AU2005205790A1 - Replacement bone tissue - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S):: Heydrick Terheyden AND Patrick H. Warnke AND Eugene Sherry ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys Level 10, 10 Barrack Street, Sydney, New South Wales, Australia, 2000 INVENTION TITLE: Replacement bone tissue The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5102 P:\WPDOCS\CRN\SEnShenAl 2591661 .doc-26/08/05 -1- REPLACEMENT BONE TISSUE

ABSTRACT

Bone replacement tissue suitable for bone grafting procedures, said bone replacement tissue being grown in a host until suitable for transplantation into a patient, and methods for manufacturing said bone replacement tissue.

FIELD OF THE INVENTION The present invention relates to replacement tissue, and in particular, to replacement bone material suitable for use in bone grafting procedures, and to methods for manufacturing said replacement bone material.

BACKGROUND OF THE INVENTION Bone tissue is composed of a matrix that primarily consists of collagen protein, but is strengthened by deposits of calcium, hydroxyl and phosphate salts, referred to as hydroxyapatite. Inside and surrounding this matrix lie the cells of bone tissue, which include osteblasts, osteocytes, osteoclasts and bone-lining cells. All four of these cell types are required for building and maintaining a healthy bone matrix, as well as remodelling of the bone under certain conditions.

Injury, disease and developmental defects can all result in bone defects that require bone grafting procedures, where new bone or a replacement material is placed in apertures around a fractured bone, or holes/defects in bone. Bone grafting assists the bone to heal, or merely provides mechanical structure to the defective bone, through the provision of artificial material that is not incorporated into a patient's own bone.

Autografting may be used where it is appropriate to take the patient's own bone tissue from another site in the body, usually the iliac crest, although the distal femur and proximal tibia may also be used. Autografting has advantages in terms of its provision of P:\WPDOCS\CRN\SETSheny\12591661.doc-26/08/05 -2osteoconductivity (ie. the graft supports the attachment of new osteoblasts and osteoprogenitor cells). Furthermore, it provides osteoinductivity, or the ability to induce nondifferentiated cells into osteoblasts.

In the context autografting for injuries such as bone fractures, the grafting procedure can be quite complex, and may fail to heal properly. Grafting for bone fractures is generally only considered when a reasonable sized portion of bone has been lost via fracture. In this context, bone grafting may be performed using the patient's own bone, usually taken from the hip, or using bone from a donor. The donor/replacement bone is usually held in place by physical means (eg. screws and pins), while the healing process occurs.

The drawbacks for autografting procedures include surgical complications (eg. acute and chronic pain, inflammation, infection) and limitations in relation to the amount of bone that can be harvested for grafting. Furthermore, complications occurring after bone grafting include fracture at the donor site after cortical graft removal, intraoperative bleeding and postoperative pain after iliac crest biopsy and stress fractures, hernias through an iliac donor site and gait problems.

An alternative procedure, allografting, where bone graft material is taken from a donor or cadaver, offer some advantages over autografting in terms of the lack of surgical complications in obtaining the bone graft material. However, there is a risk of disease transmission from the donor to the recipient of the bone graft material, which is not overcome by pre-implanation treatment of the tissue with techniques such as gamma irradiation. Furthermore, the allograft may not knit well with the patient's own bone, leading to weakness at the point of union of the graft. Also, where bone is harvested from a donor, there exist the same risks as harvesting replacement bone from the patient, as discussed above.

A variety of alternative graft materials exist, including ceramic materials, polymeric materials and chemically inert substances. These bone substitutes are often innoculated P:\WPDOCS\CRN\SET\Sherry 12591661.doc-26/08/05 -3with bone marrow and/or growth factors to provide osteoconductivity and osteoinductivity properties provided by use of autografted bone.

In the case of certain bone substitute materials, there is the disadvantage that they do not become permanently incorporated into a patient's own bone and are thus subject to breakage, loosening and erosion.

Furthermore, while in vivo bone reconstruction using a polymeric matrix has been found to have the capacity for bone regeneration (Borden et al., J Bone Joint Surg Br. 2004 Nov;86(8):1200-8; Mankani et al., Biotechnol Bioeng. 2001 Jan 5;72(1):96-107), the site of regeneration will naturally be in a weakened state until full bone mineralisation and osteoblast replacement is attained.

Accordingly, there remains a need for improved methods in bone replacement technology.

SUMMARY OF THE INVENTION The present invention is predicated on the finding that bone replacement material may be grown in vivo in certain tissues of the body, and be provided with a blood supply via angiogenesis of the bone replacement material. These blood vessels may thus be sutured to the blood vessels that vascularise the site where the bone replacement material is required Accordingly, in a first broad form, the present invention relates to a method for growing replacement bone tissue for a patient, said method comprising: a) providing a scaffold for the replacement bone tissue; b) inoculating the scaffold with osteoblast precursor cells; c) implanting the scaffold subcutaneously, or into fat or muscle tissue in a host; a) harvesting the scaffold, replacement bone and a portion of the blood supply of the replacement bone when sufficient formation and angiogenesis of replacement bone has occurred; P:\WPDOCS\CRN\SET\Sherr\ 259166 I.doc-26/08/05 -4b) transplanting the scaffold, replacement bone and the blood supply of the bone to a site where replacement bone tissue is required in the patient; and c) connecting at least part of a blood supply of the replacement bone to a blood vessel at the site where the replacement bone tissue is required.

Preferably, inoculating the scaffold further involves inserting hydroxyapatite crystals into the scaffold.

Even more preferably, the hydroxyapatite crystals are provided as bone mineral blocks.

In a further preferred form, the osteoblast precursor cells are provided in mixture of bone marrow cells.

In yet a further preferred form, the osteoblast precursor cells are derived from a mixture of bone marrow cells.

In still yet another preferred form, the osteoblast precursor cells are mesenchymal stem cells.

Preferably, the osteoblast precursor cells are haematopoietic stem cells.

Even more preferably, the haematopoietic stem cells are derived from monocyte precursor cells.

In another preferred form, the osteoblast precursor cells are adult stem cells or embryonic stem cells isolated from an embryo of the host species.

Even more preferably, the stem cells are totipotent stem cells isolated from a fertilised egg of the host species.

P:\WPDOCS\CRN\SETrSher3A\12591661.doc-26/08/05 In one form, the osteoblast precursor cells are autologous with respect to the patient's tissue.

In another form, the osteoblast precursor cells are allogenic with respect to the patient's tissue.

In yet a further form, inoculating the scaffold further includes providing at least one growth factor within the scaffold.

In still yet another preferred form, inoculating the scaffold further includes providing at least one growth factor on the outer surface of the scaffold.

Preferably, the at least one growth factor is selected from the group consisting of the bone morphogenetic protein (BMP) family members.

Even more preferably, the BMP is BMP-2.

In another preferred form, the BMP is BMP-7.

In yet another form, anatomical modelling studies are performed for shaping the scaffold to optimise the scaffold shape to fit the site where replacement bone is required in the patient.

In a preferred form, the anatomical modelling studies include computed tomography or magnetic resonance imaging.

Preferably, the anatomical modelling studies further include use of computer-aided design.

In a further preferred form, the anatomical modelling studies include three-dimensional computed tomography.

P:\WPDOCS\CRN\SETSherry\l2591661.dc-26A)8/05 -6- In yet another preferred form the anatomical modelling studies include magnetic resonance imaging (MRI).

In a further form, the scaffold is a suitable biocompatible and/or bioabsorbable material.

Preferably, the biocompatible and/or bioabsorbable material is selected from titanium, stainless steel, zirconium oxide, ceramic tricalcium phosphate, polymers.

Even more preferably, the scaffold is titanium.

In yet another preferred form, the scaffold comprises a mesh-like structure.

In another form, the muscle into which the scaffold is implanted is a trunk muscle selected from the group consisting of Trapezius, Latissimus dorsi, Pectoralis major, Pectoralis minor, Levator scapulae, Rhomboid minor, Rhomboid major, Serratus anterior, Deltoid, Supraspinatus, Infraspinatus, Teres minor, Teres major, Subscapularis, Splenius capitis, Splenius cervicis, Iliocostalis lumborum, Iliocostalis thoracis, Iliocostalis cervicis, Longissimus thoracis, Longissimus cervicis, Longissimus capitis, Spinalis thoracis, Spinalis cervicis: Spinalis capitis, Semispinalis thoracis, Semispinalis cervicis, Semispinalis capitus, Multifidus, Short rotators, Interspinalis, Intertransversi, Trapezius, Latissimus dorsi, Subclavius, Pectoralis major, Pectoralis minor, Levator scapulae, Rhomboid minor, Rhomboid major, Serratus anterior, Deltoid, Supraspinatus, Infraspinatus, Teres minor, Teres major, Subscapularis, Splenius capitis, Splenius cervicis, Iliocostalis lumborum, Iliocostalis thoracis, Iliocostalis cervicis, Longissimus thoracis, Longissimus cervicis, Longissimus capitis, Spinalis thoracis, Spinalis cervicis, Spinalis capitis, Semispinalis thoracis, Semispinalis cervicis, Semispinalis capitus, Multifidus, Long rotators, Short rotators and the Interspinalis, Intertransversi.

In yet another form, the muscle into which the scaffold is implanted is a leg muscle selected from the group consisting of Tensor fascia lata, Gluteus maximus, Gluteus medius, Gluteus minimus, Piriformis, Superior gemellus, Obturator internus, Inferior P:\WPDOCS\CRN\SET1Sherry\ 12 59166 1. doc-26)8/5 -7gemellus, Quadratus femoris, Semitendinosus, Adductor longus, Adductor brevis, Adductor magnus, Gracilis, Obturator externus, Sartorius, Rectus femoris, Vastus lateralis, Vastus intermedius, Vastus medialis, Articularis genus, Psoas major, Illiacus, Pectineus, Soleus, Plantaris, Popliteus, Flexor digitorum longus, Tibialis posterior, Flexor hallucis longus, Peroneus longus, Peroneus brevis, Tibialis anterior, Extensor hallucis longus, Extensor digitorum longus and Peroneus tertius.

In another preferred form, the muscle into which the scaffold is implanted is a head or neck muscle selected from the group consisting of Obliquus capitis inferior, Obliquus capitis superior, Rectus capitis posterior major, Rectus capitis posterior minor, Longus colli, Longus capitis, Rectus capitis anterior, Rectus capitis lateralis, Anterior scalene, Scalenus minimus (may be absent), Middle scalene, Posterior scalene, Sternocleidomastoid, Platysma, Sternohyoid, Omohyoid, Sternothyroid, Thyrohyoid, Stylohyoid, Digastric, Mylohyoid, Geniohyoid, Occipitalis (2 bellies), Frontalis (2 bellies), Orbicularis oculi, Corrugator supercilii, Levator labii superioris alaeque nasi, Levator labii superioris, Zygomaticus minor, Zygomaticus major, Risorius (may be absent), Levator anguli oris, Buccinator, Depressor anguli oris, Depressor labii inferioris, Masseter, Medial pterygoid, Lateral pterygoid, Levator palpebrae superioris, Lateral rectus, Medial rectus, Superior rectus, Superior rectus, Inferior rectus, Superior oblique and the Inferior oblique.

In still another preferred form, the muscle into which the scaffold is implanted is an arm muscle selected from the group consisting of Coracobrachialis, Biceps brachii, Brachialis, Triceps brachii, Anconeus, Pronator teres, Flexor carpi radialis, Palmaris longus, Flexor carpi ulnaris, Flexor digitorum superficialis, Flexor digitorum profundus, Flexor pollicis longus, Pronator quadratus, Brachioradialis, Extensor carpi radialis longus, Extensor carpi radialis brevis, Extensor digitorum, Extensor digiti minimi, Extensor carpi ulnaris, Supinator, Abductor pollicis longus, Extensor pollicis brevis, Extensor pollicis longus, Extensor indicis, Abductor pollicis brevis, Flexor pollicis brevis, Opponens pollicis, Adductor pollicis, Palmaris brevis, Abductor digiti minimi, Flexor digiti minimi brevis, Opponens digiti minimi, Palmar interossei, Dorsal interossei, Lumbricals, Abductor hallucis, Flexor digitorum brevis, Abductor digiti minimi, Abductor ossis metatarsi quinti, P:\WPDOCS\CNSETlSherry259166 I.dc-26/08A)S -8- Quadratus plantae, Lumbricals, Flexor hallucis brevis, Adductor hallucis, Flexor digiti minimi brevis, Plantar interossei (3 muscles), Dorsal interossei (4 muscles), Extensor hallucis brevis and Extensor digitorum brevis.

In another broad form, the patient and the host are different organisms.

In still another form, the patient and the host are the same organism.

In a further broad form, the present invention provides a kit for growing replacement bone for a patient, the kit comprising: a) a scaffold suitable for supporting bone growth subcutaneously or within fat or muscle tissue of a host; and b) a source of osteoblast precursor cells.

Preferably, the kit further includes at least one growth factor, and even more preferably, an osteoblast growth factor.

Even more preferably, the kit further includes hydroxyapatite crystals suitable for placement in the scaffold.

In another preferred form, the kit includes hydroxyapatite crystals pre-placed within the scaffold.

In yet another preferred form, the hydroxyapatite crystals of the kit are present as bone mineral blocks.

In another broad form, the present invention relates to a bone graft material, suitable for transplantation into a patient requiring said graft, said bone graft material comprising: a) a scaffold housing bone tissue; and b) a blood supply comprising one or more blood vessels for suturing into a region in the patients body where the bone graft is required; P:NWPDOCS\CRN\SE1 Sherry\ 125 9166 1. dm-26/08fO5 -9wherein said bone graft material has been manufactured according to the method of: a) providing a scaffold for the bone graft material; b) inoculating the scaffold with osteoblast precursor cells; c) implanting the scaffold subcutaneously, or into fat or muscle tissue in a host; d) harvesting the scaffold housing the bone tissue and a portion of a blood supply of the replacement bone when sufficient formation and angiogenesis of replacement bone has occurred.

Preferably, inoculating the scaffold further involves inserting hydroxyapatite crystals into the scaffold.

Even more preferably, the hydroxyapatite crystals for the bone graft material are provided as bone mineral blocks.

In another preferred form, the osteoblast precursor cells for the bone graft material are provided in a mixture of bone marrow cells.

In a further preferred form, the osteoblast precursor cells for the bone graft material are derived from a mixture of bone marrow cells.

In yet another preferred form, the osteoblast precursor cells for the bone graft material are mesenchymal stem cells.

In still yet another preferred form, the osteoblast precursor cells for the bone graft material are haematopoietic stem cells.

Preferably, the haematopoietic stem cells for the bone graft material are derived from monocyte precursor cells.

Even more preferably, the osteoblast precursor cells for the bone graft material are adult stem cells or embryonic stem cells isolated from an embryo of the host species.

P:\WPDOCS\CRNMSETSherr3A 12591661. doc-2608/05 In yet another preferred form, the stem cells for the bone graft material are totipotent stem cells isolated from a fertilised egg of the host species.

In still another form, the osteoblast precursor cells for the bone graft material are autologous with respect to the patient's tissue.

In another form, the osteoblast precursor cells for the bone graft material are allogenic with respect to the patient's tissue.

In still another preferred form, inoculating the scaffold further for the bone graft material includes providing at least one growth factor within the scaffold.

Preferably, inoculating the scaffold for the bone graft material further includes providing at least one growth factor on the outer surface of the scaffold.

Preferably, the at least one growth factor for the bone graft material is selected from the group consisting of the bone morphogenetic protein (BMP) family members.

Even more preferably, the BMP for the bone graft material is BMP-2.

In another preferred form, the BMP for the bone graft material is BMP-7.

In a particularly preferred form, anatomical modelling studies are performed for shaping the scaffold for the bone graft material to optimise the scaffold shape to fit the site where replacement bone graft material is required in the patient.

Preferably, the anatomical modelling studies for the bone graft material include computed tomography or magnetic resonance imaging.

Even more preferably, the anatomical modelling studies for the bone graft material further include use of computer-aided design.

P:\WPDOCS\CRN\SET1She~)1259166 .doc-26/08/05 11 In another preferred form, the anatomical modelling studies for the bone graft material include three-dimensional computed tomography.

Preferably, the scaffold of the bone graft material is a suitable biocompatible and/or bioabsorbale material.

Even more preferably, the biocompatible and/or bioabsorbable material for the bone graft material is selected from titanium, stainless steel, zirconium oxide, ceramic tricalcium phosphate, polymers.

In a further preferred form, the scaffold for the bone graft material is titanium.

Preferably, the scaffold for the bone graft material comprises a mesh-like structure.

Even more preferably, the patient receiving the bone graft material and the host in which the bone graft material is grown are different organisms.

In another preferred form, the patient receiving the bone graft material and the host in which the bone graft material is grown are the same organism.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1: Image showing titanium mesh scaffold for mandibular replacement FIGURE 2: Image showing titanium mesh scaffold for mandible loaded with bone growth materials.

FIGURE 3: CAD plan showing idealised mandibular scaffold (implant) P:\WPDOCS\CRMSEShcrry\12591661.do-26/08/05 12- DETAILED DESCRIPTION OF THE INVENTION Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

It has surprisingly been found by the present inventor that replacement bone material that is highly suitable for bone grafting can be grown in vivo when a bone scaffold containing osteoblast progenitor cells is placed in certain tissues in a host, thus avoiding the need for bone harvesting from a patient or donor.

Accordingly, in a first broad form, the present invention relates to a method for growing bone for a bone graft in a patient, wherein the method involves the steps of: d) providing a scaffold for the replacement bone tissue; e) inoculating the scaffold with osteoblast precursor cells; f) implanting the scaffold subcutaneously, or into fat or muscle tissue in a host; g) harvesting the scaffold, replacement bone and a portion of the blood supply of the replacement bone when sufficient formation of replacement bone has occurred; and h) transplanting the scaffold, replacement bone and the blood supply of the bone to a site where replacement bone tissue is required in the patient.

The term "patient" refers to patients of human or other mammal and includes any individual it is desired to examine or treat using the methods of the invention. However, it will be understood that "patient" does not imply that symptoms are present. Suitable mammals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals sheep, cows, horses, donkeys, pigs), laboratory test P:\WPDOCS\CRN\SETSherry\ 2591661 .doc-26/08/05 13 animals rabbits, mice, rats, guinea pigs, hamsters), companion animals cats, dogs) and captive wild animals foxes, deer, dingoes).

The term "host" as used herein refers to a mammal that may be the same organism as the patient, or a separate organism. That is, the scaffold may be implanted into subcutaneous tissue, fat tissue or muscle tissue of an animal that is the patient, or an animal that is not the patient.

Preferably, the host and the patient are the same organism. However, those skilled in the art will appreciate that bone growth within the scaffold may occur in another animals, and in particular, in another animal that has a tissue type identical with, or similar to, that of the patient. As is appreciated in the art, choosing such a host minimises the risks of tissue rejection following transplantation of the replacement bone tissue.

That is, where the host is different from the patient, it is preferable that the host has the same tissue type as the patient (eg. An identical twin), or a similar tissue type (ie. a relative, or a donor-matched tissue type). However, it is possible for the donor and patient to have significantly different tissue types, and in this situation, immunosuppressant drugs may be employed in order to reduce the risk of rejection of the bone replacement tissue.

In one embodiment, the host may be a different species from the patient. In particular, the host may be an animal such as a pig (ie. xenotransplantation). Particularly preferred are animals that are engineered to be humanised, and are thus far more suitable for xenotransplantation than non-humanised animals.

Preferably, the scaffold is filled with bone minerals (ie. hydroxyapatite), such as in the form of bone mineral crystals or blocks, which serve as hydroxyapatite carriers for cells and growth factors that are added to the cage. Suitable bone hydroxyapatite materials are known to those skilled in the art and include, for example, allograft-based bone graft substitutes in which allograft bone is used alone or along with other materials (eg. Allogro, Othroblast, Opteform, Grafton, VG1 ALIF, VG2 PLIF). Alternatively, ceramic materials P:\WPDOCS\CRN\SETSherry\12591661.doc-26/08/05 14may be used, which may be bioactive and/or resorbable (eg. calcium phosphate, bioglass plus Osteograf, Norian SRS, ProOsteon, Osteoset, Ossatura, Cerasorb, Chronos, BonePlast, Novabone, Novamin), or polymeric materials that may or may not be biodegradable, plus (eg. polymer of degradable or non-degradable synthetic collagen fiber felted mass. (eg. Cortoss, Immix, Infuse, Healos (collagen with HA coating)).

The osteoblast precursor cells that are used to inoculate the scaffold may be provided by a mixture of bone marrow cells, as bone marrow is known to contain mesenchymal stem cells and haematopoietic stem cells, both of which have an ability to differentiate into osteoblasts. Alternatively, the bone marrow mixture may be purified in order to provide a concentrated mix of either or both of these stem cells types.

The osteoblast precursor cells may be isolated from the patient and thus allogenic relative to the patient's own tissue, or may be isolated from another organism, and thus autogenous with respect to the patients own tissues.

In one embodiment, stem cells may be totipotent stem cells isolated from fertilised eggs from the host species, adult stem cells, or pluripotent stem cells which are embryonic stem cells isolated from the host species.

In a particularly preferred form, the scaffold is also inoculated with at least one osteoblast growth factor on an outer and/or inner surface of the scaffold, wherein the inner surface of the scaffold represents the surface housing the bone minerals. Preferably, the at least one growth factor is a bone morphogenetic protein (BMP). The BMPs are a group of related proteins originally identified by their presence in bone-inductive extracts of demineralized bone. Molecular cloning has revealed at least six related members of this family, which have been designated BMP-2 through BMP-7. These molecules are part of the TGF-13 superfamily.

P:\WPDOCS\CRN\SETShenrA12591661.doc-26/08/05 The BMPs can be divided into subgroups with BMP-2 and BMP-4 being 92% identical, and BMP-5, BMP-6, and BMP-7 being an average of about 90% identical. Single BMP molecules, such as BMP-2, are capable of inducing the formation of new cartilage and bone (Li et al., J Spinal Disord Tech. 2004 Oct;17(5):423-8). Whether each of the BMPs possesses the same inductive activities in an animal is the subject of ongoing research.

Studies of transgenic and knockout mice and from animals and humans with naturally occurring mutations in BMPs and related genes have shown that BMP signaling plays critical roles in heart, neural and cartilage development (Chen et al., Growth Factors. 2004 Dec;22(4):233-41).

In one preferred form, the BMP is BMP-7. In an even more preferably form, the BMP is BMP-2.

In another preferred form, anatomical modelling studies are performed prior to shaping the scaffold, in order to produce a scaffold having a shape that is optimised to fit into the region where the replacement bone material is required. A variety of techniques exist in the art and are well known by the skilled person, such as computed tomography and magnetic resonance imaging, which are preferably assisted by the use of computer-aided design.

In a preferred form, the scaffold is a suitable biocompatible and/or bioabsorbable material.

A variety of suitable biocompatible and/or bioabsorbable materials are known in the art, and include, but are not limited to, titanium, stainless steel, zirconium oxide, ceramic tricalcium phosphate and polymers. In a particularly preferred form, the scaffold is formed from titanium. Even more preferably, the scaffold comprises a mesh-like structure, and in particular, a titanium mesh-like structure.

The present invention has an advantage in that a nutrient supply is provided by placement of the bone scaffolds into areas where there is a relatively large blood supply, which is ideal for supporting growth and differentiation of osteoblast precursor cells, and the laying down of new bone within the bone scaffold.

PA\WPDOCS\CRN\SE'nShenV!259166 I.d-26)8/l5 -16- The anatomical regions into which the scaffold may be implanted include subcutaneously, into fat tissue or into a muscle.

Suitable muscles may be selected from muscles of the trunk selected from the group consisting of Trapezius, Latissimus dorsi, Pectoralis major, Pectoralis minor, Levator scapulae, Rhomboid minor, Rhomboid major, Serratus anterior, Deltoid, Supraspinatus, Infraspinatus, Teres minor, Teres major, Subscapularis, Splenius capitis, Splenius cervicis, Iliocostalis lumborum, Iliocostalis thoracis, Iliocostalis cervicis, Longissimus thoracis, Longissimus cervicis, Longissimus capitis, Spinalis thoracis, Spinalis cervicis: Spinalis capitis, Semispinalis thoracis, Semispinalis cervicis, Semispinalis capitus, Multifidus, Short rotators, Interspinalis, Intertransversi, Trapezius, Latissimus dorsi, Subclavius, Pectoralis major, Pectoralis minor, Levator scapulae, Rhomboid minor, Rhomboid major, Serratus anterior, Deltoid, Supraspinatus, Infraspinatus, Teres minor, Teres major, Subscapularis, Splenius capitis, Splenius cervicis, Iliocostalis lumborum, Iliocostalis thoracis, Iliocostalis cervicis, Longissimus thoracis, Longissimus cervicis, Longissimus capitis, Spinalis thoracis, Spinalis cervicis, Spinalis capitis, Semispinalis thoracis, Semispinalis cervicis, Semispinalis capitus, Multifidus, Long rotators, Short rotators and the Interspinalis, Intertransversi.

Alternatively, the muscle is a leg muscle selected from the group consisting of Tensor fascia lata, Gluteus maximus, Gluteus medius, Gluteus minimus, Piriformis, Superior gemellus, Obturator internus, Inferior gemellus, Quadratus femoris, Semitendinosus, Adductor longus, Adductor brevis, Adductor magnus, Gracilis, Obturator externus, Sartorius, Rectus femoris, Vastus lateralis, Vastus intermedius, Vastus medialis, Articularis genus, Psoas major, Illiacus, Pectineus, Soleus, Plantaris, Popliteus, Flexor digitorum longus, Tibialis posterior, Flexor hallucis longus, Peroneus longus, Peroneus brevis, Tibialis anterior, Extensor hallucis longus, Extensor digitorum longus and Peroneus tertius.

In another form, the muscle is a head or neck muscle selected from the group consisting of Obliquus capitis inferior, Obliquus capitis superior, Rectus capitis posterior major, Rectus P:\WPDOCS\CRNSET\Sherryl12591661.doc-26/08/05 -17capitis posterior minor, Longus colli, Longus capitis, Rectus capitis anterior, Rectus capitis lateralis, Anterior scalene, Scalenus minimus (may be absent), Middle scalene, Posterior scalene, Sternocleidomastoid, Platysma, Sterohyoid, Omohyoid, Stemothyroid, Thyrohyoid, Stylohyoid, Digastric, Mylohyoid, Geniohyoid, Occipitalis (2 bellies), Frontalis (2 bellies), Orbicularis oculi, Corrugator supercilii, Levator labii superioris alaeque nasi, Levator labii superioris, Zygomaticus minor, Zygomaticus major, Risorius (may be absent), Levator anguli oris, Buccinator, Depressor anguli oris, Depressor labii inferioris, Masseter, Medial pterygoid, Lateral pterygoid, Levator palpebrae superioris, Lateral rectus, Medial rectus, Superior rectus, Superior rectus, Inferior rectus, Superior oblique and the Inferior oblique.

In yet a further form, the muscle is an arm muscle selected from the group consisting of Coracobrachialis, Biceps brachii, Brachialis, Triceps brachii, Anconeus, Pronator teres, Flexor carpi radialis, Palmaris longus, Flexor carpi ulnaris, Flexor digitorum superficialis, Flexor digitorum profundus, Flexor pollicis longus, Pronator quadratus, Brachioradialis, Extensor carpi radialis longus, Extensor carpi radialis brevis, Extensor digitorum, Extensor digiti minimi, Extensor carpi ulnaris, Supinator, Abductor pollicis longus, Extensor pollicis brevis, Extensor pollicis longus, Extensor indicis, Abductor pollicis brevis, Flexor pollicis brevis, Opponens pollicis, Adductor pollicis, Palmaris brevis, Abductor digiti minimi, Flexor digiti minimi brevis, Opponens digiti minimi, Palmar interossei, Dorsal interossei, Lumbricals, Abductor hallucis, Flexor digitorum brevis, Abductor digiti minimi, Abductor ossis metatarsi quinti, Quadratus plantae, Lumbricals, Flexor hallucis brevis, Adductor hallucis, Flexor digiti minimi brevis, Plantar interossei (3 muscles), Dorsal interossei (4 muscles), Extensor hallucis brevis and Extensor digitorum brevis.

Additionally, the vascular supply of the subcutaneous region, the fat or the muscle containing the scaffold assists in angiogenesis of the newly grown bone. Accordingly, the newly growing bone is provided with a blood supply, and the blood vessels that grow into the bone graft tissue are harvested when sufficient angiogenesis and formation of replacement bone has occurred, and connected to blood vessels at the site where the replacement bone tissue is required in the patient.

P\WPDOCS\CRN\SET\Sherry\1259166j do-26/08105 -18- The term "sufficient bone formation" as used herein refers to the formation within the scaffold of adequate amounts of bone, as determined by extent of mineralisation and bone formation (ie. formation of osteoblasts and trabeculae) within the titanium scaffold, as determined by any suitable methods in the art, such skeletal scintillography, as x-ray analysis and computerised tomography.

In a further form, the present invention relates to use of a scaffold for supporting bone growth in the manufacture of replacement bone for a patient, wherein the scaffold is inoculated with osteoblast precursor cells and implanted subcutaneously, or into fat or muscle of a host, and the scaffold, replacement bone and a blood supply of the replacement bone is harvested when sufficient angiogenesis and formation of replacement bone tissue has occurred.

In yet a further form, the invention relates to a kit for growing replacement bone for a patient, the kit comprising: c) a scaffold suitable for supporting bone growth subcutaneously or within fat or muscle tissue of a host; and d) a source of osteblast precursor cells.

Preferably, the kit contains at least growth factor, and even more preferably, the kit further contains hydroxyapatite crystals that may be pre-placed within the scaffold within the kit.

Even more preferably, the at least one growth factor is an osteoblast growth factor.

In yet a further broad form, the present invention relates to bone graft material, suitable for transplantation into a patient requiring said graft, said bone comprising: e) a scaffold housing bone tissue; and f) a blood supply comprising one or more blood vessels for suturing into a region in the patients body where the bone graft is required; wherein said bone graft material has been manufactured according to the method of: P:\WPDOCS\CRN\SET1Sherry\l2591661.doc-26/08/05 19a) providing a scaffold for the bone graft material; b) inoculating the scaffold with osteoblast precursor cells; c) implanting the scaffold subcutaneously, or into fat or muscle tissue in a host; d) harvesting the scaffold housing the bone tissue and a portion of a blood supply of the replacement bone when sufficient formation and angiogenesis of replacement bone has occurred.

In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following nonlimiting examples.

EXAMPLES

EXAMPLE 1 GROWTH OF A MANDIBLE IN VIVO An extended mandibular discontinuity defect was replaced by way of growth of a custom bone transplant inside the latissimus dorsi muscle of an adult male patient.

Materials and Methods Ethics approval was obtained from the University of Kiel, Germany. The patient gave written consent. Three-dimensional computed tomography (CT) of the patient's head was performed for the design of an ideal virtual replacement of the missing parts of the mandible with computer-aided design (CAD) (Figure Data were directed to a CADoperated three-axes milling machine, and a teflon model was created that matched the virtual mandible exactly. A titanium mesh scaffold (MARTIN, Micromesh, Tuttlingen, Germany) was then formed onto the model, which was subsequently removed (Figure 1) and the remaining titanium mesh cage was filled with ten bone mineral blocks as carriers (BioOss-Blocks; Geistlich Biomaterials, Wolhusen, Switzerland), which were coated with 7 mg recombinant human BMP7 embedded in 1 g bovine collagen type 1 (OP-1 implant, Stryker Biotech, Hopkinton, USA; figure Finally, 20 mL bone marrow was aspirated P:\WPDOCS\CRN\SETShery\ 2591661.doc-26/08/)5 from the right iliac crest to provide undifferentiated precursor cells as a target for recombinant human BMP7. We did not undertake flow cytometry to ascertain the presence of stem cells in the aspirate. Bone marrow was mixed with 5 g natural bone mineral of bovine origin (particle size 0 .5-1 .0 mm; BioOss-Spongiosa granules; Geistlich Biomaterials) and this mixture was used to fill the gaps between the blocks inside the cage.

The titanium mesh cage (Figure 2) was then implanted into a pouch of the patient's right latissimus dorsi muscle under general anaesthesia. He developed a haematoma postoperatively that was easily drained on the second postoperative day. Prophylactic antibiotics were administered, with 1 5 g ampicillin/sulbactam three times a day for 14 days. We saw no signs of infection. The patient did not complain of pain or sleep disturbance and communicated only minor concerns about the range of motion of his right arm.

Results Four weeks postoperatively, skeletal scintigraphy was performed by intravenous injection of 600 MBq technetium-99m-oxydronate tracer. Bone remodelling with vital osteoblasts was detected inside the implant, verified by a tracer enhancement, which was the first sign of successful bone induction. Additionally, CT of the thorax provided radiographic evidence for bone formation around the implant site.

Seven weeks postoperatively, the patient underwent general anaesthesia for transplantation of the mandibular replacement. The replacement was harvested along with an adjoining part of the latissimus dorsi muscle containing the thoracodorsal artery and vein that had supplied blood for the entire transplant. This pedicled bone-muscle flap was then transplanted into the defect site via an extraoral approach. Minor bone overgrowth on the ends of the replacement was curetted to fit the transplant easily into the defect. No further correction of the shape or form of the graft was needed.

After the old titanium reconstruction plate was removed, the mandibular transplant was fixed onto the original mandible stumps with titanium micro-osteosynthesis screws, returning the contour of the patient's jaw line to roughly that present before the P:\WPDOCS\CRN\SET\Sherry 2591661.doc-26/08/05 -21 mandibulectomy. The vessel pedicle was then anastomosed onto the external carotid artery and cephalic vein by microsurgical techniques. The cephalic vein was taken from the upper arm and relocated into the neck since few usable local veins were available because of a previous radical neck dissection and irradiation. Because of the patient's non-elastic irradiated skin we could not close the wound completely in the submandibular region.

Prophylactic antibiotic cover was again provided with 1.5 g ampicillin/sulbactam three times a day. 12 days postoperatively this small area had closed spontaneously with growth of granulated tissue. This area was then covered with a full thickness skin graft. Except for a small manageable area of necrosis at the wound margin (previously irradiated skin), healing proceeded uneventfully. Vascular supply to the flap was maintained successfully.

Repeat three-dimensional CT showed that both mandible stumps were in the correct position with relation to the transplant. A second skeletal scintigraphy was performed with 600 MBq Tc99m-oxydronate tracer on the 11th postoperative day and showed continued bone remodelling and mineralisation inside the mandibular transplant, indicating undisturbed vascular perfusion and survival of the induced bone cells.

By the 4th week post-transplantation the patient was able to eat solid food for the first time since he underwent the mandibulectomy.

Persons skilled in the art will appreciate that numerous variations and modifications will become apparent. All such variations and modifications which become apparent to persons skilled in the art, should be considered to fall within the spirit and scope that the invention broadly appearing before described.

Claims (40)

1. A method for growing replacement bone tissue for a patient, said method comprising: a) providing a scaffold for the replacement bone tissue; b) inoculating the scaffold with osteoblast precursor cells; c) implanting the scaffold subcutaneously, or into fat or muscle tissue in a host; d) harvesting the scaffold, replacement bone and a portion of the blood supply of the replacement bone when sufficient formation and angiogenesis of replacement bone has occurred; e) transplanting the scaffold, replacement bone and the blood supply of the bone to a site where replacement bone tissue is required in the patient; and f) connecting at least part of a blood supply of the replacement bone to a blood vessel at the site where the replacement bone tissue is required.

2. Method according to claim 1, wherein inoculating the scaffold further involves inserting hydroxyapatite crystals into the scaffold.

3. Method according to claim 2, wherein the hydroxyapatite crystals are provided as bone mineral blocks.

4. Method according to any one of claims 1 to 3, wherein the osteoblast precursor cells are provided in a mixture of bone marrow cells. Method according to claim 1, wherein the osteoblast precursor cells are haematopoietic stem cells.

6. Method according to claim 5, wherein the haematopoietic stem cells are derived from monocyte precursor cells. P:\WPDOCS\CR\SET\Sherry2591661.doc-26/08/O -23-

7. Method according to claim 1, wherein the osteoblast precursor cells are adult stem cells or embryonic stem cells isolated from an embryo of the host species.

8. Method according to claim 1, wherein the stem cells are totipotent stem cells isolated from a fertilised egg of the host species.

9. Method according to any one of claims 1 to 8, wherein inoculating the scaffold further includes providing at least one growth factor within the scaffold.

10. Method according to claim 9, wherein the at least one growth factor is selected from the group consisting of the bone morphogenetic protein (BMP) family members.

11. Method according to claim 10, wherein the BMP is BMP-2.

12. Method according to claim 1, wherein anatomical modelling studies are performed for shaping the scaffold to optimise the scaffold shape to fit the site where replacement bone is required in the patient.

13. Method according to claim 12, wherein the anatomical modelling studies include computed tomography.

14. Method according to claim 13, wherein the anatomical modelling studies further include use of computer -aided design. Method according to claim 14, wherein the anatomical modelling studies include three-dimensional computed tomography.

16. Method according to any one of claims 1 to 15, wherein the scaffold is a suitable biocompatible and/or bioabsorbable material. P:\WPDOCS\CRN\SETShenrr)2591661 .dc-26/08/05 -24-

17. Method according to claim 16, wherein the scaffold is titanium.

18. Method according to claim 16 or claim 17, wherein the scaffold comprises a mesh- like structure.

19. Method according to any one of claims 1 to 18, wherein the patient and the host are different organisms. Method according to any one of claims 1 to 18, wherein the patient and the host are the same organism.

21. A kit for growing replacement bone for a patient, the kit comprising: a) a scaffold suitable for supporting bone growth subcutaneously or within fat or muscle tissue of a host; and b) a source of osteoblast precursor cells.

22. The kit according to claim 21, further including at least one growth factor.

23. The kit according to claim 22, further including hydroxyapatite crystals suitable for placement in the scaffold.

24. The kit according to claim 22, further including hydroxyapatite crystals pre-placed within the scaffold.

25. The kit according to claim 23 or claim 24, wherein the hydroxyapatite crystals are present as bone mineral blocks.

26. Bone graft material, suitable for transplantation into a patient requiring said graft, said bone graft material comprising: a) a scaffold housing bone tissue; and P:\WPDOCS\CRN\SETShenrry 2591661 .doc-26/08/05 b) a blood supply comprising one or more blood vessels for suturing into a region in the patients body where the bone graft is required; wherein said bone graft material has been manufactured according to the method of: a) providing a scaffold for the bone graft material; b) inoculating the scaffold with osteoblast precursor cells; c) implanting the scaffold subcutaneously, or into fat or muscle tissue in a host; d) harvesting the scaffold housing the bone tissue and a portion of a blood supply of the replacement bone when sufficient formation and angiogenesis of replacement bone has occurred.

27. Bone graft material according to claim 26, wherein inoculating the scaffold further involves inserting hydroxyapatite crystals into the scaffold.

28. Bone graft material according to claim 27, wherein the hydroxyapatite crystals are provided as bone mineral blocks.

29. Bone graft material according to any one of claims 26 to 28, wherein the osteoblast precursor cells are a mixture of bone marrow cells.

30. Bone graft material according to any one of claims 26 to 28, wherein the osteoblast precursor cells are derived from a mixture of bone marrow cells.

31. Bone graft material according to claim 30, wherein the osteoblast precursor cells are mesenchymal stem cells.

32. Bone graft material according to claim 26, wherein the osteoblast precursor cells are haematopoietic stem cells.

33. Bone graft material according to claim 32, wherein the haematopoietic stem cells are derived from monocyte precursor cells. P:\WPDOCS\CRN\SET\Sherry\12591661.doc-26/08/)5 -26-

34. Bone graft material according to claim 26, wherein the osteoblast precursor cells are adult stem cells or embryonic stem cells isolated from an embryo of the host species.

35. Bone graft material according to claim 26, wherein the stem cells are totipotent stem cells isolated from a fertilised egg of the host species.

36. Bone graft material according to any one of claim 26 to 35, wherein inoculating the scaffold further includes providing at least one growth factor within the scaffold.

37. Bone graft material according to claim 36, wherein the at least one growth factor is selected from the group consisting of the bone morphogenetic protein (BMP) family members.

38. Bone graft material according to claim 37, wherein the BMP is BMP-2.

39. Bone graft material according to claim 37, wherein the BMP is BMP-7. Bone graft material according to any one of claims 26 to 39, wherein anatomical modelling studies are performed for shaping the scaffold to optimise the scaffold shape to fit the site where replacement bone graft material is required in the patient.

41. Bone graft material according to any one of claims 26 to 40, wherein the scaffold is a suitable biocompatible and/or bioabsorbable material.

42. Bone graft material according to claim 41, wherein the scaffold is titanium.

43. Bone graft material according to claim 41 or 42, wherein the scaffold comprises a mesh-like structure. PA\PDOCS\CRN\SETShenM 1259166.doc-26108/05 -27-

44. Bone graft material according to any one of claims 26 to 43, wherein the patient and the host are different organisms. Bone graft material according to any one of claims 26 to 43, wherein the patient and the host are the same organism. DATED this 26 th day of August, 2005 EUGENE SHERRY, HEYDRICK TERHEYDEN AND PATRICK H. WARNKE by its Patent Attorneys DAVIES COLLISON CAVE