WO2011133959A1

WO2011133959A1 - Endothelial colony forming cells for enhancement of bone generation

Info

Publication number: WO2011133959A1
Application number: PCT/US2011/033698
Authority: WO
Inventors: Mervin C. Yoder; Jiliang Li
Original assignee: Indiana University Research And Technology Corporation
Priority date: 2010-04-23
Filing date: 2011-04-23
Publication date: 2011-10-27

Abstract

Devices and methods for enhancing bone generation are disclosed herein. Endothelial colony forming cells (ECFCs) associated with a biocompatible scaffold promote bone generation when implanted to a bone defect. ECFCs may also be associated with a biocompatible matrix and injected to the site of a bone defect to promote bone generation.

Description

ENDOTHELIAL COLONY FORMING CELLS FOR ENHANCEMENT OF BONE

GENERATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional

Application Serial No. 61/327,564, filed on April 23, 2010, the entire disclosure of which is incorporated herein by reference.

TECHNICAL FIELD

[0002] The present disclosure relates generally to devices and methods for bone generation. More specifically, the present disclosure relates to enhanced bone generation by the application of endothelial colony forming cells (ECFC) to fractured or defective bone. BACKGROUND AND SUMMARY

[0003] The importance of blood vessel formation in bone repair can be observed during endochondral ossification, where the cartilage template is replaced by primary bone formation. Chondrocytes close to the center of the shaft undergo hypertrophy and then apoptosis, leaving cavities in a calcified matrix. Endothelial cells play a important role, in part by being part of the blood vessels which grow into the template, allowing mesenchymal stem cells (MSCs) to enter and form osteoblasts that begin to lay osteoid matrix. Once bone formation spreads and a marrow cavity forms, vessels enter the epiphyses and more spongy bone is created at these secondary ossification areas. Fracture healing normally results in increased blood flow to the surrounding tissue. The relevance of vessel formation is further demonstrated by through the use of inhibitors of angiogenesis, which completely prevent fracture healing and callus formation. Meanwhile, factors such as vascular endothelial growth factor (VEGF) promote fracture healing, leading to a more rapid repair response.

Vascularization and angiogenesis are important components of bone repair.

[0004] Although bone has the capacity to self-heal, this is limited to a relatively minor facture. In conditions such as extensive trauma after an accident or after removal of bone tumor, the self-healing capacity is limited and requires intervention. A current method of treatment involves the use of donor bone autografts in which the patient's healthy bone from one site is excised and implanted into the defect. However, this approach is not ideal because of the limited source of bone from the individual and the potential chronic pain to the patient (Petit et al. (2000) Nat. Biotechnol. 18(9):959-63). Alternatively, processed bone allograft remains an attractive substitute for bone grafting despite the fact that allografts have a high complication rate including nonunion, fracture and infection due to lack of adequate blood supply and bone remodeling (Garbuz et al. (1998) Orthop Clin. North Am. 29(2): 199-204; Stevenson S. (1999) Orthop Clin. North Am. 30(4):543-52. In addition, alloplastic bone substitutes (synthetic bone) have not performed as well as natural bone materials because the synthetic bones have poor integration to existing bone and poor adaptation of soft tissues to the graft probably due to the difficulty to establish the blood circulation between synthetic bone and its surrounding tissues. Vascular endothelial growth factor (VEGF) protein and gene therapy have not been successful in the clinic to accelerate angiogenesis in order to improve the healing of bone grafts in patients.

[0005] Described herein is a novel stem cell therapy using endothelial colony-forming cells (ECFCs) to facilitate bone regeneration. This cell therapy can be used to treat critical size bone defects after removal of bone tumors or caused by injuries, such as battlefield blast injuries. This therapy can also be used to treat ischemic bone diseases (bone necrosis) and accelerate bone healing, including treatment of nonunion fracture and acceleration of spinal fusion. Implantation of ECFCs to a bone defect area can increase formation of new blood vessels and promote new bone formation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 shows colony formation of rat pulmonary microvascular endothelial cells (RPMVECs).

[0007] Figure 2 shows Fluorescein Lycopersicon Esculentum (tomato) Lectin (LEL) bind N-acetylglucosamine oligomers. Fluorescence and brightfield displays of ECFCs (upper panels) and of osteoblast controls (lower panels) are displayed.

[0008] Figure 3 shows that Fluorescent Acetylated Low-Density Lipoprotein (AcLDL) complexes were taken up by ECFCs.

[0009] Figure 4 shows vessel formation of ECFCs on Matrigel® following 4 hours of incubation (left panel) and 24 hours of incubation (right panel).

[0010] Figure 5 shows vessel formation of GFP-transgenic ECFCs on

collagen/fibronectin gels following 4 days of incubation (left panel) and 14 days of incubation (right panel).

[0011] Figure 6 shows growth of ECFCs in the type I collagen scaffold following 4 days of incubation (left panel) and 14 days of incubation (right panel).

[0012] Figure 7 shows growth of ECFCs in the type I collagen scaffold (left panel). A magnified view of the growth of ECFCs in the collagen scaffold is also shown (right panel). [0013] Figure 8 shows increasing growth of ECFCs in the type I collagen scaffold on days 1, 3, 5, and 7.

[0014] Figure 9 shows various views of an exemplary scaffold. The scaffold in this example is Hydroxyapatite (20%)/ β-Tricalcium phosphate (80%) with a porosity of 60%.

[0015] Figure 10 shows growth of ECFCs in the hydroxyapatite scaffold following 48 hours of incubation (Panel A) and a cross-section (Panel B).

[0016] Figure 11 shows X-ray images and μCT images of healed midshaft femur fractures at 6 weeks post-surgery. Panel (A) shows fractures in the control group (no ECFC implantation). Panel (B) shows fractures of the ECFC-treated group (with ECFC

implantation). Fractures of the ECFC-treated group demonstrate greater callus formation at the fracture site and greater callus bridging compared to the control group.

[0017] Figure 12 shows properties of healed femurs. Panel (A) shows bone mineral content (BMC) at the femoral midshaft of the control group and of the ECFC-treated group. The BMC was significantly greater in the ECFC-treated group compared to the control group. Panel (B) shows bone mineral density (BMD) at the femoral midshaft of the control group and of the ECFC-treated group. The BMD was not statistically different between the control group and the ECFC-treated group.

[0018] Figure 13 shows mechanical properties of healed femurs. Panel (A) shows ultimate force testing of the control group (scaffold-only) and of the ECFC-treated group. The ultimate force was not statistically different between the control group and the ECFC-treated group (p = 0.85) at this timepoint in this experiment. Panel (B) shows stiffness testing of the control group and of the ECFC-treated group. The stiffness was not statistically different between the control group and the ECFC-treated group (p = 0.24) at this timepoint in this experiment. Panel (C) shows energy to failure testing of the control group (scaffold-only) and of the ECFC-treated group. The ECFC-treated group demonstrated a statistically significant greater strength compared to the control group (p < 0.05).

[0019] Figure 14 shows X-ray images, 3-D μCT images, and histology images at 2 weeks after surgery. Panels Al, A2, and A3 show vascular cast following perfusion of radiopaque Microfil silicone for the control group. Panels Bl, B2, and B3 show vascular cast following perfusion of radiopaque Microfil silicone for the ECFC-treated group.

[0020] Figure 15 shows time-course X-ray images after surgery. Panel (A) shows the control group (no local transplantation of ECFCs into the defective area). In the control group, bone failed to regenerate bone in the defective area. Panel (B) shows the ECFC-treated group (with local transplantation of ECFCs into the defective area). In the ECFC-treated group, local transplantation of ECFCs into the defective area stimulated new bone formation.

[0021] Figure 16 shows properties of fibulae. Panel (A) shows bone mineral content

(BMC) at the midshaft of fibulae of the control group and of the ECFC-treated group. The BMC was significantly higher in ECFC-treated group compared to the control group

(p=0.0004). Panel (B) shows bone mineral density (BMD) at the midshaft of fibulae of the control group and of the ECFC-treated group. The BMD was significantly higher in the ECFC-treated group compared to the control group (p=0.0019).

[0022] Figure 17 shows μCT images and histological analysis of a bone defective area at 6 weeks after surgery. In the left panel, (A) shows the defective area in a control group (without ECFC implantation). (B) shows the defective area in the ECFC-treated group (with ECFC implantation). Newly formed bone tissues were observed in the ECFC-treated group compared to the control group. In the right panel, (C) shows histological analysis of the newly formed bone tissues in the ECFC-treated group. Histological analysis shows that osteocytes, osteoblasts (dashed arrows) and osteoclasts (solid arrows) were observed within the newly formed bone.

[0023] Figure 18 shows segmental defects generated surgically at rat fibula over a 6 week period. Panel (A) shows a control group (HA/TCP scaffold implantation). The control group demonstrated new bone formation at the junction of scaffold and bone. Panel (B) shows an ECFC-treated group (HA/TCP scaffold with ECFC implantation). The ECFC-treated group demonstrated enhancement of more new bone formation compared to the control group.

[0024] Figure 19 shows properties of new bone. Panel (A) shows the bone mineral content (BMC) of new bone in the control group (HA/TCP scaffold implantation) and in the ECFC-treated group (HA/TCP scaffold with ECFC implantation). The BMC was significantly higher in the ECFC-treated group compared to the control group (p=0.032). Panel (B) shows bone mineral density (BMD) of new bone in the control group and in the ECFC-treated group. The BMD was significantly higher in the ECFC-treated group compared to the control group (p=0.030).

[0025] Figure 20 shows μCT images and histological analysis of newly formed bone tissues at 6 weeks after surgery. In the left panel, (A) shows the control group (HA/TCP scaffold implantation). (B) shows the ECFC-treated group (HA/TCP scaffold with ECFC implantation). More newly formed bone tissues were observed in the ECFC-treated group compared to the control group. In the right panel, (C) shows histological analysis of the newly formed bone tissues in the ECFC-treated group. The histological section displayed in (C) corresponds to the area shown by the dashed box in (B). Calcein and alizarin labeling in (C) indicates the active bone present at the fibula (host bone), the scaffold, and the area between the fibula and the scaffold.

[0026] Figure 21 shows properties of healed femurs. Panel (A) shows bone mineral content (BMC) at the femoral midshaft of the control group (injection of a collagen matrix) and of the ECFC-treated group (injection of a collagen matrix with ECFCs). The BMC was not statistically different between the control group and the ECFC-treated group at the timepoint of 6 weeks in this experiment. Panel (B) shows bone mineral density (BMD) at the femoral midshaft of the control group and of the ECFC-treated group. The BMD was not statistically different between the control group and the ECFC-treated group at the timepoint of 6 weeks in this experiment.

[0027] Figure 22 shows mechanical properties of healed femurs. Panel (A) shows ultimate force testing of the control group (injection of a collagen matrix) and of the ECFC- treated group (injection of a collagen matrix with ECFCs). There was a trend for ultimate force to be greater in the ECFC-treated group compared to the control group. Panel (B) shows stiffness testing of the control group and of the ECFC-treated group. There was a trend for stiffness to be greater in the ECFC-treated group compared to the control group. Panel (C) shows energy to failure testing of the control group and of the ECFC-treated group. There was a trend for energy to failure to be greater in the ECFC-treated group compared to the control group.

DETAILED DESCRIPTION

[0028] While the invention is susceptible to various modifications and alternative forms, specific embodiments will herein be described in detail. It should be understood, however, that there is no intent to limit the invention to the particular forms described, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the invention.

[0029] The following terms as used throughout the specification are intended to have the meaning set forth below:

[0030] As used herein, the phrase "enhanced bone generation" refers to an increase in the quantity, mechanical properties, and/or rapidity of bone generation or repair. Enhanced bone generation includes an acceleration of the regeneration of bone following fracture or injury, such that the bone heals or regenerates sooner with application of ECFCs. The quantity of regenerated bone can be measured, for example, by determining the amount of bone mineral content and/or bone mineral density at a given time point after application of ECFCs. The mechanical properties of bone can be measured, for example, by the stiffness, or amount of force or energy required to damage the bone.

[0031] As used herein, an ECFC population "substantially free of hematopoietic stem cells" refers to ECFC that have been isolated from blood or blood vessels and cultured according to methods such that few or no hematopoietic stem cells contaminate the population of ECFCs.

[0032] In one embodiment, an implantable device comprising a biocompatible scaffold seeded with endothelial colony forming cells (ECFCs) is disclosed, wherein the device is adapted to enhance bone generation. Biocompatible scaffolds are known to those skilled in the art, and may comprise materials including, but not limited to, hydroxyapatite, tricalcium phosphate, collagen, an apatite-collagen mixture, an apatite-collagen complex, collagen gel, collagen sponge, gelatin sponge, fibrin gel, fibronectin, synthetic peptide, an extracellular matrix mixture, alginic acid, agarose, polyglycolic acid, polylactic acid, a polyglycolic acid/polylactic acid copolymer, alginic acid, laminin, entactin, and various combinations thereof.

[0033] In one illustrative embodiment, the biocompatible scaffold comprises one or more extracellular matrix proteins that are capable of adhering ECFCs.

[0034] In one illustrative embodiment, the biocompatible scaffold comprises type I collagen. In another illustrative embodiment, the biocompatible scaffold comprises hydroxyapatite. The hydroxyapatite may be associated with a stabilizer, for example, tricalcium phosphate. In one illustrative aspect, the biocompatible scaffold comprises type I collagen associated with hydroxyapatite tricalcium phosphate. For example, the type I collagen may be wrapped around a hydroxyapatite tricalcium phosphate core.

[0035] ECFCs may be derived from multiple sources using appropriate isolation and culture conditions. In one embodiment, the ECFCs are derived from blood vessels. ECFCs that reside within the intimal layer of the blood vessel wall (or those released into the circulation) are the stem cells that form new blood vessels in vivo. In one illustrative aspect, the ECFCs are derived from micro vasculature.

[0036] In one embodiment, the ECFCs are derived from umbilical cord blood.

[0037] In one embodiment, the ECFCs are derived from peripheral blood. [0038] In the adult human, ECFCs can be found in peripheral blood at a density of about one in 100 million cells. ECFCs can be found in human cord blood at a much higher density; about one in every million cells. ECFCs can also be isolated from the vascular endothelium removed from cord blood arteries and vein or adult human blood vessels.

[0039] Isolation, culture and characterization of ECFCs have been previously described both in rodent and human blood and blood vessels (Alvarez et al. (2008) Am. J. Physiol. Lung Cell Mol. Physiol. 108(16): 1933-38; Yoder et al. (2007) Blood 109(5): 1801-09, the disclosure of each of which regarding ECFC isolation and culture is hereby incorporated by reference). ECFCs from microvasculature (also referred to as resident microvascular endothelial progenitor cells (RMEPCs) may be obtained, for example, by excising lung tissue, slicing the outer edges, and dissecting out the subjacent tissue and placing it in a dish containing cold (4° C) DMEM. Next, the tissue is digested with type II collagenase, rinsed with DMEM, transferred to a T75 flask, supplemented with DMEM enriched with 20% FBS and 100 U/ml penicillin- 100 μg/ml streptomycin and incubated at 37 °C with 5% C0₂ -21% 0₂. Cell-culture medium is replaced with DMEM enriched with 10% FBS and 1%

penicillin/streptomycin .

[0040] ECFCs from human adult peripheral blood may be obtained, for example, by first collecting fresh blood samples (50-100 ml) by venipuncture and anticoagulating in citrate phosphate dextrose solution from human volunteers. ECFCs from human umbilical cord blood, may be obtained, for example, by first collecting samples (20-70 ml) from healthy newborns (38-40 weeks gestational age in sterile syringes-containing citrate phosphate dextrose solution as the anticoagulant.

[0041] Human mononuclear cells (MNCs) may then be obtained from either the adult peripheral or umbilical cord blood. Briefly, 20-100 ml of fresh blood is diluted one to one with Hanks Balanced Salt Solution (HBSS) (Invitrogen, Grand Island, NY) and overlayed onto an equivalent volume of Ficoll-Paque (Amersham Biosciences) a ficoll density gradient material. Cells are centrifuged for 30 minutes at room temperature at 1800 rpms (740xg). MNCs are isolated and washed three times with EBM-2 medium (Cambrex, Walkersville, MD) supplemented with 10-20% fetal bovine serum (Hyclone, Logan, UT), 2%

penicillin/streptomyocin (Invitrogen) and 0.25 μg/ml of amphotericin B (Invitrogen)

(complete EGM-2 medium).

[0042] Buffy coat MNCs are initially re-suspended in 12 ml of EGM-2 medium

(Cambrex) supplemented with 10% fetal bovine serum, 2% penicillin/streptomyocin and 0.25 pg/ml of amphotericin B (complete EGM-2 medium). Four milliliters of cells are then seeded onto three separate wells of a six well tissue culture plate (BD Biosciences, Bedford MA) previously coated with extra cellular matrix proteins (e.g. type I rat tail collagen (BD

Biosciences) vitronectin, fibronectin, collagen type 10, polylysine, and the like). The plate is incubated at 37° C, 5% C02 in a humidified incubator. After 24 hours of culture, the nonadherent cells and debris are carefully aspirated, and the remaining adherent cells are washed one time with 2 ml of EGM-2 medium. After washing, 4 ml of EGM2 medium is added to each well. EGM-2 medium is changed daily until day 7 of culture and then every other day until the first passage.

[0043] Colonies of cells initially appear between 5 days and 22 days of culture and can be identified as well circumscribed monolayers of cobblestone appearing cells. For passaging, cells are removed from the original collagen coated tissue culture plates using 0.05% trypsin- 0.53 mM EDTA (Invitrogen), resuspended in 10 ml of EGM-2 media and plated onto 75 cm tissue culture flasks coated with type I rat tail collagen. Monolayers of endothelial cells are subsequently passaged after becoming 90-100% confluent.

[0044] Two approaches may be used to directly isolate the endothelial cells from arterial or venous vessels. In the first approach, a 20G blunt end needle is inserted into one end of an incised vessel and the vascular contents (plasma with blood cells) are flushed out the opposite end using sterile saline. Vascular clamps are then applied to isolate each end of the vessel (3-5 cm in length). A solution of 0.1% collagenase in Hanks balanced salt solution (HBSS) is injected through the vessel wall via a 23G needle, and the vessel segments are incubated for 5 min at 3°C. The vascular clamp from one end of the vessel is then removed and the endothelial cells are expelled via infusion of a cell dissociation buffer (Gibco) (injected through the distal end of the vessel opposite the "open" end of the vessel). The vessel segments are infused with a minimum of 10 ml of cell dissociation buffer. The suspended cells are centrifuged at 350 X g and washed in EBM-2 media with 10% FBS, counted, and viability checked using Trypan blue exclusion.

[0045] The second approach is better suited for large diameter vessels (> 1 cm). The vessel is incised along the entire length and opened with the endothelial lumen exposed. Any remaining blood cells and plasma are washed away with HBSS. The endothelium is removed by firm scraping with a rubber policeman in a single end-to-end motion. The cells adhering to the rubber policemen are washed free by swirling the policemen in a solution of EBM-2 with 10% FBS in a 6 cm tissue culture well (precoated with extracellular matrix proteins). Cells are cultured with visual examination each day. Colonies of endothelium emerge in 3-10 days. The adherent endothelial colonies are removed by trypsin-EDTA and transferred to T 25 flasks that are coated with extracellular matrix proteins. Cells may be seeded in 75 cm tissue culture flasks precoated with type I rat tail collagen in complete EGM-2 medium for passage.

[0046] In contrast to previously described endothelial progenitor cells (EPCs) that are obtained from bone marrow, ECFCs do not give rise to hematopoietic cells such as monocytes and macrophages. Thus, the ECFC population described herein is substantially free of hematopoietic stem cells. ECFCs, unlike EPC populations comprised of HSCs, give rise to endothelial progeny that form blood vessels.

[0047] In one embodiment, methods for enhancing bone generation using any of the devices described above are disclosed.

[0048] In another embodiment, a method for enhancing bone generation is disclosed wherein the method comprises injecting a composition comprising ECFCs directly into, or in close proximity to, the site of a bone defect. In one aspect, the composition further comprises a biocompatible matrix. In one illustrative embodiment, the ECFCs are associated with a biocompatible matrix prior to injection. In one aspect, the ECFCs are expanded prior to association with the biocompatible matrix and/or may expand while associated with the biocompatible matrix. An advantage of injection is that the surgical procedures required for implantation of a scaffold may be avoided.

[0049] In one illustrative embodiment, the bone defect is a result of a trauma or bone disease. Illustratively, the methods and devices described herein may be used to treat ischemic bone diseases (bone necrosis) or accelerate bone healing, including treatment of nonunion fracture and acceleration of spinal fusion. In addition, these ECFCs can be used in

combination with natural and synthetic biomaterials to produce vascularized bone grafts for transplantation.

[0050] It is demonstrated herein that ECFCs can induce new blood vessel formation and stimulate new bone formation using an experimental rodent model. Because ECFCs are very rare in peripheral blood circulation in rats, ECFCs from rat pulmonary microvascular endothelial cells were used for the experiments. The ECFCs were expanded in vitro and the cultured the cells were suspended into a type I collagen scaffold for in vivo implantation.

[0051] Currently, many researchers are investigating whether endothelial progenitor cells (EPCs), which may be isolated from bone marrow, can be used to stimulate bone regeneration in vivo. Bone marrow-derived EPC may secrete proangiogenic paracrine factors that can assist ECFCs to form new blood vessels; however, bone marrow derived EPCs, which contain hematopoietic stem cells, fail to form new blood vessels directly.

EXAMPLES

[0052] EXAMPLE 1. ECFCs enhance fracture repair.

[0053] Sprague-Dawley rats (12 weeks old) were used. The femur was fractured at its midshaft by means of a transverse osteotomy. The fractures treated with ECFCs in type I collagen scaffolds displayed a better healing and repair response (Figure 11). When type I collagen scaffolds containing ECFCs were surgically wrapped around the fractured femurs of rats, newly formed bone mineral at the site of fracture was 13% greater (p = 0.01), compared to the scaffold wrapped fractures without ECFCs (Figure 12). Bending test measurements performed 6 weeks following fracture demonstrated that energy to failure of the healed femurs treated with ECFCs was 46% greater (p = 0.01) than the healed femurs without ECFC treatment (Figure 13). These data demonstrate that ECFCs enhance bone formation at the fracture sites and significantly improve the recovery of mechanical properties, indicating that ECFCs enhance fracture healing.

[0054] EXAMPLE 2. ECFCs in Type I collagen scaffold induce formation of blood vessel and stimulate new bone formation in segment bone defects.

[0055] Sprague-Dawley rats (12 weeks old) were used. A five- millimeter segmental defect was produced at the right fibula. ECFCs (1 million cells) were seeded into Type I collagen sponge (Zimmer Dental, CA) and implanted into the defective area during surgery in the ECFC group. Only collagen scaffold without cells was implanted into the defective areas in the Control group.

[0056] Significantly more new blood vessels were found in the ECFC-treated group compared to the Control group at 2 weeks postoperatively (Figure 14). Furthermore, significant amount of new bone was observed (Figure 15) and higher bone density was detected in the ECFC-treated group in comparison with the Control group (Figure 16).

Histological tissue sections showed that newly formed bone tissues contain osteocytes, osteoblast and osteoclasts (Figure 17).

[0057] These data suggest that ECFCs can induce vasculogenesis and angiogenesis at the defect area and subsequently stimulate bone regeneration in the bone defect that bone normally fails to regenerate.

[0058] EXAMPLE 3. ECFCs within hydroxyapatite ( HA )/tri- calcium phosphate

(TCP) scaffold enhance new bone formation. [0059] Sprague-Dawley rats (12 weeks old) were used. A five- millimeter segmental defect was produced in the right fibula. In half of the animals (HA/TCP control group), a HA/TCP scaffold was placed into the bone gap during surgery. HA/TCP scaffold is a 5 mm long ceramic scaffold with 60% porosity. In the other animals (HA/TCP ECFC group), ECFCs were cultured into HA/TCP scaffold for one day before the scaffold was inserted into the bone gap during surgery.

[0060] Imaging by X-ray and microCT discovered that significantly more new bone formed in HA/TCP ECFC group compared to the HA/TCP Control group (Figure 18).

Significantly higher bone mineral content and density was detected in HA/TCP ECFC treated animals in comparison with pure HA/TCP treated animals (Figure 19). Undecalcified histological tissue sections showed fluorescent labels within HA/TCP scaffold, suggesting active new bone formation in animals implanted with HA/TCP and ECFCs (Figure 20). These data suggest that ECFCs within HA/TCP scaffold can enhance new bone formation. In addition, infusion of a contrast agent into the vasculature of the treated animals revealed greater numbers and diameter of vessels in the HA/TCP ECFC group than in the contralateral control leg or the leg of the HA/TCP treated animal.

[0061] Overall, we have demonstrated that ECFCs can induce angiogenesis and subsequently stimulate and enhance new bone formation in both Type I collagen scaffold and hydroxyapatite/tri-calcium phosphate scaffold.

[0062] EXAMPLE 4. Injection of ECFCs in collagen matrix improves fracture healing

[0063] Twelve weeks old Sprague-Dawley rats were divided into 2 groups: Matrix

Control and Matrix ECFC groups. The recipient rats were anesthetized via intraperitoneal injection with a combination of Ketamine and Xylazine. An osteotomy was made at the mid- diaphysis of the femur. To stabilize the facture site, a 0.5 mm diameter pin is inserted into the medullary cavity. One week following fracture, in the Matrix ECFC group, immediately after ECFCs (10⁶ cells) were suspended in 500 μΐ collagen matrix solution on ice, the cellularized solution is injected around the fracture site. For the Matrix Control group, only the collagen matrix solution was injected.

[0064] The collagen matrix solution contained 0.5 to 3.5 mg/ml (final collagen concentration) rat tail type I collagen (BD Biosciences), 100 ng/ml human fibronectin

(Millipore), 1.5 mg/ml sodium bicarbonate (Sigma- Aldrich), 10% FBS, 25mM HEPES, 30% complete EGM-2, and EBM-2 (Lanza). ECFCs were suspended in the solution on ice just prior to injection using a pre-cooled syringe. [0065] The study was terminated at 6 weeks after fracture. There was no difference in bone mineral content (BMC) and bone mineral density (BMD) between Matrix control and matrix ECFC groups (Figure 21) at the timepoint of 6 weeks. Four points bending test showed ultimate force, stiffness and energy to failure were slightly greater in Matrix ECFC group, compared to the Matrix control group (Figure 22). These data suggest that injection of ECFC is a useful way to improve fracture repair.

[0066] While the invention has been illustrated and described in detail in the foregoing description, such an illustration and description is to be considered as exemplary and not restrictive in character, it being understood that only the illustrative embodiments have been described and that all changes and modifications that come within the scope of the invention are desired to be protected. Those of ordinary skill in the art may readily devise their own implementations that incorporate one or more of the features described herein, and thus fall within the scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. An implantable device comprising a biocompatible scaffold seeded with endothelial colony forming cells (ECFCs) wherein the device is adapted to enhance bone generation.

2. The device according to claim 1 wherein the biocompatible scaffold comprises one or more extracellular matrix proteins capable of adhering the ECFCs.

3. The device according to any of claims 1 to 2 wherein the biocompatible scaffold comprises type I collagen.

4. The device according to any of claims 1 to 3 wherein the biocompatible scaffold comprises hydroxyapatite.

5. The device according to any of claims 1 to 4 further comprising a stabilizer.

6. The device according to claim 5 wherein the stabilizer is tricalcium phosphate.

7. The device according to any of claims 1 to 6 wherein the ECFCs are derived from a source selected from the group consisting of cord blood, peripheral blood, blood vessels, and a combination thereof.

8. A method for enhancing bone generation, the method comprising implanting a device according to any of claims 1 to 7 to a bone defect.

9. The method of claim 8 wherein the bone defect is associated with a bone disease.

10. The method of claim 8 wherein the bone defect is associated with a trauma.

11. A method for enhancing bone generation, the method comprising injecting a composition comprising a biocompatible matrix and ECFCs, wherein the injection is to a bone defect.

12. The method of claim 11 wherein the bone defect is associated with a bone disease.

13. The method of claim 11 wherein the bone defect is associated with a trauma.