WO2011158025A1

WO2011158025A1 - In vitro bone model and distraction device

Info

Publication number: WO2011158025A1
Application number: PCT/GB2011/051114
Authority: WO
Inventors: Cynthia Chang; Philippa Hulley; James Triffitt
Original assignee: Oxford University Innovation Ltd; US Department of Health and Human Services
Current assignee: Oxford University Innovation Ltd; US Department of Health and Human Services
Priority date: 2010-06-14
Filing date: 2011-06-14
Publication date: 2011-12-22
Anticipated expiration: 2012-12-14
Also published as: GB201009939D0

Abstract

The present invention relates to an in vitro three dimensional bone tissue model and uses thereof, for example to study the process of bone breakage and healing or bone regeneration. In particular, the invention includes the use of the bone tissue model to study distraction osteogenesis. The tissue model of the present invention can be used in combination with single- and multiple-sample distraction devices and apparatus. Single- and multiple-sample distraction devices and apparatus as described herein also form part of the present invention.

Description

IN VITRO BONE MODEL AND DISTRACTION DEVICE The present invention relates to an in vitro three dimensional bone tissue model and uses thereof, for example to study the process of bone breakage and healing or bone regeneration. In particular, the invention includes the use of the bone tissue model to study distraction osteogenesis. The tissue model of the present invention can be used in combination with single- and multiple-sample distraction devices and apparatus. Single- and multiple-sample distraction devices and apparatus as described herein also form part of the present invention.

A bone fracture is a medical condition in which there is a break in the continuity of the bone. A bone fracture can be the result of high force impact or stress, or trivial injury as a result of certain medical conditions that weaken the bones, such as osteoporosis, bone cancer, or osteogenesis imperfecta, wherein the fracture is then termed a pathological fracture. Bone fractures occur frequently and range in severity, but they can be debilitating to the patient and fracture healing can be a lengthy process.

The process of fracture healing involves several phases of recovery. The length of the process depends on the extent of the injury, and can take two to three weeks for the reparation of most upper bodily fractures and anywhere above four weeks for lower body fractures.

The process of the entire regeneration of the bone can depend on the angle of dislocation or fracture. While immobilization and surgery may facilitate healing, a fracture ultimately heals through physiological processes. The healing process is mainly determined by the periosteum (the connective tissue membrane covering the bone). The periosteum is one source of precursor cells which develop into chondroblasts and osteoblasts that are essential to the healing of bone. The bone marrow (when present), endosteum, small blood vessels, and fibroblasts are other sources of precursor cells.

There are three major phases of fracture healing. The first phase is the reactive phase which includes a fracture and inflammatory phase and a phase of granulation tissue formation. The second phase is the reparative phase in which a callus is formed and lamellar bone is deposited. The final remodelling phase involves the remodelling of the bone to the original bone contour. After fracture, the first change seen by light and electron microscopy is the presence of blood cells within the tissues which are adjacent to the injury site. Soon after fracture, the blood vessels constrict, stopping any further bleeding. Within a few hours after fracture, the extravascular blood cells form a blood clot or hematoma. All of the cells within the blood clot degenerate and die. Some of the cells outside of the blood clot, but adjacent to the injury site, also degenerate and die. Within this same area, the fibroblasts survive and replicate. They form a loose aggregate of cells, interspersed with small blood vessels, known as granulation tissue.

Days after fracture, the cells of the periosteum replicate and transform. In most fractures, the periosteal cells proximal to the fracture gap develop into chondroblasts and form hyaline cartilage. The periosteal cells distal to the fracture gap develop into osteoblasts and form woven bone. The fibroblasts within the granulation tissue also develop into chondroblasts and form hyaline cartilage. These two new tissues grow in size until they unite with their counterparts from other pieces of the fracture. This process forms the fracture callus. Eventually, the fracture gap is bridged by the hyaline cartilage and woven bone, restoring some of its original strength. In some cases, bone heals by intramembranous ossification, in which the bone ends are fixed tightly together such as with plates or screws, and bone matrix is directly formed (bypassing the cartilage template of endochondral ossification).

The next phase is the replacement of the hyaline cartilage and woven bone with lamellar bone. The replacement process is known as endochondral ossification with respect to the hyaline cartilage and "bony substitution" with respect to the woven bone. Substitution of the woven bone with lamellar bone precedes the substitution of the hyaline cartilage with lamellar bone. The lamellar bone begins forming soon after the collagen matrix of either tissue becomes mineralized. At this point, "vascular channels" with many accompanying osteoblasts penetrate the mineralized matrix. The osteoblasts form new lamellar bone upon the recently exposed surface of the mineralized matrix. This new lamellar bone is in the form of trabecular bone. Eventually, all of the woven bone and cartilage of the original fracture callus is replaced by trabecular bone, restoring most of the bone's original strength.

The remodeling process reshapes the bony callus by substituting trabecular bone with compact bone to form the hard cortical shell where anatomically appropriate. The trabecular bone is first resorbed by osteoclasts, creating a shallow resorption pit known as a "Howship's lacuna". Then osteoblasts deposit compact bone within the resorption pit. Eventually, the fracture callus is remodelled into a new shape which closely duplicates the bone's original shape and strength. Whilst fractured bone can often be healed in terms of reuniting the ends of the broken bone, sometimes the bones do not completely join, or join in a faulty position after a fracture. In this situation, a technique named "distraction osteogenesis" can be employed in order to correct the defective bone. Distraction osteogenesis refers to a surgical bone-lengthening procedure, also called callus distraction, callotasis and osteodistraction. It is a surgical process which can be used to reconstruct skeletal deformities and lengthen the long bones of the body. A corticotomy is used to fracture the bone into two segments, and the two ends of the bone are gradually moved apart during the distraction phase, allowing new bone to form in the gap. When the bone fragments are stably fixed and strain is the primary mechanical force, intramembranous ossification occurs. Some endochondral ossification forms when instability and compressive forces are transmitted to the tissues. When the desired or possible length is reached, a consolidation phase follows in which the bone is allowed to keep healing. Distraction osteogenesis has the benefit of simultaneously increasing bone length and the volume of surrounding soft tissues. Distraction osteogenesis in patients usually involves installing an external frame, which holds the bone in place whilst an osteotomy is performed. The cut bone is allowed to heal for several days in the latency phase. In the distraction phase, the two ends of the cut bone are gradually pulled apart at a rate of approximately half a mm to one mm each day until the desired bone length is achieved. In the final consolidation phase, the external frame is left in place until the bone has healed completely and bridged the gap between the two lengths of cut bone.

Distraction equipment usually involves an external frame and pins connecting to the bone. Force is applied to the pins in order to distract the bone ends.

Although distraction osteogenesis is most often used in the treatment of post-traumatic injuries, it is increasingly used to correct limb discrepancies caused by congenital conditions and old injuries. Uses of distraction osteogenesis include the correction of congenital deformities such as congenital short femur, fibular hemimelia, hemiatrophy and Ollier's disease. Developmental deformities such as neurofibromatosis and bow legs can also be treated with distraction osteogenesis. Post-traumatic injuries such as growth plate fractures, malunion or non-union, shortening and deformity and bone defects can also be treated. Applications in craniofacial surgery and dental surgery are becoming increasingly common. Distraction osteogenesis can also be used after infections and diseases such as osteoarthritis and septic arthritis and polio. Patients with short stature and achondroplasia and constitutional short stature can also benefit from distraction osteogensis. Thus it can be seen that distraction osteogenesis has many uses, and thus it is important to understand the cellular and molecular nature of the process in order to maximize patient benefit from this process.

In this regard, whilst there has been a certain amount of prior study, the field of research into bone fracture and regeneration and distraction osteogenesis has previously been hampered by a lack of a suitable three dimensional in vitro tissue model in which to carry out studies into the molecular and cellular nature of bone regeneration.

Previous clinical research studies with experimental animals in vivo have been informative in elucidating some of the biological events which occur during distraction osteogenesis. For example, mouse models have previously been used to study distraction osteogenesis, but the molecular and cellular events that occur during the process are still largely unknown. Previously used experimental models are not completely in vitro, and involve an in vivo element, for example Matsuno et al. (The Journal of Craniofacial Surgery 2000 1 1 (4):303-7) developed a model involving rat tibial organ culture, and Heller et al. (Plast Reconstr Surg 2007 1 19(7):2037- 45) have stretched rat calvarial structures, but as both explant methods still require animal care and surgery, these models are not entirely in vitro models. It is desirable to minimize the use of mice and other experimental animals in experimental work for ethical and economic reasons

Thus, whilst distraction osteogenesis has been used in orthopaedic surgery for several decades, there is limited knowledge regarding the molecular and cellular events that result in new, functional bone. There is a need for a new in vitro tissue model in which to study these processes in an economical and straightforward manner.

The present invention addresses this need, in that it provides an in vitro three dimensional tissue model of a fractured or broken bone, i.e. it can mimic a naturally-healing or regenerating bone. The tissue model of the present invention mimics a broken bone in an entirely in vitro environment, thus negating the need to use experimental animals. The present model is therefore more economical and more straightforward in that no animal care or surgery is required.

The tissue model of the present invention includes both mineral and soft tissue components; other three dimensional models in the art do not include both of these components of the distraction environment. For example, the commercially-available Flexercell system (Flexercell Corp., McKeesport, PA) uses cells cultured on a membrane to deliver different magnitudes of strain to different locations on the flexible membrane. This model does not comprise both mineral and soft tissue components. Also, the cyclical nature of the load may not accurately model the constant tension produced by distraction osteogenesis. Gabbay et al. (Tissue

Engineering 2006, 12(1 1 ):3055-65) have stretched cells in collagen gel to mimic the distraction gap, but have not modelled the hard bone cortex. The inventors also developed single and multiple sample distraction devices and apparatus that can be used to distract the tissue model of the invention or other models and thereby mimic in vivo distraction osteogenesis. The distraction apparatus and devices provide a means of distracting a sample in vitro, and also allow the sample to be imaged using a variety of imaging protocols. This is the first distraction apparatus to be developed that allows imaging, for example real time imaging, of distracted samples without removal of the sample from the distraction apparatus. This is an important feature as it allows imaging of live cells without disturbing the configuration and format of the distracted sample. Distraction apparatus known in the art does not allow the sample to be imaged in this way. The distraction apparatus of the present invention thus provides a valuable tool to those wishing to study sub-cellular processes during distraction osteogenesis. Furthermore, the distraction controller of the invention allows the simultaneous distraction of more than one sample at a time. The controller provides a means of applying a uniform distraction strain to each sample. Such apparatus has not previously been described, and thus the present invention fulfils a need for a way of distracting and imaging a plurality of distracted samples in a controlled manner. In one embodiment, the invention provides a method for analysing tissue development in vitro, the method comprising the steps:

(i) retaining first and second hard porous scaffold components adjacent to one another;

(ii) introducing a biocompatible matrix into the gap between the first and second hard scaffold components;

wherein one or more of the hard scaffold components and the biocompatible matrix comprise one or more types of cells involved in bone growth, repair and/or regeneration,

(iii) culturing the first and second hard scaffold components and biocompatible matrix in vitro under conditions suitable for maintaining the growth and/or differentiation of the cells;

(iv) optionally incrementally increasing or decreasing the gap between the first and second hard scaffold components;

(v) analysing the cell and/or tissue development in the first and/or second hard scaffold components and/or biocompatible matrix;

and optionally repeating steps (iii)-(v). ln a further embodiment, the invention provides an in vitro three dimensional bone tissue model, comprising:

(i) a first hard porous scaffold component,

(ii) a biocompatible matrix, and

(iii) a second hard porous scaffold component

wherein the hard scaffold components are porous materials having interconnected pores, wherein the first and second hard scaffold components are located adjacent to one another and wherein the biocompatible matrix is present in a gap between the first and second hard scaffold.

Preferably, the bone tissue model is for analysing tissue development.

In some embodiments, the bone tissue model is for use in a method as defined herein. In a further embodiment, the invention provides a method for analysing tissue development in vitro, the method comprising the steps:

(i) retaining first and second hard scaffold components adjacent to one another in a distraction device which is capable of incrementally increasing or decreasing the gap between the first and second hard scaffold components;

(v) analysing the cell and/or tissue development in the first and/or second hard scaffold components and/or biocompatible matrix whilst the first and second hard scaffold components and the biocompatible matrix are retained in situ in the distraction device; and optionally repeating steps (iii)-(v). ln yet a further embodiment, the invention provides an apparatus for distraction of bone or bone mimetics comprising:

(i) means for retaining a first hard scaffold component;

(ii) means for retaining a second hard scaffold component;

(iii) means for locating the first hard scaffold component at a position adjacent to the second hard scaffold component and for incrementally varying the gap between the first and second hard scaffold components,

wherein the apparatus is sized and configured such that the gap between the first and second hard scaffold components and, in use, any material comprised therein, is capable of being analysed by microscopic imaging means whilst being retained in the apparatus.

In some embodiments, the apparatus is for use in a method as defined herein.

The invention further provides a distraction device for studying tissue development and/or tissue regeneration in vitro, said device comprising distraction means for incrementally distracting a first and a second hard scaffold component wherein the distraction device is sized and configured such that the distraction gap and, in use, any material comprised therein, is capable of being analysed by microscopic imaging means whilst being retained in the device. In some embodiments, the device is for use in a method as defined herein.

In a preferred embodiment, the invention provides an apparatus or device for use in a method as defined herein comprising first and second U-shaped frames, each U-shaped frame comprising two arms and a connecting base, wherein the arms of each U-shaped frame each comprise adjustable retaining pins capable of retaining first and second scaffold components between the arms, wherein the first and second U-shaped frames are retained substantially parallel to each other by one or more threaded shafts which are located between the adjacent arms of the first and second U-shaped frames, wherein the threaded shafts are capable of retaining the first and second frames at a predetermined, incrementally-variable distance from one another, and thereby capable of incrementally varying the distraction gap between any first and second scaffold components retained therein.

In preferred embodiments, the connecting base of the U-shaped frame is 10-50mm, preferably 15-35mm and most preferably 15-30mm in length. In other embodiments, each arm of the U- shaped frame is 5-30mm, preferably 5-20mm and most preferably 5-18mm in length. ln other embodiments, the distance between the first and second U-shaped frames is 5-30mm, preferably 5-20mm and most preferably 5-18mm in length. Preferably, there are 1 -3 threaded shafts between each adjacent arm, most preferably 2 shafts. Nuts or similar locking means are used on the shafts to lock and retain the frames at the desired positions. Preferably, the threaded shafts do not extend significantly beyond the end of the apparatus or device. The invention also provides a distraction controller which is capable of simultaneously controlling the distraction gap in a plurality of apparatus or distraction devices as defined herein.

In yet a further embodiment, the invention provides a method for analysing tissue development in vitro, the method comprising the steps:

(i) retaining, in a plurality of distraction devices, a plurality of pairs of first and second hard porous scaffold components adjacent to one another, wherein each pair of first and second hard porous scaffold components are separated by a distraction gap;

(ii) introducing a biocompatible matrix into the gap between at least one pair of first and second hard porous scaffold components;

wherein one or more of the hard porous scaffold components and the biocompatible matrix, when present, in each device comprise one or more types of cells involved in bone growth, repair and/or regeneration,

(iii) culturing each of the first and second hard scaffold components and biocompatible matrix in vitro under conditions suitable for maintaining the growth and/or differentiation of the cells;

(iv) optionally incrementally increasing or decreasing the distraction gap between the first and second hard scaffold components in each device;

(v) analysing the cell and/or tissue development in the first and/or second hard porous scaffold components and/or biocompatible matrix in at least one of the devices whilst the first and second hard scaffold components and the biocompatible matrix in that device are retained in situ;

and optionally repeating steps (iii)-(v), wherein the distraction gaps between the first and second hard porous scaffold components in each of the devices are controlled simultaneously by a distraction controller. Thus in one embodiment, the invention relates to a method or apparatus for analysis of tissue development. Preferably, the tissue development is bone generation or regeneration, for example by callus distraction.

The three dimensional bone tissue model of the present invention comprises two hard porous scaffold components, between which is a biocompatible matrix. The model mimics two bone ends and the healing bone callus in between the bone ends, although as discussed below, the model can also mimic other aspects of bone growth and regeneration, for example the growth plate.

The hard porous scaffold components are bone-mimics or mimetics, i.e. they are intended to mimic the hard mineral component of bone. The hard porous scaffold is rigid and provides a physical support on which cells may grow in a three dimensional construct.

The hard porous scaffold components may comprise any hard porous material which is suitable for the growth and/or maintenance of cells. Those of skill in the art will appreciate the nature of suitable scaffolds which are capable of supporting the growth of cells. The hard porous scaffold should allow cells to adhere thereto and allow the survival of cells when placed in a liquid medium. It should not be cytotoxic.

One or both of the hard scaffolds may be a porous matrix. The pores of the hard porous scaffold are interconnected or substantially interconnected. The hard scaffold may be sufficiently porous to allow seeding, growth, and migration of cells. In one embodiment all or substantially all of the pores are at least, or about, 40 μηη to at least, or about δθθμηη in diameter. For example, the pores are at least or about 40, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 220, 240, 260, 280 or 300 μηη in diameter. Preferably, the pores are 200- 700 μηη for osteogenesis and vascular infiltration, but may be between 40 to 800 μηη for other cell types. In some embodiments, the pore sizes are about 400 μηη or about 500 μηη.

The hard porous scaffold may be obtained from a natural or naturally-derived material or a synthetic material. ln some embodiments, the hard porous scaffold is an inert material. In some embodiments, it is a non-organic material.

Examples of natural or naturally-derived materials include bone, bone from which the mineral component has been dissolved, bone which has been demineralized and coral. In some embodiments of the invention, the hard scaffold does not include natural, i.e. unmodified, bone.

Examples of suitable synthetic scaffold materials include mineral scaffolds, polymeric scaffolds, porous or sintered glass, porous or sintered ceramic, and porous or sintered metals.

Many polymers may be used in the manufacture of suitable scaffolds. Polymers, both natural and synthetic, are well known in the art and include, but are not limited to, polylactides,

polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates,

polyalkylene succinates, poly(malic acid), poly(amino acids), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, chitin, chitosan, poly(L-lactic acid), poly(lactide-co-glycolide), poly(hydroxybutyrate-co-valerate), and copolymers, terppolymers, or combinations or mixtures of the above materials.

Preferred polymers include collagen, hydroxyapatite, polylactic acid (PLA), polyglycolic acid (PGA) and polycaprolactone (PCL); and mixtures thereof.

In some embodiments, the scaffold comprises hydroxyapatite or tricalcium phosphate, or a mixture thereof. In a particularly preferred embodiment, the scaffold comprises 50-70% hydroxyapaptite and 30-50% tricalcium phosphate, most preferably about 60% hydroxyapatite and about 40% tricalcium phosphate.

Additionally, fibrous scaffolds such as electrospun nanofiber scaffolds could be used, if made suitably rigid and capable of withstanding the distraction process without tearing (e.g. see Li WJ. J Biomed. Mater. Res. 60: 613-621 , 2002).

The hard porous scaffold is preferably capable of being sterilized, e.g. by autoclaving, ethylene oxide, radiation or liquid. Methods of making scaffolds used herein are well known in the art. These methods include phase separation, gas foaming and solvent casting, textiles, electrospinning and freeform fabrication. If desired, the scaffolds may undergo additional surface modification to improve their interaction with cells. One or more surfaces of the hard porous scaffolds may be modifiable. In particular, one or both of the adjacent surfaces of the two hard porous scaffolds may be surface modified.

For example, arginine-glycine-aspartate (RGD) peptides may be chemically attached to the surface, or the scaffolds may be subjected to plasma treatment in order to modify the scaffold surface chemical structure to adjust biocompatibility. Different areas or surfaces of the scaffolds could have different surface modifications, such as with RGD peptides collagen coating, or fibronectin coating, or a cell-repelling coating (e.g. silicone) to produce patterns on the scaffold. Different surface "patterns" could produce different patterns of cell adhesion on the scaffolds.

Suitable scaffolds are obtainable from and manufactured by Biocetis (Cournonsec, France); another suitable scaffold is the Triosite product by Zimmer Ltd. (Swindon, UK).

The scaffold may be cut to an appropriate size according to the intended use of the three dimensional tissue model, for example in the uses discussed below. The sizes of the hard scaffold components are selected such that the overall three dimensional bone tissue model, apparatus or device of the invention may fit in a suitable imaging (e.g. microscopic visualisation) apparatus. For example, the scaffold sizes are selected such that the model fits between the objective lens and stage of a light microscope.

The hard scaffolds are preferably in the form of cuboidal blocks. Preferably, each side is at least 1 mm, more preferably at least 2mm or at least 3mm.

In particular, the volume of each of the hard scaffold components may be 5-1000mm³, preferably 10-200mm³, most preferably 50-150mm³ and particularly preferably about 100mm³.

Preferably, the areas of the adjacent surfaces of the first and second scaffold components (which define the gap for the biocompatible matrix) are 5-50mm², most preferably, 5-25mm², and particularly preferably about 10mm². ln some embodiments, the hard porous scaffold components are both cuboidal blocks having dimensions of 1 -5mm x 1 -5mm x 5-20mm.

In one preferred embodiment of the invention, each piece of the hard scaffold has the dimensions of about 3mm x about 3mm x about 10mm.

The sizes and shapes of the first and second hard porous scaffolds may be the same or different. One or both or parts thereof of the hard scaffold components may be seeded with any suitable type of cell, depending on the intended use.

Preferably, the cells are cells involved in bone growth, repair and/or regeneration. For example, cells of the mesenchymal stem cell lineage, including bone cells, cartilage cells and fat cells, more specifically, osteoblasts, fibroblasts, chondrocytes, adipocytes and/or vascular lineage cells, may be used. Cell lines which are known to those of skill in the art may also be used with the present invention, for example osteoblast cells such as mouse 2T3 osteoblasts. Cells from any organism may be used, for example mammalian and particularly human cells. Also, cells from patients may be used. Preferably, one or both scaffold components are seeded with one or more or combinations of cells involved in bone growth, repair and/or regeneration. Examples of such cells include osteoblasts, bone lining cells such as inactive osteoblasts, osteocytes and osteoclasts. It is envisioned that other cells involved in bone growth and formation, for example stem cells, fibroblasts, adipocytes, cells of the bone marrow, pericytes and vascular cells may also be used. The cells are preferably those which are capable of responding to mechanical factors, such as matrix elasticity and mechanical loading, and of surviving in culture in the model.

The cells may be seeded asymmetrically. Different types, mixtures or combinations may be seeded in each scaffold component, depending on the desired outcome of the experiment. The scaffold components may be seeded before or after the scaffold components are retained. In particular, the scaffold components may be seeded before or after the scaffold components are retained in an apparatus or device of the invention. The seeding may be carried out by direct application of the seeding cells onto one or both scaffold components, or the seeding may be carried out by indirect means, e.g. by immersion of one or more scaffold components in a culture comprising seed cells.

In other embodiments, the scaffold may be filled, packed, infused, adsorbed with, and/or absorbed with one or more agents, for example a bioactive agent, or an inert agent, and combinations thereof.

Examples of suitable bioactive agents include bone marrow, platelet-rich plasma, bone morphogenetic proteins (BMPs), vascular endothelial growth factors (VEGF), connective tissue growth factors (CTGFs), osteoprotegerin, growth differentiation factors (GDFs), cartilage- derived morphogenic proteins (CDMPs), LIM mineralisation proteins (LMPs), transforming growth factor beta, antibiotics, immunosuppresive agents and combinations thereof.

Examples of inert agents include cell culture medium, carriers, excipients, sterilizing solution, labelling solution and other suitable agents.

The first and second scaffolds may be the same or different. In particular, the first and second hard scaffolds may be different materials, different porosities, different sizes, different seeds and/or different agents therein. Preferably, however, the first and second scaffolds are the same.

The gap (also known as the distraction gap) is defined by the two adjacent surfaces of the first and second scaffold components.

The gap between the two hard porous scaffold components of the tissue model may initially be up to 0.1 mm, 0.5mm, 1 mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm or 10mm, or indeed any suitable length. The length of the gap will increase in distraction conditions and decrease in compression conditions.

The biocompatible matrix component of the tissue model mimics the callus or clot environment between the two bone ends in healing or growing or regenerating bone. The biocompatible matrix resides between two sections or surfaces of hard porous scaffold. It is not cytotoxic.

The biocompatible matrix is preferably a matrix of synthetic, natural, organic or protein-based origin, which may be further modified to increase tissue integration. The matrix should allow cells to be incorporated therein and for the cells to be able to survive, proliferate, and/or migrate into it and from it to one or more of the hard porous scaffolds. It should be able to adhere mechanically or chemically to the hard scaffolds.

The matrix should be sufficiently elastic in order to adhere to the hard porous scaffolds and remain adhered thereto upon distraction or compression conditions.

The biocompatible matrix is preferably a natural or synthetic, polymerisable or cross-linkable material, for example, fibrinogen, collagen, alginate, chitosan, silk, hyaluronic acid, aggrecan, fibronectin, laminin, gelatin; any proteoglycan, glycoprotein, glycosaminoglycan or hyaluronan; and synthetic polymers such as PCL, PLA, PLGA, and copolymers thereof. Any combination or mixture of these materials are also acceptable. In some embodiments, the biocompatible matrix is a biological material, for example comprising one or more types of peptide, polypeptide or protein. Examples include fibrinogen, fibrin, collagen, silk, and polysaccharides (e.g. chitosan, hyaluronic acid, dextran, cellulose).

In other embodiments of the invention, the biocompatible matrix is a gel, for example a collagen gel or a fibrin gel.

In yet other embodiments, the soft matrix may comprise a fibrin-based material, for example, Tisseel (produced by Baxter). The fibrin-based material may comprise solutions of fibrinogen and thrombin which, when mixed, polymerise into a fibrin gel.

The soft matrix may also comprise a synthetic polymer, for example PLA, PLGA, and

polycaprolactone.

The biocompatible matrix may also comprise or consist of one or more extracellular matrix components, e.g. Matrigel (BD Biosciences), proteins, glycoproteins, glycosaminoglycans, lipids, laminin, fibronectin, decorin, aggrecan, vimentin, any collagen subtypes, any small leucine-rich glycoproteins, any proteoglycans, any glycoproteins, any glycosaminoglycans, hyaluronans and hyaluronic acids. The biocompatible matrix may also or alternatively comprise a surgical adhesive (e.g. Tisseel) or other fibrin sealants or glues; platelet-rich concentrate; platelet rich plasma; cyanocrylates (such as Histoacryl-TissueSeal Inc., Ann Arbor, Ml or Dermabond - Ethicon, Inc., Somerville, NJ); Indermil; Liquiband; High Viscosity Dermabond; elastomeric materials such as modified, gecko-inspired poly(glycerol sebacate acrylate) (PGSA) (see A. Mahdavi, PNAS February 19, 2008 vol. 105 no. 7 2307-2312); oxidized cellulose; gelatin; collagen-based hemostatic materials; polyethylene glycol-based sealants; and /or glutaraldehyde-albumin based sealants (e.g. Wheat, JC, Urologic Clinics of North America, Volume 36, Issue 2, Pages 265-275 (May 2009)).

In a preferred embodiment of the invention, the biocompatible matrix is seeded with cells. The cells can be any of those described herein in the context of the hard scaffold, depending on the particular use being considered, particularly cells involved in bone growth, repair and/or regeneration. Various combinations and numbers of cells may be seeded, as described above. Also, bioactive agents as described above may be included in the biocompatible matrix.

The three dimensional bone tissue model or apparatus or device of the invention may be located in a vessel suitable for tissue culture, for example a standard tissue culture plate, jar, dish, tube or flask; and suitable tissue culture media added.

In a preferred embodiment of the invention, the model or apparatus or device of the invention is placed in a standard 6-well tissue culture plate. A suitably-sized tissue culture vessel and culture medium, for example standard media, e.g. DMEM or F12, may be used. One skilled in the art will appreciate which medium and culture conditions would be suitable depending on the assay in which the tissue culture model is used.

For example, osteogenic medium can be used in studies where ossification is desirable.

Chondrogenic (for cartilage), stem cell, vasculogenic (for smooth muscle and endothelial), adipogenic (for brown and white fat), haematopoietic (for blood lineages and marrow stroma), neurogenic media, or any medium appropriate to the optimal differentiation of the tissue under study may be used.

The bone tissue model may also be placed in a bioreactor, for example a dynamic flow bioreactor, depending on the nature of the study. The distraction device of the invention comprises distraction means for incrementally distracting a first and a second hard scaffold component. In the context of the present invention, the term "distracting" refers to the gradual and/or incremental moving apart (or together, i.e. compression) of the first and second hard porous scaffolds from a first position to a second position.

Furthermore, the apparatus of the invention comprises means for locating the first hard scaffold component at a position adjacent to the second hard scaffold component and for incrementally varying the distance/gap between the first and second hard scaffold components. In both cases, the new positions of the first and second scaffolds are then retained or fixed, optionally for a set period of time, thus allowing new bone to form in the gap.

Distraction means and distractors are well known in the art and include ratchet and thread- based distraction mechanisms.

The distraction apparatus comprises means for independently retaining a first and a second hard scaffold component. The hard scaffolds are immobilised in positions adjacent to one another.

The retaining means (retainer) may for example be one or more pins which extend from the frame of the apparatus and are capable of being extended into the hard scaffolds. A plurality of pins may be used in order to immobilise the hard scaffold components, for example, 1 , 2, 3, 4, 5, or 6 pins. The pins may extend into or through the hard scaffolds. Preferably, 4 pins are used, particularly preferably two pins on each side of each hard scaffold component, which immobilise the hard scaffolds. Alternatively, the hard scaffolds are retained in position by other means, e.g. a clamp or compression device or other retaining/immobilising device.

The distraction apparatus or device may be produced from any suitable material which has enough rigidity to retain the hard scaffolds in the desired spatial relationship during the distraction/compression process.

It is not necessary that the distraction apparatus or device be as rigid as distractors which are used in vivo (which are generally metal) in view of its use in vitro. Suitable materials will be known to one of skill in the art. Examples includes stainless steel or other metal alloys, for example titanium alloys or aluminium alloys, and rigid polymers, e.g. polypropylene. When the present inventors were studying distraction osteogenesis, they found that the available in vivo distraction devices, such as those used for distracting mouse leg bones in vivo, were not suitable for imaging without first removing the distracted sample from the distraction device due to the configuration of the distraction hardware. Removal of the sample caused changes or alterations in the formation of the distracted sample, and thus experimental results obtained in this way were not always reliable.

The apparatus and device of the invention are therefore both sized and configured in such a way that the gap between the first and second hard scaffold components and, in use, any material comprised therein such as the biocompatible matrix and any cells contained therein, are capable of being analysed by microscopic imaging means whilst being retained in the apparatus or device. It should be noted, however, that the invention is not limited to uses involving microscopic imaging. The apparatus and device must merely be of a size and configuration which are suitable for use in microscopic imaging. In other words, the dimensions of the apparatus and device of the invention are such that the complete distraction apparatus/device is capable of being placed in an analysis or imaging device, for example on a microscope stage, without the need to remove the scaffold/matrix construct from the apparatus/device. Preferably, the apparatus or device has an essentially-flat profile, thus allowing it to be placed on a flat surface, such as that of a microscope stage.

In some embodiments, the apparatus or device is capable of being mounted such that all or part of the distraction gap (and, in use, the biocompatible matrix and any cells contained therein) is capable of being imaged using a light microscope. Preferably, the light microscope is one which is fitted with an objective lens having a focal length of 1 -50mm.

In other embodiments, the apparatus or device is capable of being mounted such that all or part of the distraction gap (and, in use, the biocompatible matrix and any cells contained therein) is capable of being located within the focal length of the objective lens of a light microscope. ln other embodiments, the apparatus or device is capable of being mounted such that all or part of the distraction gap (and, in use, the biocompatible matrix and any cells contained therein) is capable of being mounted within 1 -50mm of the objective lens of a light microscope.

The apparatus and device of the invention are both configured in such a way that light is capable of being transmitted from one side of the apparatus or device to the other side through the distraction gap (e.g. for analysis or imaging purposes) without being impeded by elements of the apparatus or device.

In particular, the apparatus and device of the invention are both configured in such a way that light is capable of being passed from a first side of the apparatus or device to a second side of the apparatus or device through the distraction gap in at least part of a plane perpendicular to the nearest point of contact between the first and second scaffold components or perpendicular to the distraction gap.

In use, this allows for the imaging of the biocompatible matrix and any cells contained therein, at any depth in the distraction gap. In this context, the term "light" includes visible and non-visible wavelengths.

Thus, this property of the present invention provides a key distinguishing feature over the prior art distraction devices, which do not allow imaging, for example in real time, of the tissue samples, due to their size and configuration of the elements of the distraction device (e.g. the distractor, retainer, frame, etc.) with respect to being placed on a microscope stage.

The apparatus and device of the invention may additionally comprise a frame, to which is attached the retaining and locating means (retainer and locator), and the distraction means (distractor).

In some embodiments, the frame is generally U-shaped. In other embodiments, the frame is not C-shaped.

A particular advantage of the apparatus or device of the invention is that it allows for the imaging or visualisation of the bone model in situ, i.e. without the need for the bone model to be removed from the distraction apparatus or device. This allows the bone model to be imaged whilst the scaffold and biocompatible matrix components are retained in a particular spatial configuration. The bone model may then be returned to suitable culture conditions allowing for further tissue regeneration for a set time, and then re-analysed, optionally with further distraction or compression. This cycle of tissue regeneration/analysis may be repeated for any desired number of times, for example 2-5, 2-10 or 2-20 times.

The apparatus or device of the present invention is of a suitable size and configuration to be used in conjunction with a variety of analytical and/or imaging devices. These properties of the apparatus and device allow the researcher to study the cells in situ in the distraction device without the need for removing the tissue from the distraction device.

The bone model or the cell and/or tissue development may be analysed, viewed or imaged by any suitable analytical or imaging technique, preferably a microscopic technique. For example, it may be analysed, viewed or imaged by transmission, fluorescent, upright, inverted, multiphoton or confocal microscopy, or any appropriate imaging technique, without removing the tissue sample from the device or apparatus. Suitable dyes or stains may be applied to the sample if necessary and appropriate for the particular experimental protocol in which the apparatus or device is used.

For example, the scaffold can be visualised using a suitable dye, for example red fluorescent bisphosphonate, such as with 50μΜ rhodamine-risedronate or other calcium phosphate dye.

The cells may be visualised using standard assays, for example the Live/Dead assay.

Other imaging techniques include micro and nanoCT, MRI, ultrasound and X-ray.

In a preferred embodiment, only vital stains or dyes or genetically-modified intracellular markers such as GFP or YFP, are applied to the scaffold or biocompatible matrix and any cells or material contained therein; and real-time imaging of the live cells may be conducted.

An inverted microscope may be used to image three dimensional tissue constructs in real time; the real time response of cells and tissues can therefore be assessed. Digital image correlation technology can be used to provide strain-mapping of the three dimensional construct, which would be very difficult if alternative mechanical-loading devices other than the apparatus or device of the present invention are used. Whilst other devices may exist for applying mechanical load to in vitro tissue constructs, the apparatus and device of the present invention provide a means of imaging tissue constructs without removal of the construct from the apparatus or device.

The apparatus or device, in combination with tissue or a three dimensional tissue model such as that of the present invention, could reduce or replace aspects of animal testing, for example animal testing which involves bone healing and bone imaging. The invention further provides an apparatus or device as defined herein additionally comprising first and second hard porous scaffold components as defined herein, optionally with a biocompatible matrix as defined herein located in the distraction gap between the first and second hard porous scaffold components. In one embodiment of the invention, a coverslip or other suitable support may be placed underneath the hard scaffolds or biocompatible matrix or distraction gap in order to provide extra support for the matrix and to prevent medium that may be present in the scaffold or matrix from leaking into the imaging device. The methods of the invention are carried out in vitro, i.e. they are not carried out on the human or animal body.

The above-described in vitro three dimensional tissue model, the apparatus and the device of the invention may be used for a wide variety of purposes and studies. The model provides an environment which mimics that of in vivo broken, healing, growing or regenerating bone tissue, and hence the bone tissue model can be used in any studies wherein it is desired to study any of these issues.

In particular the invention includes the use of the tissue model in combination with a distractor, for example a device or apparatus of the present invention. The three dimensional tissue model of the invention is not limited to use in the apparatus or device of the invention; the tissue model can be used with any suitable distraction device and can be sized appropriately. The tissue model in combination with a distractor, for example the distraction apparatus or device described herein, can be used for the study of distraction osteogenesis, and the effects of mechanical strain on cells. For example, cellular and molecular effects can be studied. The tissue model can be combined with suitable distraction apparatus and the model may be distracted (or compressed) for example 0.1 , 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mm per day, or distraction (or compression) may be more than 1 mm per day, or may be any suitable length. During distraction, a distraction device (e.g. a thread-, screw- or ratchet-based mechanism) is used to apply force to one or both of the scaffold pieces in order that the scaffold pieces become further apart from each other, and thus the biocompatible matrix clot mimic is stretched. In general during distraction, the length of the gap in which the biocompatible matrix component of the tissue model resides is stretched or lengthened.

Once the sample has been distracted, various assays known in the art can be performed, in order to elucidate signalling and cellular responses to distraction. For example, the effects of distraction on collagen fibre alignment can be assessed. Effects on signalling events, for example pathways involving nuclear c-fos, nuclear phospho-ERK and phospho-FAK can be studied. The tissue model and apparatus and device of the present invention can thus be used to investigate cellular responses to mechanical strain, for example cytoskeletal responses such as actin stress fibre formation and integrin-mediated responses. These pathways can be manipulated with substances such as cytochalasin D, statins, ROCK inhibitor, RGD peptides and srd inhibitor.

Thus, the present invention can particularly be used to study the process of bone healing and/or growth during distraction. An important feature of cells in the distraction gap is to lay down matrix with the appropriate alignment and mechanical properties.

The effects of distraction on collagen fibre alignment can be evaluated.

Mineral production can also be analysed.

Whether the cells align in response to distraction can be studied; cells may align parallel to the direction of the strain or the cells may align perpendicularly. The tissue model provides the skilled person with a model environment which is ideally suited to investigating the cellular and molecular effects of strain and distraction on bone tissue.

However, the use of the three dimensional tissue model of the present invention is not limited to use in studying the effects of strain or distraction osteogenesis.

The effects of compression on bone cells or other kinds of cells which have been seeded onto the scaffolds can also be studied using the tissue model, apparatus or device of the present invention, or alternatively the effects of compression/distraction regimens can be studied, wherein compression is alternated with distraction. Suitable protocols will be apparent to the skilled person.

The tissue model of the present invention, apparatus and device of the invention can be used to study angiogenesis, which denotes the process of blood vessel formation. Angiogenesis may occur during bone regeneration and growth following breakage of the bone, and the present invention allows the study of such processes. In this case bone and/or other appropriate cells can be co-cultured with vascular pre-cursor cells and angiogenesis can be investigated. The tissue model apparatus and device of the invention provides an in vitro model in which to study this process in the context of bone repair/regeneration.

Differentiation of bone precursor cells, for example mesenchymal stem cells and bone marrow stromal cells, can also be studied. Migration of cells from the hard scaffold to the soft scaffold, or vice versa, can also be studied in the tissue model of the present invention. Also, the tissue model can be designed to mimic a skeletal growth plate. In that case, the hard scaffold is seeded with osteoblasts and the soft matrix is seeded with chondrocytes. This distribution of cells mimics the skeletal growth plate, and thus the tissue model, apparatus and device of the invention provides an in vitro three dimensional environment in which cellular and molecular events at the growth plate can be studied.

The present invention provides a model for the sophisticated examination of the effects of regulatory factors on the cells within the tissue model. Regulatory factors, for example growth factors, hormones, signalling molecules etc. can be applied to the scaffold; different regulatory factors may be applied to different sections of the scaffold, or different regulatory factors can be applied to each component of the tissue model. Thus, the present invention allows for a multitude of combinations of regulatory factors, or other molecules, to be applied to the cells within the model, and the effects of such molecules can be examined.

The invention may be used to test the efficacy of drugs or biological molecules in promoting various aspects of bone healing, such as cell migration, orientation, protein expression, extracellular matrix synthesis, mineralisation, angiogenesis and/or cell differentiation.

The invention mimics the effect of distraction on bone cells and can be used to screen for drugs or biological therapeutics which may enhance or accelerate the bone-regeneration process.

In a further embodiment, the invention provides a distraction controller which is capable of simultaneously controlling the distraction gap in a plurality of apparatus or distraction devices as defined herein. Preferably, the distraction controller comprises a (linear) actuator, e.g. a micrometer or screw- based or threaded means. In some embodiments, a micrometer screw device is incorporated into the frame in order to apply identical strain to the distraction apparatus or device.

The distraction controller may also comprise one or more connectors, capable of transmitting the distraction or compression force from the actuator to each of the individual apparatus or distraction devices.

In some embodiments, the distraction controller additionally comprises a frame capable of accommodating the plurality of apparatus or distraction devices. In one embodiment the distraction controller comprises a frame, preferably a metal frame, which can be positioned over, for example, a standard tissue culture six well plate. The frame may extend into each well of the six well plate and attach to apparatus or devices of the invention. Each apparatus or device may be suspended horizontally in a well of the six well plate, held in place by means for locating the apparatus or device in the controller frame.

In some embodiments, each device or apparatus is individually capable of being removed or disconnected from the distraction controller. In other embodiments, each device or apparatus is individually capable of being connected to the distraction controller. In yet other embodiments, each device or apparatus is individually capable of being removed and reconnected to the distraction controller. Preferably, the distraction gap in the removed apparatus or device is retained upon removal or disconnection of the apparatus or device from the distraction controller. This distraction gap between the first and second hard porous scaffolds may be retained by any suitable means, for example, mechanical or magnetic means.

In other embodiments, a clutch-based system may be used to reduce temporarily the retaining force on the scaffold components for a duration and amount which allows the distraction controller to move the scaffold components a sufficient amount to produce a new desired distraction gap.

In general, the same distraction or compression force is applied by the distraction controller to each of the apparatus or devices, hence the distraction gaps in between the pairs of first and second hard porous scaffold components in each apparatus or device is increased or reduced by the same amount.

In some embodiments, the distraction controller is capable of simultaneously controlling the distraction gap in a 2-12, preferably 2-8, and most preferably 2-6 apparatus or distraction devices as defined herein.

The invention also provides a distraction system, comprising a distraction controller of the invention and a plurality of apparatus or devices of the invention.

The system may also comprise a three dimensional bone tissue model of the invention, optionally configured for use within one or more of the apparatus or devices of the invention.

The invention further provides a kit comprising:

(i) one or more apparatus or devices of the invention; and

(ii) a plurality of hard porous scaffold components and/or a biocompatible matrix.

Preferably, the kit additionally comprises (iii) a distraction controller as defined herein. Preferably, the kit is suitable for use in a method as defined below. The invention further provides a method for analysing tissue development in vitro, the method comprising the steps:

and optionally repeating steps (iii)-(v),

wherein the distraction gaps between the first and second hard porous scaffold components in each of the devices are controlled simultaneously by a distraction controller.

Preferably step (v) comprises removing the distraction device from control of the distraction controller; analysing the cell and/or tissue development; and then reconnecting the distraction device to the distraction controller.

The distraction controller therefore has the advantage over single distractors that it enables simultaneous distraction of several samples, thereby administering uniform strain/compression to said bone model samples.

In general, the individual distraction apparatus and devices comprised within the distraction controller can be used for any of the purposes as discussed above for the single distraction apparatus and devices. For example, the distraction controller allows for study of the effects of various stimuli, including mechanical stimuli, on cells and tissue or tissue constructs in a three dimensional environment. The distraction controller, in combination with three dimensional tissue or a tissue model or construct or apparatus or device of the invention, can be used to study and screen for drugs or therapeutic molecules, for example which may enhance or accelerate bone regeneration.

In a preferred embodiment, the distraction controller is used in combination with the three dimensional tissue model of the present invention or apparatus or device of the invention, although the invention is not limited to the use of the distraction controller with the tissue model of the present invention or apparatus or device of the invention.

In yet a further embodiment, the invention provides an apparatus or device for use in a method as defined herein, comprising:

first (101 ) and second (102) mounting bars,

wherein the bars (101 , 102) each comprise a plurality of retaining devices (104) mounted thereon, the retaining devices (104) comprising adjustable retainers (105) capable of retaining first and second scaffold components between the bars,

wherein the first and second bars (101 , 102) are retained substantially parallel to each other by one or more actuators (106),

wherein the actuators (106) are capable of retaining the first and second bars (101 , 102) at a predetermined, incrementally-variable distance from one another,

and thereby capable of uniformly incrementally-varying the distraction gaps between all first and second scaffold components which are retained in adjacent retaining devices (104). In yet further embodiments, the invention provides an apparatus or device for use in a method as defined herein, comprising:

first (101 ), second (102) and third (103) substantially parallel mounting bars, wherein the bars (101 , 102, 103) each comprise a plurality of retaining devices (104) mounted thereon, the retaining devices (104) comprising adjustable retainers (105) capable of retaining first and second scaffold components between adjacent bars, wherein the first and second bars (101 , 102) are retained substantially parallel to each other by one or more actuators (106),

wherein the said actuators (106) are capable of retaining the first and second bars (101 , 102) at a predetermined, incrementally-variable distance from one another, and thereby capable of uniformly incrementally-varying the distraction gaps between all first and second scaffold components which are retained in adjacent retaining devices (104) between the first and second bars (101 , 102),

wherein the second (102) and third (103) bars are retained substantially parallel to each other by one or more actuators (107),

wherein the said actuators (107) are capable of retaining the second (102) and third (103) bars at a predetermined, incrementally-variable distance from one another,

and thereby capable of uniformly incrementally-varying the distraction gaps between all first and second scaffold components which are retained in adjacent retaining devices (104) between the second (102) and third (103) bars.

In some preferred embodiments of the invention the retaining devices (108) which are mounted on the second bar (102) comprise adjustable retainers (109) which are capable of retaining first and second scaffold components between the first (101 ) and second (102) bars and adjustable retainers (1 10) which are capable of retaining first and second scaffold components between the second (102) and third (103) bars.

The mounting bars generally substantially parallel. The mounting bars are adapted to receive a plurality of retaining devices, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 or more retaining devices. The retaining devices are preferably removably mounted on the mounting bars.

The mounting bars are preferably made from metal, e.g. sheet metal. The bars may, for example, be 3-7 mm thick, preferably 4-6 mm thick, most preferably about 4.9 mm thick. The actuators are preferably linear actuators, e.g. a micrometer or screw-based or threaded means. The actuators are located on the mounting bars. The may act independently or the actuators on any one mounting bar may act co-ordinately, i.e. together. The actuators are capable of providing micrometer increments. The retaining devices comprise adjustable retainers capable of retaining first and second scaffold components (preferably hard porous scaffold components) between adjacent bars. The adjustable retainers are preferably adjustable retaining pins.

In some embodiments, the adjustable retainers are integral (i.e. part of) the retaining devices. In some preferred embodiments, the adjustable retainers are retained in the retaining devices by magnetic means; this enables the removal of the adjustable retainers (optionally with the associated scaffold components) from the retaining devices. The magnets are preferably neodymium magnets. The retaining devices are generally present in pairs which are adjacent/opposite to each other, e.g. with retaining devices on the first bar being positioned opposite retaining devices on the second bar; and, where present, retaining devices on the third bar being positioned opposite retaining devices on the second bar. In this way, the first of each pair of the retaining devices will retain the first scaffold component and locate it at a position which is a defined distance away from the second scaffold component which is retained by the second in the pair of the retaining devices.

The first and second scaffold components are preferably held in an end to end orientation a defined distance apart by the first and second retaining devices. The scaffold components may be retained perpendicular to the mounting bars, although other configurations are possible.

In these embodiments, the distraction gaps between all of the first and second scaffolds positioned between adjacent bars can be increased/decreased uniformly, i.e. in unison. This provides advantages in terms of the number of osteogenesis models which can be carried out and also in terms of being able to compare models under defined, possibly closely-related, conditions.

The invention further relates to the use of an apparatus or device of the invention in a method of the invention.

The invention further provides a kit comprising:

(i) first (101 ) and second (102) mounting bars,

(ii) a plurality of retaining devices (104) capable of being mounted on the mounting bars, and optionally,

(iii) scaffold components,

wherein the retaining devices (104) comprising adjustable retainers (105) capable of retaining first and second scaffold components between the bars,

wherein the first and second bars (101 , 102) are retained substantially parallel to each other by one or more actuators (106), wherein the actuators (106) are capable of retaining the first and second bars (101 , 102) at a predetermined, incrementally-variable distance from one another,

and are thereby capable of uniformly incrementally-varying the distraction gaps between all first and second scaffold components which may be retained in adjacent retaining devices (104).

The invention further provides a kit comprising:

(i) first (101 ), second (102) and third (103) substantially parallel mounting bars,

(iii) scaffold components,

wherein the retaining devices (104) comprising adjustable retainers (105) capable of retaining first and second scaffold components between adjacent bars,

wherein the said actuators (106) are capable of retaining the first and second bars (101 , 102) at a predetermined, incrementally-variable distance from one another,

and thereby capable of uniformly incrementally-varying the distraction gaps between all first and second scaffold components which may be retained in the retaining devices (104) between the first and second bars (101 , 102),

and thereby capable of uniformly incrementally-varying the distraction gaps between all first and second scaffold components which may be retained in adjacent retaining devices (104) between the second (102) and third (103) bars.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows that 2T3 mouse osteoblasts survive and proliferate in fibrin clots. The Figure shows cell seeded fibrin clots that were cultured and harvested at the indicated time points and stained with Live/Dead reagent and imaged with fluorescent microscopy (4x).

Figure 2 shows that alkaline phosphatase staining increases over 7 days. The Figure shows cell-seeded fibrin clots that were cryosectioned and stained for alkaline phosphatase activity (4x). The top panel shows cells that were grown in control medium, and the bottom panel shows cells grown in osteogenic medium.

Figure 3 shows fibrin-scaffold constructs that were assembled and cultured for 7 days.

Live/Dead stain showed viable cells populating the fibrin and lining the pores of the scaffold using fluorescence (top and middle panels 4x, bottom panel 10x).

Figure 4 shows the fibrin-scaffold constructs of Figure 3 imaged with confocal microscopy showing cells in a single plane.

Figure 5 shows cell seeded constructs that were cultured and distracted 0.5mm on day 3. Rhodamine-risedronate was combined with Live/Dead stain to visualise the live cells in relation to the mineral scaffold using fluorescence microscopy. The Figure shows constructs 1 day after distraction (top and middle panels 4x, bottom panel 10x).

Figure 6 shows confocal microscopy of the constructs of Figure 5.

Figure 7 shows the distraction apparatus. The top panel shows the non-distracted construct with a 1 mm cell-seeded fibrin gap, and the bottom panel shows the construct which has been distracted 1.5mm. The construct was cultured for 3 days then distracted 0.5mm on day 3.

Figure 8 shows a graphical representation of the three dimensional tissue model.

Figures 9A-9D and 10A-10B show examples of the apparatus or device of the invention.

Figure 1 1 : Sequence of images of fibrin construct in flat distractor undergoing distraction, analysed to produce the vector and strain fields shown in Figure 12. Images are at 2x, acquired on an Olympus microscope. Scale bar = 1 mm and applies to all images. Black speckles were produced by applying photocopier toner on the surface. Top row (0): fibrin gap at original pre- distraction 0.9 mm length, before (left) and after (right) application of toner. Numbers indicate the order of distraction, with each the gap in each image lengthened by 0.075 mm compared to the previous image. For example, (1 ) shows a 0.075 mm distraction compared to (0), (6) shows a total 0.45 mm distraction compared to (0), and (12) shows a 0.9 mm distraction compared to (0). Figure 12: Deformation vector fields (left column) and strain maps of maximum normal strain (right column) of one scaffold-fibrin construct. The vector fields and strain maps were calculated from corresponding image sequences. The scales indicate vector length (left) and percentage strain.

Figure 13: Live/Dead staining of fibrin-scaffold constructs at the indicated time points after a single 0.45 mm distraction. Non-distracted control "Ctrl" samples were stained at the same time points indicated as the corresponding distracted samples. 4x (left columns) and 10x (right columns) images were acquired with a Nikon inverted fluorescence microscope.

Figure 14: Alkaline phosphatase staining of a fibrin-scaffold construct at indicated time points after a single 0.45 mm distraction. 2x scale bar = 1 mm, 4x scale bar = 500 μηη. Constructs at specified time points are the same as those shown in Figure 13. 2x (left column) and 4x (right column) images are shown. The 4-hour post-distraction sample was damaged during processing. 2x images are shown in the left column and 4x images are in the right column.

Figures 15-17: Embodiments/parts of the multi-distractor of the invention.

The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The disclosure of each reference set forth herein is incorporated herein by reference in its entirety. EXAMPLES

Example 1 : Three dimensional tissue model assembly

Hard scaffolds were obtained from Dr J. Lu of Biocetis, Village Hannibal, 34660, Cournonsec, France. The scaffolds are a 60% hydroxyapatite, 40% tricalcium phosphate composite material with interconnected pores. The supplier provided them pre-cut to the dimensions of 3mm x 3mm x 10mm.

The Tisseel kit, sold for surgical sealant use by Baxter Healthcare Ltd (Norfolk, UK), provided the materials for producing the fibrin soft matrix. Tisseel powder (containing fibrinogen) was dissolved in aprotinin solution at a concentration of 57.5 mg/ml, under sterile conditions and according to the manufacturer's recommendations. Thrombin powder was dissolved in calcium chloride solution at a concentration of 250 lU/ml. Solutions were maintained at 33-37^sC until use. 2T3 mouse osteoblast cells were used between passage 19-40. They were resuspended in fibrinogen solution at a concentration of 300,000 cells/mL immediately prior to fibrin

polymerization.

Control medium consisted of alpha-MEM (Gibco, Paisley, UK) supplemented with 7% fetal bovine serum (Biosera, Ringmer, UK), 100 U/ml penicillin and 100 mg/ml streptomycin.

Osteogenic medium consisted of control medium supplemented with 10mM beta- glycerophosphate, 50 Mg/mL L-ascorbic acid and 10-8M dexamethasone. Supplements were from Sigma-Aldrich unless otherwise stated. Two 3mm x 3mm x 10mm blocks of hard scaffold were installed in a mouse fixator, provided by Rocky Tuan (University of Pittsburgh Medical Center) using pins and screws such that the middle ends of both blocks were in contact. The movable ring of the fixator was then moved 1 mm away from the bottom ring by turning the nuts 1 full turn. This produced a 1 mm gap in between the 2 scaffolds. Equal volumes of thrombin solution and fibrinogen solution (containing cells) were mixed by pipetting in the gap and allowed to polymerize into fibrin gel for 10 minutes in air. The scaffold-fibrin construct was then transferred into control medium or osteogenic medium and cultured in a humidified environment containing 20% oxygen and 5% carbon dioxide. Medium was changed every 2-3 days for up to 7 days in culture. Viability testing

The Live/Dead Viability/Cytotoxicity assay solution (Molecular Probes, Eugene, Oregon) was prepared for a final concentration of 4 μΜ Calcein AM and 2μΜ ethidium homodimer-1 in phosphate buffered saline, abbreviated PBS (Lonza, Basel, Switzerland). On days 1 , 3, and 7 after assembly, the fixator containing the scaffold-fibrin construct was removed from the culture medium. The construct was removed from the fixator and washed in PBS, then immersed in Live/Dead stain for 30 minutes at room temperature in the dark. The stained construct was then washed again and placed on a glass slide for imaging. An Olympus upright fluorescence microscope and a Zeiss upright confocal microscope were used to detect images at 4x and 10x magnification of green and red fluorescence in the stained samples. Live cells appeared green and dead nuclei appeared red.

Alkaline phosphatase activity

Cell-seeded fibrin clots were produced by mixing fibrinogen solution and thrombin as described above, in the absence of the hard scaffolds. Clots were cultured in control or osteogenic medium for up to 7 days. Samples were harvested at 1 , 2, 3, and 7 days after seeding and placed in Cryo-M-Bed (TAAB Laboratories Equipment Ltd, Aldermaston, UK) then frozen at - 20 °C. Samples were thawed and cryosectioned or subjected to whole mount staining.

Cryosectioned samples were cut into 10 μηη sections and air-dried on glass slides, then stored at -80 °C until use.

For the alkaline phosphatase activity stain, samples were thawed and rinsed in PBS, then post- fixed in 95% cold industrial methylated spirits (IMS) for 1 minute. The staining solution, 0.01 % naphthol AS-MX phosphate alkaline solution (Sigma) and 240 g mL fast violet B salt (Sigma), was applied to the samples for 20 minutes at room temperature. Samples were rinsed with water, and selected clots and sections were counterstained with 10 g/mL DAPI blue fluorescent nuclear stain (4', 6-diamidino-2-phenylindole, Molecular Probes, Eugene, OR) for 20 minutes. An Olympus upright microscope was used to capture light images and fluorescence images. Example 2: Distraction

This Example provides details of experiments performed using a prior art distractor wherein the scaffold/matrix construct had to be removed from the distractor prior to imaging. The prior art distractor is shown in Figures 7-8. Cell-seeded constructs were cultured in the mouse fixators for 3 days (see Figures 7 and 8). To apply a 0.5mm distraction of the fibrin gap on Day 3 after seeding, the fixator was removed from the culture vessel and medium. The movable ring was moved 0.5 mm away from the bottom ring by turning the nuts 0.5 turn each. This created a 0.5mm increase in the gap between the two hard scaffold ends, thus stretching the cell-seeded fibrin clot. The fixators were returned to culture in medium.

Distraction imaging

On Day 4, one day after distraction, the distracted constructs were harvested for imaging.

Live/dead staining was applied as described above. Selected constructs were stained with a combination stain, containing both live/dead reagents and the red fluorescent bisphosphonate, rhodamine-risedronate (10 μΜ, provided by Prof Graham Russell). The rhodamine-risedronate binds tightly to mineral, and hence the hard scaffold was visible on confocal microscopy images. Fluorescence and confocal microscopy imaging was used as described.

Viability

2T3 mouse osteoblasts survive and proliferate in fibrin clots over 7 days, as shown in the increase in number of green fluorescent cells (see Figure 1 ). Fewer dead red nuclei were evident at each time point than live green cells. The same trend was evident for cells in the scaffold-fibrin construct, which showed live green cells in the fibrin clot and lining the pores of the scaffold (see Figures 3 and 4).

Alkaline phosphatase activity

Alkaline phosphatase activity increases in cell-seeded fibrin clots over 7 days, with clots cultured in osteogenic medium showing more purple staining at each time point than those cultured in control medium. Combined alkaline phosphatase and DAPI stain show that areas containing more nuclei also show more intense staining (see Figure 2).

Distraction

Live green fluorescent cells are visible in both the fibrin clot and the scaffold. The fibrin-bridged gap is clearly visible (see Figures 5 and 6). Samples stained with rhodamine-risedronate allow visualization of the mineral scaffold surface in addition to the cells. Example 3: Distractor of the invention

Figures 9A-9D and 10A-10B show examples of a distraction apparatus or device of the invention. The distractor comprises two U-shaped steel frames of dimensions 30mm x 15mm which are held spatially apart by threaded bolt shafts, thus allowing the distance between the first and second steel frames to be incrementally varied.

Retainer means are also attached to each of the frames in the form of sharp metal pins which extend into and act to immobilise the hard scaffolds. In the Figures, the scaffold components consist of four 3mm x 3mm x 10mm blocks of 60% hydroxyapatite/40% tricalcium phosphate.

All threaded parts have thread size 2-56. Example 4: Further distractor of the invention

Methods

Hard scaffolds were assembled with a 0.9 mm gap in the flat distractors using a Delrin^® mould which was designed to assist the fibrinogen-thrombin mixing process by providing a groove to hold the hard scaffolds in place and producing a mixing chamber between the scaffold edges when they were separated. Fibrinogen was prepared at a final 28.75 mg/ml concentration, and thrombin at a final 5 lU/ml concentration. For the cell-seeded experiments, the fixator-scaffold assemblies were autoclaved prior to pre-incubation, and 2T3 mouse osteoblast cells were resuspended in the fibrinogen solution at a final density of 45 ^χ 10⁴ cells/ml. Scaffold assemblies were pre-incubated in warm PBS (strain mapping studies) or warm sterile medium (cell studies) for 1 hour. To produce the fibrin clot, the casting mould was used to mix 20 μΙ fibrinogen solution and 20 μΙ thrombin solution between the scaffold ends.

Results

Incremental distraction of toner-speckled fibrin-scaffold constructs

Raw images of the incremental distraction process of photocopier toner-speckled constructs were collected by light microscopy for digital image correlation analysis. In the cell experiments, the fibrin gap was distracted by 50% of the original length (0.45 mm length increase,) so for the strain analysis it was important to achieve at least this magnitude of distraction. Indeed, for two of the four strain mapping samples, the fibrin gap was distracted by twice this length (0.9 mm at 12 incremental distractions.) A representative image sequence is shown in Figure 1 1. The pre- distraction 0.9 mm fibrin gap (0), and the succession of 0.075 mm incremental distractions up to a total increase in length of 0.9 mm (12) is shown in Figure 1 1 . One sample was distracted up to 1 .8 mm (four times the amount applied in the cell studies), with some tearing of the clot evident after 1 .35 mm of distraction. The fourth sample contained a bubble and exhibited early signs of tearing after 0.225 mm of distraction, with greater tearing evident at 0.45 mm. In each sequence, note that while the x-y position of the construct varies as a result of manually removing the fixator to effect the distractions, the surface is always in focus (indicating a constant z-position). DIC strain analysis

The light microscope images in tagged image file format were converted to 8-bit files in ImageJ (NIH, Bethesda, MD,) and imported into DaVis 7.2 Software (LaVision GmbH, Gottingen, Germany) for DIC analysis. Results of DIC analysis for one of the four construct samples are given in Figure 12. In the left column, the deformation vector field is superimposed on the raw image used in the analysis. The vectors are displayed as arrows pointing in the direction of deformation. Magnitude is indicated by vector length as well as by the shade of the background. The scale indicates the vector length in pixels, with the minimum set at 0 and the maximum set at the maximum value of the vector field sequences of each sample. Longer vectors indicate larger displacements. In all vector fields, the vectors indicate that the scaffold edges are moving apart in the vertical direction, as expected.

In the right column, maximum normal strain percentage (also known as the principal strain percentage), calculated from the associated vector field, is indicated at the corresponding region by shade. For each sample sequence, the shade scale minimum was set at the minimum strain value in the series. Negative strain values indicate compression. The scale maximum was set at either 15% or 25%, whichever showed the greatest detail in the gap region. The outline of the scaffold and fibrin gap may be discerned in the images. Areas of high strain may be observed to mapped to the background regions of the image. These, and areas at the edges of the image, should not be considered reliable. The results show that a central region of high strain is evident in the gap, with unevenly distributed strains approximately ranging between 1 % to 15%. Most areas of the gap exhibit strains approximately between 2.5% and 7.5%. In inhomogeneous areas, strains of 20% or greater are evident.

Due to the rather large displacements of the constructs between images, shift and rotation correction was not accurate for the entire image sequence for any of the samples. Hence, only the results from the first 6 incremental distractions (equivalent to 0.45 mm total distraction) are reported, as this distraction amount is immediately relevant to the cell studies. The extracted regions were manually selected to account for differences in construct geometry and orientation in the image sequences, and therefore the analyzed region varies in size among the samples.

Each numbered vector field corresponds to the deformation (or difference) between two consecutive images such that the number displayed matches the largest number of incremental distractions represented in the two images analysed. That is, the vector field shown in Figure 12(1 ) represents the deformation between the original pre-distraction gap (0) and the gap after one 0.075 mm distraction (1 ). Vector field (2) represents the deformation in the images representing the first 0.075 mm incremental distraction (1 ) and the second 0.075 mm distraction (2). As the strain maps are calculated from the vector fields, the numbered strain maps correspond to the raw images as described above. Time course profiling of a single distraction on an in vitro model of DO

Results of the Live/Dead staining of cell-seeded distraction experiments are presented in Figure 13. Within each experiment, 6 constructs were harvested for the end point assay at the indicated time points after the 0.45 mm distraction of the fibrin gap. Non-distracted controls were also included, harvested at the same time as the distracted samples at the indicated time points. Each construct tested is represented in the figure, with one image at 4x magnification showing the entire gap with scaffold edges, and one image at 10x to show a representative area in greater detail. The green "live" stain allows much crisper visualization of cell morphology than Dil. Red "dead" nuclei are evident, but are greatly outnumbered by live cells. Results of the alkaline phosphatase stained constructs are presented in Figure 14. Alkaline phosphatase staining was carried out after live/dead staining. This shows the same experiment and scaffolds as Figure 13. Alkaline phosphatase activity is indicated by reddish-purple cell- associated staining. Images at 2x are shown on the left and at 4x on the right. The scaffolds are on the left and right of the distraction gap. Note that the 4-hour distraction sample is not presented as it was damaged during processing.

Summary

In total, the studies described above characterize this model as a valid model of early distraction osteogenesis. Mechanically, the strains calculated within the gap are comparable to those in an in vivo model (Waanders 1998, Aronson 1989.) Biologically, alignment and alkaline phosphatase patterns at 24 hours are similar to the elongated columns of mineralizing tissue in the mineralization zone shown in in vivo studies. In terms of the materials, the enhanced differentiation of cells on the scaffold compared to within the gap is an example of the benefits of incorporating both hard and soft substrates, as this more accurately models the in vivo situation. These experiments demonstrate the utility and versatility of the model in fulfilling the design purpose of creating a system that allows thorough investigation of the early distraction osteogenesis process.

Example 5: Multi-distractor over 6-well cell culture plate

This multi-distractor is based on the concept of a moving frame. This relies on the side parts of the frame (cut from a metal sheet and 4.9 mm thick) moving away from the centre part of the frame. It is illustrated in Figures 15-17.

Distraction is controlled by micrometer heads. On the side part, the stem of the micrometer sits in and is secured with a stem locknut. On the centre part, the spindle is proposed to sit in an available bearing. The point is to enable rotation, but to block the movement along axis.

Therefore, for example, thin O-rings could be used.

The dimensions of the bearing used in the model may be adjusted for specific designs. Sizes of screws are not given. Those in the current design are M1.6 to fit holes and threads and should be as long as necessary. For mounting, M3 of sufficient length could be used and secured with a nut. This final design allows about 4 mm of distraction, which is enough for the scaffold and fibrin used at the moment (1 mm fibrin length) Assemblies A3 (Figure 15B) and C1 (Figure 16B) rest on frame parts, and their inner width is exactly equal to the width of the flat parts of the frame. The reason is that a very tight fit is needed. Circular depressions in these parts are designed to fit 1 mm diameter flat neodymium magnets. A2s (Figure 15A) are placed on the assemblies so that magnets are aligned as shown in A3 and C1 .

B3 (Figure 16A) is a tool for removing A2 parts while remaining their relative arrangement (and hence the hard scaffold-fibrin assembly held in place by the pins.) B3 includes a threaded rod which allows the adjustment of the position of the magnets on the block at the end of the rod for this purpose.

Materials can be any appropriate. They should as light as possible, but allow for relevant manufacturing. Parts that are submerged in solutions may be made of stainless steel.

References:

1 . Waanders NA et al. 1998. Clin Orthop Relat Res (349) pp. 225-34.

2. Aronson et al. 1989. Clin Orthop Relat Res (241 ) pp. 106-16.

Claims

1 . A method for analysing tissue development in vitro, the method comprising the steps:

(v) analysing the cell and/or tissue development in the first and/or second hard scaffold components and/or biocompatible matrix whilst the first and second hard scaffold components and the biocompatible matrix are retained in situ in the distraction device; and optionally repeating steps (iii)-(v).

2. A method for analysing tissue development in vitro, the method comprising the steps:

and optionally repeating steps (iii)-(v).

3. A method for analysing tissue development in vitro, the method comprising the steps: (i) retaining, in a plurality of distraction devices, a plurality of pairs of first and second hard porous scaffold components adjacent to one another, wherein each pair of first and second hard porous scaffold components are separated by a distraction gap;

and optionally repeating steps (iii)-(v),

4. A method as claimed in any one of the preceding claims, wherein the tissue

development is bone generation or bone regeneration.

5. A method as claimed in any one of the preceding claims, wherein the hard porous scaffold components are bone mimics.

6. A method as claimed in any one of the preceding claims, wherein the hard porous scaffold comprises hydroxyapatite and/or tricalcium phosphate.

7. A method as claimed in any one of the preceding claims, wherein one or both of the adjacent surfaces of the hard porous scaffold have been modified.

8. A method as claimed in any one of the preceding claims, wherein the hard porous scaffolds are in the form of cuboidal blocks, each having a volume of 5-1000mm³.

9. A method as claimed in any one of the preceding claims, wherein the cells are osteoblasts, osteocytes and/or osteoclasts.

10. A method as claimed in any one of the preceding claims, wherein the distraction gap is initially 0.1 -10mm.

1 1 . A method as claimed in any one of the preceding claims, wherein the biocompatible matrix is a natural or synthetic, polymerisable or cross-linkable material.

12. A method as claimed in any one of the preceding claims, wherein the biocompatible matrix comprises a gel, preferably a collagen gel or fibrin gel.

13. A method as claimed in any one of the preceding claims, wherein the analysing the cell and/or tissue development is carried out using a microscopic technique.

14. A method as claimed in any one of the preceding claims, wherein the analysing the cell and/or tissue development is carried out using transmission, fluorescent, upright, inverted, multiphoton or confocal microscopy.

15. An in vitro three dimensional bone tissue model for use in a method as defined in any one of claims 1 to 14, comprising:

(i) a first hard porous scaffold component,

(ii) a biocompatible matrix, and

(iii) a second hard porous scaffold component

16. Apparatus for use in a method as defined in any one of claims 1 to 14, comprising:

(i) means for retaining a first hard scaffold component;

(ii) means for retaining a second hard scaffold component; (iii) means for locating the first hard scaffold component at a position adjacent to the second hard scaffold component and for incrementally varying the gap between the first and second hard scaffold components,

17. A distraction device for use in a method as defined in any one of claims 1 to 14, said device comprising distraction means for incrementally distracting a first and a second hard scaffold component wherein the distraction device is sized and configured such that the distraction gap and, in use, any material comprised therein, is capable of being analysed by microscopic imaging means whilst being retained in the device.

18. Apparatus or device for use in a method as defined in any one of claims 1 to 14, comprising:

first and second U-shaped frames, each U-shaped frame comprising two arms and a connecting base, wherein the arms of each U-shaped frame each comprise adjustable retaining pins capable of retaining first and second scaffold components between the arms, wherein the first and second U-shaped frames are retained substantially parallel to each other by one or more threaded shafts which are located between the adjacent arms of the first and second U-shaped frames, wherein the threaded shafts are capable of retaining the first and second frames at a predetermined, incrementally-variable distance from one another, and thereby capable of incrementally-varying the distraction gap between any first and second scaffold components retained therein.

19. Apparatus or device as claimed in claim 18 wherein the length of the base is 10-50mm, each arm is 5-30mm and the length between each U-shaped frame is 5-30mm.

20. A distraction controller which is capable of simultaneously controlling the distraction gap in a plurality of apparatus or distraction devices as claimed in any one of claims 16 to 19.

21 . A kit comprising:

(i) one or more apparatus or devices as claimed in any one of claims 16 to 19;

22. A kit as claimed in claim 21 , additionally comprising:

(iii) a distraction controller as defined in claim 20.

23. Apparatus or device for use in a method as defined in any one of claims 1 to 14, comprising:

first (101 ) and second (102) mounting bars,

and thereby capable of uniformly incrementally-varying the distraction gaps between all first and second scaffold components which are retained in adjacent retaining devices (104).

24. Apparatus or device for use in a method as defined in any one of claims 1 to 14, comprising:

first (101 ), second (102) and third (103) substantially parallel mounting bars,

wherein the bars (101 , 102, 103) each comprise a plurality of retaining devices (104) mounted thereon, the retaining devices (104) comprising adjustable retainers (105) capable of retaining first and second scaffold components between adjacent bars,

and thereby capable of uniformly incrementally-varying the distraction gaps between all first and second scaffold components which are retained in adjacent retaining devices (104) between the first and second bars (101 , 102),

wherein the said actuators (107) are capable of retaining the second (102) and third (103) bars at a predetermined, incrementally-variable distance from one another, and thereby capable of uniformly incrementally-varying the distraction gaps between all first and second scaffold components which are retained in adjacent retaining devices (104) between the second (102) and third (103) bars.

25. Apparatus or device as claimed in claim 23 or 24, wherein the retaining devices (108) which are mounted on the second bar (102) comprise adjustable retainers (109) which are capable of retaining first and second scaffold components between the first (101 ) and second (102) bars and adjustable retainers (1 10) which are capable of retaining first and second scaffold components between the second (102) and third (103) bars.

26. A kit comprising:

(i) first (101 ) and second (102) mounting bars,

(iii) scaffold components,

27. A kit comprising:

(iii) scaffold components,

wherein the said actuators (106) are capable of retaining the first and second bars (101 , 102) at a predetermined, incrementally-variable distance from one another, and thereby capable of uniformly incrementally-varying the distraction gaps between all first and second scaffold components which may be retained in the retaining devices (104) between the first and second bars (101 , 102),