAGACCTCTCAGGGAAGGGGACAAGAGAACTGGCAGCTAGTTTCTGCA GTGAGCCGTGAATCTGCCAAACTCCATGCCTGCTTGAACCTCAGCTGCCTCGATT ATCC ACGCAAGGGAGC AACAGTTAAACCTC AATGTGCAATGTCTCTTTTAGC AAA GGGGGTTCGCTGTTCCCCTTATCCCTCACTCATGGGGGCCCTGCGTGCTACTTCCT GGCTCGCCTAGTGGTCCTACCGCGCTGGGTCGGCTGCCCCGCGCTCACACTGTGC ACCGAAGCCCGCCTTGCGCTAACTAATGTTTAACTCAGGCCGAAACTTGGCGGCG AGCTCAGGGTGACTGCGTGCAGAGCAGCCGCGCAGGACGTCCGTCTCTGCGCGC AGTGACTTCTGCCTGCGCTCAAGCTTCAGAGTTCAGTTTCAAGGAGCCGCCCGAC CATGAAGACACCGTGGAAGGTTCTTCTGGGACTGCTTGGTGTCGCTGCGCTTGTC ACCATCATCACCGTGCCAATAGTTCTGCTGAGCAAAGATGAAGCGGCAGCTGACA GCCGCAGAACGTATTCACTAGCTGACTATTTAAAGAGTACCTTTCGGGTCAAGTC CTACTCTTTGTGGTGGGTTTCAGACTTTGAATACCTCTACAAACAAGAGAACAAT ATCTTGCTGCTCAATGCTGAACATGGAAACAGCTCCATTTTCTTGGAGAACAGTA CCTTTGAAAGCTTTGGATATCATTCAGTGTCACCTGACCGACTGTTTGTTCTCTTG GAATACAACTACGTGAAGCAATGGAGACATTCCTACACAGCTTCATACAACATTT ATGATGTGAATAAAAGACAGCTGATCACAGAAGAGAAGATTCCAAATAATACAC AGTGGATCACATGGTCACCAGAAGGTCATAAGTTGGCATATGTCTGGAAGAATG ATATTTACGTTAAAGTTGAACCACACTTACCTAGTCATAGGATCACATCGACAGG AGAAGAAAATGTAATATATAATGGAATAACTGACTGGGTTTATGAAGAGGAAGT CTTCGGTGCCTACTCTGCACTGTGGTGGTCTCCAAACAACACGTTTCTAGCTTATG CCCAGTTTAACGACACAGGAGTGCCGCTCATTGAATACTCCTTCTATTCTGATGA GTCACTGCAGTACCCCAAGACAGTGTGGATTCCATACCCAAAGGCAGGAGCTGTG AATCCAACTGTAAAGTTCTTTATTGTAAATATAGACTCTCTCAGCTCATCCTCTAG TGCGGCTCCCATCCAAATCCCTGCTCCTGCATCTGTGGCAAGAGGGGATCACTAT TTATGTGATGTGGTGTGGGCTACAGAAGAAAGAATTTCACTACAGTGGCTCAGGA GGATTCAGAACTATTCCGTGATGGCTATCTGTGACTATGATAAGATCAACCTAAC GTGGAACTGTCCATCCGAGCAGCAGCATGTTGAAATGAGTACCACAGGCTGGGTC GGAAGATTTAGGCCCGCAGAACCTCACTTCACCTCTGATGGAAGCAGCTTCTATA AGATC ATCAGCGAC AAAGATGGCTACAAAC ACATCTGCC ACTTCCCGAAAGATA AGAAAGACTGTACATTTATTACAAAAGGAGCCTGGGAAGTCATTAGTATCGAAG CTCTGACCAGCGATTATCTATACTACATTAGTAACCAATATAAAGAAATGCCAGG AGGAAGAAATCTCTATAAAATTCAACTTACTGACCACACAAATGTGAAGTGCCTT AGTTGTGACCTGAATCCAGAAAGATGTCAGTATTATGCGGTATCATTTAGTAAAG AGGCAAAGTACTATCAGCTGGGATGTTGGGGCCCCGGTCTGCCCCTCTAC ACTCT ACATCGTAGCACGGATCATAAAGAGCTGCGAGTCCTGGAAGACAATTCTGCTTTG GATAGAATGCTGCAGGATGTCCAGATGCCTTCAAAAAAATTGGACTTCATTGTTT TGAATGAAACAAGATTTTGGTATCAAATGATCTTGCCCCCTCATTTTGATAAATCC AAGAAATATCCTCTACTATTAGATGTATATGCAGGTCCCTGTAGTCAAAAAGCAG ATGCTTCCTTCAGACTGAACTGGGCCACTTACCTTGCAAGTACAGAAAACATCAT AGTAGCTAGCTTTGACGGCAGAGGAAGTGGTTACCAAGGAGATAAGATCATGCA TGCAATCAACAGAAGATTGGGAACACTGGAAGTTGAAGATCAAATTGAAGCAGC CAGGCAATTTGTAAAAATGGGATTTGTGGATAGCAAGCGAGTTGCAATTTGGGGC TGGTCATATGGAGGGTATGTAACCTCAATGGTCCTGGGATCGGGAAGTGGCGTGT TCAAGTGCGGAATAGCTGTGGCACCTGTGTCACGGTGGGAGTACTATGACTCAGT GTACACAGAGCGTTACATGGGTCTCCCAATTCCAGAAGACAACCTTGACCATTAC AGGAACTCAACAGTCATGAGCAGAGCTGAACATTTTAAACAAGTTGAGTACCTCC TTATTCATGGAACGGCAGATGATAATGTTCACTTTCAGCAGTCAGCTCAGATCTC CAAAGCCCTGGTGGATGCTGGTGTGGATTTCCAAGCAATGTGGTACACGGATGAA GACCACGGGATCGCTAGCAGCACAGCTCACCAGCACATCTATTCCCACATGAGCC ATTTCCTCCAGCAGTGCTTCTCCTTACACTAGCACAGCATAGCTCTCCATAGCTTA TTTAAGACCACATTTGTTCTCATTATCTCAAAAGTGCACTGTTAAGATGAAGATCT TAATAATGTTGCATTGAGACATTTCAGGCTGCTTTCTCCAGTTTTACACCTGCAAT CCTAACTAAGGATGCCTGTCCCCAGAACAGATTACTACCTTAGAGCAATTTGGAT TTTCCCCTCTGTTTTGTTTATCATTTAAAATCATTTCCACATCAGCTGCTGAAACA ACAAATATAAATTATTTTTGCAAGAGCTATGCATAGATTTCCTGAGCAGAATTTC AATTTTTTTTTTCCTTACTAGACTGGTCCAAATCTCGTTTCCCTTATTTAAGTGGGT GACAAGATGTGGGTAATGATGTCATTAGGGCAGCAACAAGAGAAGAGGGAGCAG GGAGTATGGCTAGAAACCCAGGTCCAAGCATACAAACCAACCAGGCTACTGTCA GCTCGCCTCGAGAAGAGCTGTTCACTGCCAGACTGGCACAGTTTTCTGAGAAAGA CTATTCAAACAGTCTCAGGAAATCATATATGCAAAGCACTGACTTCTAAGTAAAA CCACAGCAGTTGAATAGACTCCAAAGAAATGCAAGGGACGCTGCCAGCAATGTA AGGGCCCCAGGTGCC AGTTATGGCTATAGGTGCTAC AAAAAC ACAGCAAGGGTG ATGGGAAAGCATTGTAAATGTGCTTTTGAAAATTACCGATGTCTCCTAGTGAAAA GAGGCAGCTTGGAACATATCGACTTGCCCTGTTAAAAGTTGAAAATATTTGTGTC ACAAATTCTAACATGAAGGAATACTTGCATTAGTTTTTCCTACTTCATTTCTTTGA GCATTTTAATTAAAATATTTTAACTTCATTAGCTTTTTAATGGAAAATTAAATGAG AATGTTTTGTGTTATTATTTCTATTCTAC ACACTGGAACTTTGCCTGGTC ATTTAGT AAATATGCTTCCATTTTTCAGAGGTAATGGGTTATGTCTTGAATCAAACTTACACC GCATTGACATATGGACACATTTGTTCAAAGGTTCTTGTTTAACTTGTTAGACATCC AAGACTGTCTTGTAAACATGGAAAGAGTTCAGCTTTTTTTTAAAAAAAATTAGAT ACATAAAACTGTTTAAACTTAAATGATTCATAGGGGTTTCTCTAATACCCCCTGA AATGTCTATTTTCACACTTATCACATAGCTTTGGAGAAATCAATTAACAATAATTT GGTCATTTAAATACTATGGAATTCATAATATAATTTAATCTAGGGGAGCATGTGA GGCTCACAGCATGGGACAGAACACTATCAGCAGAGTCGTTTTGGTTGACTTGTCT TTCCAGGGTTTTCTGTATTGTTTTGTCTCTGGTCTCTTCTTGTGGGGGGGTGATATA ATAGAAAACATACATTCTGATATTGAACTTGACTTAGCAATGTTTGGATAACTTA GAAAATACTACGTTTTGTTTCAATTATTTATAACACAACTGCATTTTTAAAATGGA CTTCACACATTTAAATGCAGTTTCAGACCAGAGTGCTAGATAAAATTATTCTGGA CAGGTGAGCCACAGCATGAATTGCAAACGTGGCAGAAATGGAAACTACCTATCA AATAAGGGTGGGATTAGATATTCATGAGCTTGATGCCACTTCCCAGGCCCCCCCA CGGATTCACTCCACCTTAAGTAAGTCTCAAGTCCTATTCTTCCATTGCAAGCACTG CTGGTAAGTAAGCCTGTAGGTTTCTGGCTATAATCCTATTTTAGAATACAGCACTT TCACCATGTGCAGCAGTATCCACCTGCAAAGTTCTAGATCAAGGGACAGAGGTGA CATGTTCCTGTCAGTGTGAAAACATAAACAGAAATTACCCGTTGTGAATTCATCA ATCTAAACCCCATTATATAATAATCTAGATTTTATTAATATATCTTATATGATGTA TCACATAATATTTATATAATAGCCAAACCATGAATTTTGTGTTCCTTTTGTTTCTA AATATGTTGTTTCTGGAGCCTGAATTAATCAAATATCACAGCACACATTTTTCAGG CAATGATGGTTTCAAATGGTGATAATGTGAATAAATTCAGTTTTTATTTGACTCTC ATAGCACAAGTTTTACTGTTCCGTAAGAGTTGTGAATTAGATTAAGAACCTGATG CGGAAGTTCTAAGTGAACTCAAGAGTAAGTTTGAAAAGCACAAGAGCTAGTAAC GTCACTAAGGGACATAGACCTGTCTCTGAGGGACATATGCATTCTCTGGAAGCAC ATGGATCGAGCTTCCTTCTCAGAGCACACCAGCATCAGGGGCGCATGCGCTAAGG GAAGGCCGCCCTGACGCTTCACTATCCAGGATCACTTCAGTCTGTGCCATTCCGG GCACTGTGGTTCTCGGAGTATTAGCAGTTCCACCTCTTGGCGCTTATTGCTCTTGC TCTAAGTTGGAGTGAGAATGCCGGTTTATAAAATTACTAGATTTGTAC AATATTT AAGATTCAATAAATTCTAAGTGAAAAAACA

FURTHER ASPECTS AND EMBODIMENTS

In a further aspect, the invention relates to a method of treating a bone fracture in a subject in need thereof, comprising administering an effective amount of a DPP-4 inhibitor to the subject. In a further aspect, the invention relates to a menthod of preventing non-union of a bone fracture or of preventing healing complications following bone fracture in a subject in need thereof, comprising administering an effective amount of a DPP-4 inhibitor to the subject. In a further aspect, the invention relates to a method of promoting fracture healing in a subject in need thereof, comprising administering an effective amount of a DPP-4 inhibitor to the subject.

In an embodiment of the methods according to the above aspects, the bone fracture is a non-union bone fracture, a compound fracture or a delayed fracture healing. In a further embodiment the DPP-4 inhibitor is administered systemically. In a further embodiment, the DPP-4 inhibitor is administered locally. In a further embodiment, the DPP-4 inhibitor is administered to the site of fracture. In a further embodiment the DPP-4 inhibitor is administered in one or more doses over a period of less than 6 months. In a further embodiment, the DPP-4 inhibitor is administered in one or more doses over a period of less than 3 months. In a further embodiment, the DPP-4 inhibitor is administered in one or more doses over a period of less than 1 month. In a further embodiment, prior to diagnosis of the bone fracture, the subject was not receiving a DPP-4 inhibitor. In a further embodiment, the subject in need thereof was not, prior to the bone fracture, diagnosed with or treated for Type II diabetes. In a further embodiment, the subject in need thereof is over age 65. In a further embodiment, the subject in need thereof is under age 40. In a further embodiment, the subject in need thereof has a BMI less than 25. In a further embodiment, the subject in need thereof has a BMI of greater than or equal to 25 and less than 30. In a further embodiment, the subject in need thereof has a BMI of equal to or greater than 30. In a further embodiment, the DPP-4 inhibitor is administered in one or more doses, and wherein at least one of the doses is administered locally during surgery to set the fracture. In a further embodiment, the subject in need thereof is a human subject. In a further embodiment, the healing complications are osteoporosis related healing complications to bone fracture. In a further embodiment, the DPP-4 inhibitor is selected from one or more of alogliptin, linagliptin, saxagliptin, sitagliptin, vildapliptin, gemigliptin, or teneligliptin. In a further embodiment, the method further comprises administering metformin, in the same or a different formulation as the DPP-4 inhibitor. In a further embodiment, the method further comprises administering one or more other therapeutic agent.

In a further aspect the invention relates to a method for reducing the inhibitory effects of marrow adipose tissue (MAT) on fracture healing in a subject in need thereof, comprising administering an effective amount of a DPP-4 inhibitor to the subject.

In a further aspect the invention relates to a method of preparing cells for transplantation, comprising providing a cell culture comprising osteogenic progenitor cells (OPCs) and/or mesenchymal stem cells, contacting the cells with a DPP-4 inhibitor in an amount effective to increase osteogenic gene expression, or osteo-/ chondrogenic cell differentiation, or osteogenic lineage commitment, thereby generating a culture comprising DPP-4 treated cells, and transplanting cells from the culture to a fracture in a subject in need thereof.

In a further aspect the invention relates to a method of preparing cells for transplantation, comprising providing a cell culture comprising CD45^"CD31^"Scal⁺CD24⁺ multipotent cells, treating the cell culture to promote lineage commitment and/or differentiation into osteogenic progenitor cells (OPCs) and/or mature cells of the osteogenic lineage/bone tissues, sorting the cells to isolate or enrich for OPCs, and transplanting the OPCs to a fracture in a subject in need thereof.

In a further aspect the invention relates to a method for preventing lose of bone mineral density (BMD) in an astronaut or other individuals exposed to an altered gravity environment, said method comprising administering to the astronaut or individual an effective amount of a DPP-4 inhibitor. In an embodiment of this method, the astronaut is exposed to an altered gravity environment for greater than one week, and the DPP-4 inhibitor is administered prior to and/or during and/or after the exposure.

In a further aspect the invention relates to a method of preventing loss of bone mineral density (BMD) in a subject in need thereof, comprising administering to the subject an effective amount of a DPP-4 inhibitor.

Claims

1. A DPP-4 inhibitor for use in treating a bone fracture in a subject in need thereof.

2. A DPP-4 inhibitor for use in preventing non-union of a bone fracture or preventing healing complications following bone fracture in a subject in need thereof.

3. A DPP-4 inhibitor for use in promoting fracture healing in a subject in need thereof.

4. A pharmaceutical composition comprising a DPP-4 inhibitor for use in treating a bone fracture in a subject in need thereof, wherein said pharmaceutical composition further comprises a pharmaceutically-acceptable diluent, excipient, or carrier, and wherein the DPP-4 inhibitor is present in an effective amount to treat or prevent bone fractures.

5. A pharmaceutical composition comprising a DPP-4 inhibitor for use in preventing nonunion of a bone fracture or in preventing healing complications following bone fracture in a subject in need thereof, wherein said pharmaceutical composition further comprises a pharmaceutically-acceptable diluent, excipient, or carrier, and wherein the DPP-4 inhibitor is present in an effective amount to treat or prevent bone fractures.

6. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-5, wherein the bone fracture is a non-union bone fracture, a compound fracture, or a fracture with delayed healing.

7. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-6, wherein the DPP-4 inhibitor is administered systemically.

8. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-6, wherein the DPP-4 inhibitor is administered locally.

9. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-6, wherein the DPP-4 inhibitor is administered to the site of fracture.

10. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-9, wherein the DPP-4 inhibitor is administered in one or more doses over a period of less than 6 months.

11. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-10, wherein the DPP-4 inhibitor is administered in one or more doses over a period of less than 3 months.

12. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-11, wherein the DPP-4 inhibitor is administered in one or more doses over a period of less than 1 month.

13. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-12, wherein, prior to diagnosis of the bone fracture, the subject was not receiving a DPP-4 inhibitor.

14. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-13, wherein the subject in need thereof was not, prior to the bone fracture, diagnosed with or treated for Type II diabetes.

15. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-14, wherein the subject in need thereof is over age 65.

16. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-15, wherein the subject in need thereof is under age 40.

17. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-16, wherein the subject in need thereof has a BMI less than 25.

18. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-16, wherein the subject in need thereof has a BMI of greater than or equal to 25 and less than 30, or wherein the subject in need thereof has a BMI of equal to or greater than 30.

19. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-18, wherein the DPP-4 inhibitor is administered in one or more doses, and wherein at least one of the doses is administered locally during surgery to set the fracture.

20. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-19, wherein the subject in need thereof is a human subject.

21. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-20, wherein the healing complications are osteoporosis related healing complications to bone fracture.

22. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-21, wherein the DPP-4 inhibitor is selected from one or more of alogliptin, linagliptin, saxagliptin, sitagliptin, vildapliptin, gemigliptin, or teneligliptin.

23. The DPP-4 inhibitor or pharmaceutical composition for use of claim 22, wherein the use further comprises administering metformin, in the same or a different formulation as the DPP-4 inhibitor.

24. The DPP-4 inhibitor or pharmaceutical composition for use of any of claims 1-22, wherein the use further comprises administering one or more other therapeutic agent.

25. A DPP-4 inhibitor for use in reducing the inhibitory effects of marrow adipose tissue (MAT) on fracture healing in a subject in need thereof.

26. A method of preparing cells for transplantation, comprising

providing a cell culture comprising osteogenic progenitor cells (OPCs) and/or mesenchymal stem cells,

contacting the cells with a DPP-4 inhibitor in an amount effective to increase osteogenic gene expression, or osteo-/ chondrogenic cell differentiation, or osteogenic lineage commitment, thereby generating a culture comprising DPP-4 treated cells.

27. A method of preparing cells for transplantation, comprising providing a cell culture comprising CD45^~CD31^~Scal⁺CD24⁺ multipotent cells, treating the cell culture to promote lineage commitment and/or differentiation into osteogenic progenitor cells (OPCs) and/or mature cells of the osteogenic lineage/bone tissues, and

sorting the cells to isolate or enrich for OPCs.

28. DPP-4 treated cells prepared by the method of claim 26 or 27 for use in transplantation to a fracture in a subject in need thereof.

29. A DPP-4 inhibitor for use in preventing loss of bone mineral density (BMD) in an astronaut or other individuals exposed to an altered gravity environment.

30. The DPP-4 inhibitor for use of claim 28, wherein the astronaut is exposed to an altered gravity environment for greater than one week, and the DPP-4 inhibitor is administered to the astronaut prior to and/or during and/or after the exposure.

31. A DPP-4 inhibitor for use in preventing loss of bone mineral density (BMD) in a subject in need thereof.