WO2021064139A1 - Biomaterial construct and methods of production thereof - Google Patents

Abstract

The present invention relates to a biomaterial construct, a method of producing a biomaterial construct, a tissue equivalent implant comprising or consisting of said biomaterial construct and a method of treatment of a damaged tissue in an individual using said biomaterial construct. The method of producing the biomaterial construct comprises steps of providing a vessel containing a hydrogel, and simultaneously rotating the vessel about both a first axis of rotation and a second axis of rotation to apply a centrifugal force to the hydrogel, thereby causing plastic compaction of the hydrogel to form the biomaterial construct by expulsion of at least some of a fluid phase of the hydrogel through a surface of the hydrogel. Biomaterial constructions formed according to this method show improved mechanical properties in comparison to biomaterial constructions formed by conventional methods of hydrogel compaction, due to more homogeneous plastic compaction of the hydrogel achievable by the method of the present invention.

Description

Biomaterial construct and methods of production thereof

Field

The present invention relates to biomaterial constructs and methods of production thereof. Particularly, although not exclusively, it relates to biomaterial constructs having particular applicability for tendon and other tissue repair grafts, and methods of making such constructs.

Background

A major challenge in tissue engineering is to fabricate biomimetic scaffolds which are suitable for replacement of native human or animal tissues. Over the last decade, many synthetic and natural origin scaffolds have been used to serve this purpose [1], [2] Synthetic polymers have good physical and structural properties, but the limitation is that they have a poor biological function. Tissue engineering with natural polymers has inherent advantages over synthetic polymers due to their improved biological function [3], [4]

Collagen type 1 is a naturally occurring polymer commonly used in tissue engineering applications. Fabrication of cell-seeded collagen gels in tissue engineering has being widely used since it was reported [5], [6] To date, various groups have studied and used collagen gels with cells as a 3D model to mimic various tissues in vitro and in vivo [7], [8], [9]

However collagen hydrogels typically have low mechanical strength, and accordingly may not be suitable for use in all applications without further processing. In 2005, Brown et al developed a method for rapid compression of collagen hydrogel by a technique known as ‘plastic compression’, using vertical static compression (VSC). Here, a collagen gel is compressed by a static weight placed on top, to extract water from the gel and to form a collagen sheet having improved strength. The degree of compression is directly proportionate to the applied weight [10] this process is shown schematically in Fig. 1. The collagen gel 1 is compressed by a weight W to form a collagen sheet 2. The collagen sheet 2 is rolled to form a biomaterial construct 3. In 2012, Braziulis et al modified this technique by using T-shaped compression stamps and used this as skin substitute [11]

However, fabricating constructs which replicate native tissue types having a complex nature (such as tendons) is challenging.

US9101693 discusses the plastic compression method developed by Brown et al. in detail. This discloses a technique of rolling a compressed biomaterial sheet formed using VSC to form a three- dimensional biomaterial construct. However, constructs formed in this manner suffer from a number of problems. One problem is that voids/gaps may be present between consecutive rolled layers of the construct. This can lead to lower structural strength of the construct. Additionally, cells seeded on or within the construct tend to ‘fill the gap’ and migrate or proliferate in such void regions. Accordingly constructs formed in this manner tend to present a heterogeneous cell distribution when seeded with cells.

The present invention has been devised in light of the above considerations.

Summary

The present inventors have developed a method for producing compressed collagen with properties suitable for tissue engineering purposes. Collagen constructs produced according to this method may find particular use in fabrication of biomimetic materials for tendon, blood vessels and bladder tissue engineering.

Accordingly, in a first aspect, the present invention provides a method of producing a biomaterial construct comprising steps of providing a vessel containing a hydrogel; and simultaneously rotating the vessel about both a first axis of rotation and a second axis of rotation to apply a centrifugal force to the hydrogel, thereby causing plastic compaction of the hydrogel to form the biomaterial construct.

The hydrogel comprises a fluid phase and the centrifugal force applied by rotation about the first and second axis expels at least some of the fluid phase out of the hydrogel through a surface of the hydrogel (which may be referred to as a fluid leaving surface). The expulsion of fluid phase causes the hydrogel to plastically compact.

By applying simultaneously rotating the vessel about both a first axis of rotation and a second axis of rotation to apply the centrifugal force to the hydrogel, plastic compaction of the hydrogel may occur in a more homogeneous manner than in typical known methods of plastic compaction.

Rotation about the first axis may apply a radial centrifugal force to the hydrogel (i.e. a force directed outwards from the centre of the hydrogel to the surface), and the rotation about the second axis may distribute the radial centrifugal force over the surface of the hydrogel. The radial centrifugal force may be 1 N or more, preferably between about 1 N and about 5 N. The magnitude of the force applied may be selected by appropriate selection of the rotational speed. This allows the expulsion of the fluid phase over the surface of the hydrogel (i.e. it increases the proportion of the hydrogel surface that acts as a fluid leaving surface). The fluid leaving surface accordingly may be the entire perimeter of the hydrogel, thereby resulting in more even plastic compression of the hydrogel, which may be symmetrical about a longitudinal axis of the hydrogel. Where rotation about a second axis of rotation is not provided, the fluid leaving surface will comprise only a portion of the perimeter of the hydrogel, thereby resulting in uneven or asymmetrical plastic compression of the hydrogel.

Plastic compaction involves deforming an object such as a gel (here, a hydrogel) to reduce its volume, such that the object substantially retains its new volume, even after the cause of compaction is removed. Plastic compaction is a rapid, cell-independent process which results from subjecting the gel to a physical treatment, such as an external force or pressure, which expels interstitial liquid (i.e. a fluid phase) from the gel, such that it does not return on removal of the load: i.e. the gel undergoes a plastic compaction. Gels comprise a scaffold matrix, which in an untreated gel, is generally in a gross, hydrated form. This scaffold structure collapses during plastic compaction without loss of structural detail, dehydrating the scaffold in the gel, and leading to increased density and strength. Plastic compaction is distinct from the slow process of cell-driven contraction, which occurs through the intrinsic action of cells growing within the gel i.e. plastic compaction is not cell-mediated and does not occur through the action of cells which are cultured within the gel. Plastic compaction may have a vector in one, two or more defined directions and the direction, rate and extent of the compaction is controllable.

The amount or extent of compaction may be varied, depending on the intended use of the resulting biomaterial construct. Compaction of the hydrogel, may result in a reduction in the thickness, for example the diameter, of the gel of at least 5 fold, at least 10 fold or at least 20 fold. For example, the volume of the hydrogel may be reduced by 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 99.9% or more by plastic compaction. This reduction in volume may be partially or entirely due to expulsion of the fluid phase of the hydrogel. For example, the amount of fluid lost or removed from the hydrogel by plastic compaction may be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or at least 99.9% of the original fluid content of the gel. Preferably, some interstitial fluid remains after compaction, for example at least 10%, at least 1% or at least 0.1% of the original fluid content of the gel.

The vessel containing the hydrogel may be any suitable container. The vessel may have a substantially circular cross-section. Preferably, vessel may have a substantially circular cross-section in a direction perpendicular to the second axis of rotation. In this way, it may be possible to produce substantially cylindrical biomaterial constructs. Additionally provision of a substantially circular cross section may encourage substantially symmetrical plastic compression of the hydrogel. Conveniently, the vessel may be e.g. a centrifuge tube. The volume of the vessel is not particularly limited and may be selected based on the volume of hydrogel intended for plastic compaction. However in some embodiments, the volume of the vessel may be from 10 ml to 200 ml, for example about 50 ml.

The vessel may comprise a region for collection of the expelled fluid phase of the hydrogel. The region may be a region of the vessel which does not contain hydrogel. Such region may be provided by e.g. only partially filling the vessel with the hydrogel. Such a region may be formed during rotation of the vessel as a result of plastic compaction of the hydrogel thereby reducing the volume of the hydrogel within the vessel, thereby providing a region of the vessel which does not contain hydrogel. In some embodiments, the vessel may comprise a compression chamber region for containing the hydrogel, and a fluid catchment region for containing fluid expelled from the hydrogel. The fluid catchment region may be disposed vertically below the compression chamber region during rotation of the vessel, so that fluid expelled from the hydrogel can run down from the compression chamber region into the fluid catchment region under gravity. There may be a filter member disposed between the compression chamber region and the fluid catchment region to support the hydrogel and/or the resulting biomaterial, and prevent the hydrogel and/or the resulting biomaterial construct from slipping into the fluid catchment region of the vessel. The filter member may be a porous filter member. The filter member may be a mesh. The region for collection of the expelled fluid phase of the hydrogel may contain an absorbent medium for absorption of the expelled fluid phase. Conveniently, the fluid collection region may be provided around an inner periphery of the vessel. Accordingly, a layer of an absorbent medium may be provided around an inner periphery of the vessel. The absorbent medium may be any material that absorbs a liquid phase. For example, the absorbent medium may comprise a paper medium (e.g. blotting paper), a sponge or mesh material (e.g. a polymer sponge or mesh). In some arrangements, an intermediate layer may be disposed between the absorbent medium and the hydrogel, for example a nylon mesh. Providing such an intermediate lay can help to prevent the hydrogel from sticking to the absorbent medium during compression.

In some preferred embodiments, the first axis of rotation may be offset from the centre of the vessel. The second axis of rotation may pass through the centre of the vessel, in other words, the vessel may spin about the second axis. Preferably the first axis of rotation and the second axis of rotation are parallel, although in alternative arrangements, the first axis of rotation and the second axis of rotation may not be parallel (they may be arranged at an angle to one another).

Rotation about the first axis of rotation may be at a sufficient speed to expel fluid phase from the hydrogel into the vessel without damaging the hydrogel or its components. For example, rotation about the first axis of rotation may be at a speed of from 100-1000 revolutions per minute (RPM), preferably 100-500 RPM. Rotation at higher speeds may cause damage to the hydrogel and/or the resultant biomaterial construct. Lower speeds may not result in plastic compaction of the hydrogel.

The speed of rotation about the second axis of rotation may be proportional to the speed of rotation about the first axis of rotation. Preferably, the ratio of RPM about the first axis of rotation to RPM about the second axis of rotation r is from 0.005 to 0.5, preferably 0.05 to 0.2. When r is 0.005, a full rotation about the second axis of rotation occurs once every 10 seconds when the speed of rotation about the first axis rotation is 1200 RPM. When r is 0.5, the speed of rotation about the second axis of rotation is half of the speed of rotation about the first axis of rotation. Providing r in this range can help to ensure a suitable distribution of the centrifugal force across the fluid leaving surface of the hydrogel, thereby allowing for more even plastic compaction of the hydrogel in comparison to a hydrogel compacted e.g. by rotation around a first axis of rotation only.

The vessel may be configured to be held by a rotatable body (e.g. a rotatable drum). Such rotatable body may be configured to support a plurality of vessels, so that a plurality of biomaterial constructs can be produced simultaneously. For example, the rotatable body may comprise a one, two or three or more slots arranged circumferentially around the rotatable body. Preferably, the rotatable body is configured to support the plurality of vessels such that the vessels are symmetrically disposed about the circumference of the rotatable body. This may help to ensure a suitable weight distribution to prevent damage to the rotatable body during rotation.

Rotation of the vessel about the primary axis of rotation and the secondary axis of rotation may be provided by a gearing system. The gearing system may be a planetary gearing system comprising a sun gear and one or more planetary gears, wherein the vessel is connected to a planetary gear. Calculation for the number of teeth on each of the gears adheres to the following formula where R is ring gear teeth, P is planet gear teeth, and S is sun gear teeth. R=2xP+S

The ratio r_w in this gearing system is given by: r_w =

Alternatively, the gearing system may comprise a gear train having one or more double gears, for example a gear train comprising first, second, third and fourth gears, wherein the second and third gear form a double gear, with the first gear being in meshing connection with the second gear, and the third gear being in meshing connection with the fourth gear. In this way, the first axis of rotation is located at the axis of rotation of the first gear. The second axis of rotation is located at the axis of rotation of the fourth gear. The vessel may be connected to the fourth gear.

The ratio r_w in this gearing system is given by: r_w

X «c

N_b Np

N_A is the number of teeth of the first gear, N_B is the number of teeth of the second gear, Nc is the number of teeth of the third gear, and N_D is the number of teeth of the fourth gear.

It may be possible to more easily provide a low ratio r using a gear train configuration as compared with a planetary gearing system.

The gearing system may be powered by a motor. Alternatively in some arrangements, the gearing system may be powered by manual input, e.g. manual rotation of one or more drive shafts. Torque may be transferred from the motor to the gearing system via one or more drive shafts. The voltage supplied to the motor may be controlled by a control module. The voltage supplied to the motor may be controlled using pulse-width modulation to thereby control the speed of rotation of the gearing system. Pulse width modulation is the control of voltage feed to a DC motor by driving the motor with a series of “ON-OFF” signals. Varying the duty cycle (fraction of time the output voltage is “ON” compared to “OFF”) of these signals while keeping the frequency constant can allow control the power output of the motor, by controlling the average voltage supplied to the motor. For example, a series of narrow pulses at a frequency T will provide a lower average voltage than a series of wide pulses at a frequency T. Power output of the motor may therefore be controlled by varying the width of applied voltage pulses applied to the motor terminals. Using pulse width modulation to control the voltage supply to the motor may result in low power loss, due to reduced power dissipation. Furthermore, it may result in better speed stability.

The centrifugal force may be a variable centrifugal force. In some embodiments, the centrifugal force applied to the hydrogel may vary with time. For example, the centrifugal force applied to the hydrogel may be increased during rotation of the vessel, by e.g. increasing the RPM about the first and/or second axis of rotation. This may be advantageous to help ensure suitable expulsion of the fluid phase of the hydrogel at higher degrees of compression. Alternatively, the centrifugal force applied to the hydrogel may be decreased during rotation of the vessel, by e.g. decreasing the RPM about the first and/or second axis of rotation. The applied centrifugal force will be proportional to the square of the revolution speed.

Alternatively, the centrifugal force on the hydrogel may be kept substantially constant overtime. As the centrifugal force on an object is proportional to the mass of the objection, and because the mass of the hydrogel decreases overtime as at least some of a fluid phase is expelled through a surface of the hydrogel, the centrifugal force on the hydrogel may be kept substantially constant by increasing the RPM about the first and/or second axis of rotation during rotation of the vessel. Accordingly, in some embodiments, the RPM about the first and/or second axis of rotation during rotation of the vessel may be varied based on the mass of the hydrogel. The mass of the hydrogel at a specified point in time during compression may be determined by e.g. monitoring the volume of fluid expelled from the hydrogel at that point in time, or by otherwise monitoring the degree of compression of the hydrogel by optical or other means. The centrifugal force may also be varied based on the weight of the biomaterial construct. The centrifugal force may varied proportionally to the weight of the biomaterial construct. During plastic compaction of the hydrogel to form the biomaterial construct, the weight of the biomaterial construct will decrease due to expulsion of the fluid phase overtime. In preferred embodiments, the centrifugal force may be increased as the weight of the biomaterial construct decreases due to plastic compaction and expulsion of the fluid phase. However, in some embodiments it may be desirable to decrease the centrifugal force as the weight of the biomaterial construct decreases due to plastic compaction and expulsion of the fluid phase. As above, the centrifugal force may be increased by increasing the RPM about the about first and/or second axis of rotation. The centrifugal force may be decreased by decreasing the RPM about the about first and/or second axis of rotation.

The centrifugal force may be applied for a time between 1 minute and 2 hours, preferably between 10 minutes and 30 minutes. The time for which the centrifugal force is applied is not particularly limited, and may be selected to balance factors such as energy usage vs extent of compressions. For example, applying the centrifugal force for a longer time may result in greater plastic compaction of the hydrogel, although may use more energy.

The step of simultaneously rotating the vessel about both a first axis of rotation and a second axis of rotation to apply a centrifugal force to the hydrogel may be repeated as part of a multi-step compression sequence. In other words there may be multiple discrete compression stages. This may be advantageous where it is desired to more closely monitor the degree of compression of the hydrogel over time. Alternatively, an automated monitoring mechanism may be employed to monitor the degree of compression of the hydrogel overtime. For example, a camera or other suitable monitoring device may be used to monitor the degree of the compression of the hydrogel, by observing the change in size of the hydrogel over time. The change in size of hydrogel over time will be proportional to the amount of fluid lost from the hydrogel. The specific hydrogel material is not particularly limited, and the method disclosed herein may be suitable for use with a wide variety of hydrogels. Hydrogels comprise a matrix of scaffold fibrils or fibres and an interstitial fluid. Gels may be formed by the coalescence and elongation of fibrils, as the fibrils form a continuous network around the interstitial liquid which originally held the monomers. The interstitial liquid in a hydrogel is typically an aqueous liquid. For example, the liquid may be water with solutes such as salts and proteins dissolved therein. In some embodiments, the interstitial liquid is a cell culture medium suitable for the growth and proliferation of cells. Any hydrated polymer material may be suitable for use in a hydrogel described herein, including naturally occurring polymers, for example proteins, such as silk, fibrin, fibrinogen, fibronectin, laminin, elastin, albumin or collagen (e.g. collagen type I), glycoproteins such as fibronectin, or polysaccharides such as glycosaminogylcans, chitin, cellulose, or methylcellulose. In some preferred embodiments, the fibrils or fibres of the hydrogel are made from collagen (i.e. the hydrogel may be a collagen gel). Native fibril forming collagen types are preferred including collagen types are I, II, III, V, VI, IX and XI and combinations of these (e.g. I, III V or II, IX, XI).

Other suitable materials for the fibrils or fibres of the hydrogel may include synthetic polymers i.e. polymers that are not naturally present in the human or animal body. Suitable polymers include organic polymers such as polylactones (e.g. PLA, PGA and PCL), polyethylene glycol (PEG), inorganic polymers such as phosphate glass and synthetic, gelling polypeptide gels. Alternatively or additionally other fibrelike or fibre-forming materials could be used, e.g carbon nanotubes or composites i.e. combinations of synthetic or natural polymers.

In some embodiments, the hydrogel may comprise two or more different types of fibril or fibre. For example, the hydrogel may comprise: fibronectin and collagen; collagen and polylactide; collagen and albumin; fibrin and collagen; collagen and carbon-nanotubes; fibrinogen and collagen; fibrinogen, collagen and fibronectin or fibrin, collagen and fibronectin.

Techniques for formulating and casting gels for use as biomaterials are well-known in the art (see, for example, W02006/003442; W02007/060459; Marenzana et al 2006 Exp Cell Res 312423-433;

Tomasek et al (2002) Nat Rev Mol Cell Biol 3 349-363; Harris et al Nature 290 (1981) 249-251 ; Elsdale et al 1972 J Cell Biol. 54626-637; Kolodney et al J Cell Biol. (1992) 117 73-82; Eastwood et al Biochem Biophys Acta 1201 (1994) 186-192).

The hydrogel may further comprise viable cells, the hydrogel being plastically compacted to produce a biomaterial comprising the viable cells. In some preferred embodiments, cells are seeded uniformly throughout the gel before plastic compaction.

Preferably the cells are human or other mammalian cells. The viable cells may be cells that confer tissue functionality and/or provide structures which replace or facilitate the repair of endogenous tissue. For example, such cells may comprise one or more of: muscle cells to provide contractile structures, tenocytes for tendon structures, vascular and/or neural cells to provide conductive elements, metabolically active secretory cells, such as liver cells, hormone synthesising cells, sebaceous cells, pancreatic islet cells or adrenal cortex cells to provide secretory structures, stem cells, such as bone marrow-derived or embryonic stem cells, dermal fibroblasts, skin keratinocytes, (and combination layers of the two), Schwann cells for nerve implants, intestinal and lung epithelial cells, smooth muscle cells, pericytes, mesenchymal stem cells, and endothelial cells for vessel structures, urothelial and smooth muscle cells for bladder/urethra structures; cholangiocytes and biliary cells; osteoblast, osteoclast and osteocytes for bone structures; oesophageal and tracheal cells; tenocytes for tendon structures; and chondrocytes for cartilage structures. Such cells may be provided as single cells, spheroids and/or organoids. In a second aspect, the present invention provides a biomaterial construct produced according to the method of the first aspect. The biomaterial construct may be substantially cylindrical.

The biomaterial construct may be formed from any of the materials discussed above in relation to the first aspect. However, in preferred embodiments, the construct comprises collagen fibres or fibrils.

The biomaterial construct may comprise a substantially symmetrical (e.g. symmetrical about a longitudinal axis of the biomaterial construct) fibre or fibril distribution, for example a substantially symmetrical collagen distribution. Constructs having a substantially symmetrical fibre or fibril distribution may be advantageous as they may have improved mechanical properties compared to constructs having a non-symmetrical fibre or fibril distribution. In some embodiments, the density of the fibre or fibril distribution (e.g. the collagen) may increase progressively along a radius of the construct from the centre to the edge of the construct.

The size and shape of the result biomaterial construct is not particularly limited, although may depend in part on the size and shape of the initial hydrogel, and the degree of plastic compression of the hydrogel. It may be possible to form biomaterial constructs having a length of between e.g. 1 mm and 500 mm, and a diameter of between e.g. 0.5 mm and 100 mm.

Biomaterial constructs produced according to the present may find use in a wide range of applications, for example in production of tissue equivalent implants, for use in e.g. tendon, nerve, ureter and/or blood vessel engineering.

In a third aspect, the present invention provides a tissue equivalent implant comprising or consisting of a biomaterial construct according to the second aspect of the invention.

A tissue equivalent implant is a device for implantation into an individual to repair or replace endogenous tissue, which, for example, may be damaged or diseased. Examples of diseased tissues which may be repaired or replaced by tissue equivalent implants include nerve, tendons, cartilage, skin, bone, urogenital elements, liver, cardiopulmonary tissues, kidney, ocular tissues, blood vessels, intestine, and glands. For example, some potential applications of the tissue equivalent implants described herein include use as a tendon graft, as a nerve graft, as a ureteral graft, and/or as a vascular graft.

Tissue equivalent implants produced according to the present invention may be bioactive, biocompatible, reproducible, customizable, biodegradable, and mechanically comparable to native tissue.

The tissue equivalent implant is preferably fixable at a site of tissue damage. For example, the implant may be fixable such that the entry end is located adjacent the proximal stump of a damaged tissue and the exit end is located adjacent the distal stump of a damaged tissue. The tissue equivalent implant may be fixed by any convenient technique. For example, it may be sutured or glued in place.

In a fourth aspect, the present invention provides a method of treatment of a damaged tissue in an individual comprising producing a tissue equivalent implant using a method described herein and fixing said implant to said damaged tissue to repair and/or replace said tissue. In a fifth aspect, the present invention provides a system for performing a method according to the first aspect of the invention. The system may comprise a rotating drum for supporting one or more vessels, and configured to simultaneously rotate the vessel about both a first axis of rotation and a second axis of rotation to apply a centrifugal force to an object (for example, a hydrogel) contained within the vessel.

The rotating drum may be driven by a gearing system. The gearing system may be powered by a motor. The motor may be controlled by a control module, which may be configured to control power supply to the motor to thereby control the speed of revolution (RPM) of the gearing system. The control module may be operable by input into a graphical user interface (GUI). The rotating drum may be supporting by a support frame. Other features of the system - in particular, e.g. features relating to a gearing system suitable for use in such a system are set out above in relation to the first aspect of the invention.

In a sixth aspect, the present invention provides a kit comprising a system according to the fifth aspect of the invention and a hydrogel, or a hydrogel precursor solution. The hydrogel precursor solution may be a collagen solution.

Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of and the aspects and embodiments described above with the term “comprising” replaced by the term ’’consisting essentially of.

It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/- 10%.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Brief Description of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1 is a schematic diagram showing the plastic compaction process previously described by Brown et al. [10].

Figure 2 is a schematic diagram of showing a method according to the present invention.

Figure 3 shows (a) an example apparatus suitable for performing a method according to the present invention; and (b) detailed view of the gear train arrangement of this apparatus.

Figure 4 shows (a) an uncompressed collagen gel; (b) constructs formed by centrifugation of a collagen hydrogel about a first axis of rotation only (not according to the invention); and (c) constructs formed by centrifugation of a collagen hydrogel by a first axis and a second axis of rotation

Figure 5 shows (a) a biomaterial construct formed by centrifugation of a collagen hydrogel around a first axis of rotation only (not according to the present invention), and (b) a biomaterial construct formed by centrifugation of a collagen hydrogel around both a first axis of rotation and a second axis of rotation, according to the present invention.

Figure 6 is a graph showing the force strain response of a biomaterial construct according to the present invention.

Figure 7 is a bar chart comparing the average sample break force (mN) of biomaterial constructs according to the present invention with that of comparative constructs.

Figure 8 is a bar chart comparing the wet gel mass percentage decrease of biomaterial constructs according to the present invention with that of comparative constructs.

Figure 9 is a bar chart comparing the mean percentage compression of biomaterial constructs according to the present invention (CDC) with that of comparative constructs (VSC).

Figure 10 is a bar chart comparing the mechanical properties of cellular and acellular biomaterial constructs according to the present invention (CDC). Figure 11 shows the different collagen distribution in the CDC gels (biomaterial constructs according to the present invention) as compared with the VSC gels (biomaterial constructs not according to the present invention).

Figure 12 consists of various SEM images showing: (A) rolled VSC construct; (B) cellular unrolled VSC with trapped cells;(C) Fibril alignment in a VSC construct; (D) CDC intact construct; (E) a CDC construct having aligned cells in between fibrils after cyclic loading; (F) aligned fibrils in a CDC construct; (G) fibril arrangement in a CDC construct formed from hydrogel set under standard room conditions; (H) fibril arrangement in a CDC construct formed from hydrogel set under an applied centrifugal force; and (I) fibril alignment of a native tendon.

Figure 13 shows live/dead percentage in the CDC gels (biomaterial constructs according to the present invention) as compared with the VSC gels (biomaterial constructs not according to the present invention) on days 1 , 3 and 5.

Figure 14 shows cell proliferation for the the CDC gels (biomaterial constructs according to the present invention) as compared with the VSC gels (biomaterial constructs not according to the present invention) after 1 , 3 and 5 days.

Figure 15 shows cellular alignment in (A) VSC gels (biomaterial constructs not according to the present invention) as compared with (B) CDC gels (biomaterial constructs according to the present invention).

Detailed Description

The present invention will now be described, by way of example only, with reference to the figures.

Two different sets of example biomaterial constructs were produced by methods according to the present invention. For both biomaterial constructs according to the present invention, initially cylindrical collagen hydrogels were used to form the construct. Comparative control gels were also produced.

The term “compressed gel(s)” used below refers to a biomaterial construct. A “CDC gel” is a biomaterial construct produced according to the present invention. A “VSC gel” is a comparative biomaterial construct compressed by vertical static compression (VSC) method as previously described by Brown et al [10].

Casting of cylindrical and control collagen hydrogels

The collagen hydrogel was cast using 4 ml (80%) rat tail collagen type 1 (First Link, Birmingham, UK), 0.5 ml (10%) minimum essential medium (MEM) (Invitrogen, Paisley, United Kingdom), neutralized by 5 M and 1 M NaOH (Sigma-Aldrich, Dorset, United Kingdom) and added 0.5 ml (10%) DMEM. The gel was cast by pouring 5ml of this neutralised collagen solution into a centrifuge tube of 1 5cm diameter and spun for 30 minutes at 2000 rpm in a centrifuge to form a cylindrical hydrogel by fibro-genesis mechanism. The control gels were cast in a rectangular mold (33 mm x 13 mm x 4 mm) and placed in an incubator for 20 minutes (37°C, 5% CO2), and subsequently compressed by vertical static compression (VSC) method as previously described by Brown et al [10]

Example 1

Cylindrical dynamic compression (CPC)

As shown schematically in Fig. 2, the fabricated cylindrical hydrogel 5 was placed in a centrifuge tube 7 of radius 2cm, which was then supported by an apparatus as shown in Fig. 3(a). In this example, the centrifuge tube was split into a compression chamber region and a fluid catchment region (not shown), with a flat filter element disposed between the compression chamber region and fluid catchment region. The flat filter provided a flat base to support the collagen whilst being porous to allow water to leave the compression chamber and flow into the fluid catchment region. A layer of blotting paper was provided around the inner periphery of the centrifuge tube to provide additional absorption of the expelled fluid phase of the hydrogel.

The centrifuge tube was supported by a rotatable drum 9 connected to a a gear train comprising first, second, third and fourth gears, labelled respectively as gears A, B, C and D in Fig. 3 (b), the second and third gear (gears B and C) forming a double gear, with the first gear A being in meshing connection with the second gear B, and the third gear C being in meshing connection with the fourth gear D. The centrifuge tube was supported on the fourth gear to thereby provide rotation about the first axis of rotation Oi (the axis of rotation of the first gear) and about a second axis of rotation O2 (the axis of rotation of the fourth gear).

The sizes of the gears were limited by the drum’s diameter and hence, the gear’s radius combined was selected to be equal to the distance between the tube slot and drum centre which is 0.1 m. The gear’s radius follows the following formula where rA, rB, rC and rD is the radius of each respective gear’s pitch circle. rA+rB+rC+rD=0A m

The following gear specifications were used:

Table 1 : Gear specifications for Example 1

This gear train orientation gives a ratio r_w = i which is a low ratio, signifying the tubes whirl once every 9 drum revolutions.

The rotatable drum 9 of this apparatus is supported by a frame 11 , which is configured to also support a motor 13. The motor is arranged to drive a central main shaft 15 to thereby rotate the rotatable drum 9. Here, the rotatable drum is configured to hold three centrifuge tubes disposed circumferentially around the drum, and circumferentially spaced by approximately 120° to allow for even load distribution during centrifugation.

Each sample was spun for 15 minutes at 300 RPM about the first axis of rotation, and also about the second axis of rotation at an RPM of 33.33 due to the gearing ratio. The direction of rotation of the rotating drum and the vessels are indicated by arrows in Fig. 2. The applied centrifugal force is indicated by arrow F, and is a force acting radially outwards. Rotation of the hydrogel resulted in plastic compaction by expulsion of at least some of a fluid phase of the hydrogel, thereby reducing the overall volume of the hydrogel, as shown schematically in Fig. 2, to thereby produce a cylindrical biomaterial construct 11 .

Comparative samples were also produced. Each comparative sample was spun at 300 RPM about a first axis of rotation only.

Sample shape and profile

Figs. 4 show that the shape of the samples made with and without rotation about the secondary axis of rotation differ. Fig. 4 shows (a) an uncompressed collagen gel; (b) constructs formed by centrifugation of a collagen hydrogel about a first axis of rotation only (not according to the invention); and (c) constructs formed by centrifugation of a collagen hydrogel about a first axis and a second axis of rotation. It can be seen that by centrifugation of a collagen hydrogel about both a first axis and a second axis of rotation, more effective compression of the collagen gel can be achieved. The resultant constructs are denser (appear ‘whiter’), and are more cylindrical in shape.

Fig. 5 (a) is a collagen construct formed by centrifugation of a collagen hydrogel around a first axis of rotation only, i.e. not according to the present invention. Fig. 5 (b) is a collagen construct formed by centrifugation of a collagen hydrogel around both a first axis of rotation and a second axis of rotation.

Samples made without rotation about a secondary axis of rotation (i.e. by rotation around a single axis of rotation only) have profiles resembling a circle’s segment as seen in Fig. 5(a) as opposed to samples made according to the present invention which have a more symmetrical collagen distribution and an approximately circular profile, as seen in Fig. 5(b). Without rotation about a second axis of rotation, only one side of the sample is pressed against the outer edges of the vessel, during centrifugation, thus leading to an asymmetrical collagen distribution in the resulting biomaterial construct.

The colour intensity of the profile also signifies how condensed the collagen is in the area. In Fig. 5(b) collagen is densely packed all around the perimeter of the profile. But in Fig. 5(a), collagen is densely packed on one half of the perimeter of the profile. This is because the fluid leaving surface in normal operation is the perimeter of the profile of the hydrogel, therefore collagen is continually compressed all around its edges. Compression by rotation about a single axis of rotation only allows fluid to leave through one side of the collagen, and hence it is only densely packed around one half of the profile. Uniaxial Tensile Testing

Each sample was loaded into the Zwick-Roell uniaxial tensile testing machine. The test on each sample comprised two parts: preconditioning cycle and stretch test to destruction. Readings of the sample’s force response to strain was recorded.

The preconditioning cycle was performed due to the complex structure soft biological tissues. Fibres of the collagen graft are initially ‘tangled’ when first fabricated and the preconditioning cycle serves to align the fibres in the best orientation, thereby reducing the hysteretic effect. With enough cycles to a subfailure load, samples will produce a repeatable mechanical response. Ultimately, preconditioning allows more repeatable testing results to be achieved. The preconditioning cycle for this experiment involved loading and unloading the samples to 370 mN for 5 cycles. Samples were then tested to destruction by continually stretching the samples until a tear appears, at which point the force response of the sample is considered to be its break force.

Fig. 6 depicts the force strain response of a sample. The figure shows the load-deformation curves shift to the right during the preconditioning cycle and the strain at which the sample fails and its corresponding break force. The highest force response is considered to the sample’s break force, and by calculating the gradient of the graph, the stiffness of the sample can be determined.

When tested to destruction, each sample failed at approximately 5 mm of elongation which is approximately 100 % strain of each sample. The average break force of the comparative samples compressed with rotation about 1 axis are compared with the average break force of the sample compressed with rotation about 2 axes (according to the invention) in Fig. 7. The average break force of samples according to the invention was 738 ± 214 mN and for the comparative samples was 167 ± 45 mN. Break force of the samples is an indicator of the tensile strength of the graft samples.

Fig. 7 shows that the samples according to the invention have a break force which is on average, 440 % times the break force of samples not according to the invention. This is a significant result, and it can be concluded with certainty that samples compressed according to the invention by rotation about both first and second axes of rotation have greater tensile strengths than samples compressed by rotation about a single axis of rotation only.

Stiffness of the samples was calculated by obtaining the gradient of the linear region of the load-strain curve as seen in Fig. 6. The average stiffness of the collagen samples was 0.317 N mnr¹. By comparing this against the stiffness of some mammalian tendons as can be seen in Table 2, below, it is seen that the average stiffness of the pure collagen samples is less than the stiffness of mammalian tendons. Accordingly, it may be desirable to manufacture composite material tendon grafts to more accurately mimic the tendon stiffness of native tissues.

Table 2: Comparison of sample stiffness with stiffness of mammalian tendons Fluid loss

The wet gel mass percentage decrease of samples according to the invention and comparative samples are compared in Fig. 8. The average fluid loss of samples according to the invention was 91 ± 4.6 % and for the comparative samples was 95.9 ± 1.7 %. The percentage of fluid loss is an indicator for the degree of compression of both devices. It was found that the average fluid loss of the comparative samples was greater. Without wishing to be bound by theory, the inventors believe that this may be due to the difference in the FLS areas. In samples according to the invention, because the FLS is the entire periphery of the construct, during compression, the FLS may gradually become blocked by the condensed fibres which constricts the flow of fluid out of the gel.

Example 2

Cylindrical dynamic compression (CPC)

The fabricated cylindrical hydrogel was placed in a centrifuge tube of radius 2cm, supported by a rotatable drum of diameter 22.5cm and 11.5 cm height. The centrifuge tube was placed against the wall of the drum, standing upright. A multi-step compression sequence was performed, as summarized in Table 3, below. During the first two steps of compression, a nylon mesh was wrapped around the hydrogel and placed in the tube, which contained blotting paper on the wall for absorption of a fluid phase of the hydrogel during compaction. During the last four steps, the nylon mesh and the blotting paper were both wrapped around the hydrogel and then placed in the tube. The angular velocity of the cylindrical spinner was 300 rpm (about the first axis of rotation). The ratio of first axis rotation to second axis rotation was 9:1 (1 rotation around the 2^nd axis per 9 rotations around the 1^st axis).

Here, the compression was performed for convenience as a multi-step compression sequence to allow for monitoring of the degree of compression of the hydrogel overtime, and to replace the nylon mesh and blotting paper with smaller lengths throughout the process as the hydrogel reduced in size due to plastic compression. However, it is not necessary to perform the compression in multiple stages.

Table 3: CDC compression process.

The amount of water loss of the collagen gel during compression was calculated by weight of the gels before and after compression. By assessing the weight it was possible to measure the percentage of decreased wet gel mass by compression.

The fluid loss from the cyclically dynamically compressed hydrogels (CDC gels, which are biomaterial constructs according to the present invention) was compared against the water loss from VSC gels (biomaterial constructs not according to the present invention, as shown in Fig. 9.

The mean weight after compression in VSC gels was 0.05 ± 0.01 grams, versus 0.20± 0.04 grams for the CDC gels. The percentages of decrease of wet gel mass of the VSC gels was 98.82 ± 0.33% and for the CDC gels it was 95.86 ± 1.65%. The decrease was significantly higher in the VSC gels, with p=0.027 (p<0.05). Without wishing to be bound by theory, the present inventors suggest that this may have been caused by blockage of the fluid leaving surface by the condensed collagen fibres at the fluid leaving surface.

Cell Seeding

Lapine posterior tibial tendon was extracted with standard tenotomy procedure. Digested with collagenase type 1 (Sigma-Aldrich, Dorset, UK) and incubated at 37°C for 3 hours in the water bath, after digestion mixture was filtered with 70 pm cell strainer to remove undigested ECM and cultured in the T225 flask with standard cell culture protocol. The cultured tendon fibroblasts of passage 1 with cell concentration of a million cells per ml in DMEM were inflexed longitudinally in the compressed CDC gels with 1 ml syringe and 23G needle (BD Microlance, Spain).

Cyclic loading

The CDC gels (biomaterial constructs) were wrapped in a 4.5 cm long nylon mesh and was sutured to ‘A’ frames as described in Cheema et al. 2005 [12] by prolene 4-0 sutures (Ethicon Ltd Edinburgh United Kingdom). These ‘A’ frames were attached above the stepper motors (Parker Irwin, PA, USA) in a plastic mold with 5 ml DMEM. The loading and unloading of the CDC gels were programmed by Parker Hannifin EaSI- V software (Parker Irwin, PA, USA) with two cycles per hour and 10% of cyclic strain (n=5).

Both acellular and cell-loaded (cellular) constructs were cyclically loaded under 10% strain for 7 days. Mechanical testing was performed on days, 1 , 3 and 7 of loading. For the mechanical testing, the CDC gels were kept at room temperature in the PBS prior to the testing and each construct was cut in the average size of 2cm and clamped with steel mesh of 2mm on the each side to provide grip. The mechanical testing was performed on the CDC gels (n=10) by the dynamic mechanical analyser (DMA) (DMA-7e Perkin Elmer, Buckinghamshire, UK) by uniaxial tension with probe speed of 200 mN per minute. The data was acquired and analysed by Pyris version 5.02 software (DMA Perkin Elmer, Buckinghamshire, UK)

Fig. 10 shows that the mechanical properties of the constructs for the day 1 CDC cellular gels (70.77 ± 18.47 mN) were less to the CDC acellular gels (167.35±45.54 mN). This was because of cellular attachment and movement, which had made the construct weak. However, for day 3, CDC cellular break strength [311.33 ± 37.11 mN (p<0.05)] was increased significantly to the CDC acellular [251 ± 32.51 mN (p<0.05)], as cells were able to respond external applied force. It also initiated remodelling of the matrix, which had an increase in the break strength significantly at end of 7 days [1123.39 ± 152.25mN (p<0.05)] to the CDC acellular [620 ± 31 60mN (p<0.05)].

Collagen Distribution Analysis

The constructs were fixed in a 10% formalin saline for one hour and embedded in paraffin wax. Sections of 5 pm were de-waxed and re-hydrated in series of xylene and ethanol. The constructs were stained with Sirius red [Sirius direct red 80] (Sigma-Aldrich Dorset, UK) for an hour to measure collagen intensity. The sections were dehydrated in series of ethanol and cleared in xylene and mounted on a light electronic microscope (Olympus BH-2/PM-ADF, Japan). Fig. 11 shows the different collagen distribution in the CDC gels as compared with the VSC gels.

The collagen intensity across the transverse section (TS) of the construct was unsystematic in the VSC due to the spiral rolling of the constructs, which had caused discontinuity pattern. Plotting these pixel intensities which had recorded interval between 90 to 150 gray values in the pixel distance of 1500 pixels. In the CDC, there was approximately constant distribution of the collagen intensity at an average of 100 gray value across the construct, however, on the edge, there was the exponential peak of collagen density, which was due to fluid leaving surface (FLS) where collagen deposition was high.

Scanning electron microscopy

The compressed gels (CDC gel, VSC gel) were left in 3% glutaraldehyde in 0.1% Caocodylate buffer for 10 minutes and washed in the deionised water to eliminate the excess of glutaraldehyde. The gels were dried over cover slip for 15 hours and put on the stub and sputter-coated with gold -palladium. All images were obtained using a secondary electron detector in a Philips XL 30 Field Emission SEM, operated at 5 kV, and average working distance was of 10 mm.

Fig. 12 shows that in the VSC rolled gels at 80X (Fig. 12(A)), there were interstice regions in the construct. This was due to rolling. Conversely, in the CDC gels (Fig. 12(D)) there were no gaps and the construct had a more homogeneous structure. At higher magnification in the unrolled cellular VSC gels (Fig. 12(B)), cells were trapped inside the construct, giving them less mobility. On the other hand, in the CDC gels (Fig. 12(E)), cells were seen in between collagen fibre which was biomimetic. At the collagen fibril level, there was a random architecture of collagen fibrils in the VSC gels (Fig. 12(C)). However, in the CDC gels (Fig. (F)), there was a unidirectional alignment of the collagen fibrils. The inventors theorise that this was in part due to cyclic loading of the constructs. This unidirectional alignment of the collagen fibril is same as native tendon (Fig. 12(1)). The fibril arrangement without setting the CDC hydrogel gel inside centrifuge (Fig. 12(G)) where fibril was random and less dense, but this is arrangement was changed after setting CDC gel inside a centrifuge (Fig. 12 (H)), due to centrifugal force exerted on the gel.

Cell viability

Cell viability (live/dead) assay was carried out at 1 , 3 and 5 days. For the live/dead assay (Invitrogen, UK) 20 mI of calcein and 17 mI of ethidium homodimer was added in 5 ml of PBS and covered over gels, which were then incubated for 30 minutes and mounted on the glass slides, observed under confocal laser microscope (Bio-Rad, UK) fitted with Olympus BX51 upright microscope. Images were obtained and interpreted by LaserVoxfor 3D volume rendering software.

The Cell’s survival of the seeded constructs was analysed by the live/dead assay for Day 1 , 3 and 5 for the VSC and CDC gels respectively, as shown in Fig. 13. In the VSC gel cell survival was 97%, 92%and 80% respectively and cells were seen more in clumps with randomly stretched pattern. This was because they were immobilised inside the matrix. In the CDC gel, cell survival rate over the same periods were 99%, 96% and 90% respectively. In other words, the CDC gels showed improved performance. Proliferation assay

A proliferation assay was performed for day 1 , 3 and 5 by Alamar blue dye which is a colourimetric method to measure cell proliferation and cell toxicity via cellular metabolism. The constructs were washed in PBS and incubated in DMEM (without L-glutamine and phenol red) (Sigma — Aldrich) with 10% Alamar blue dye (AbD Serotec, Oxon, UK) for 2 hours in a dark and inside incubator (37C,5% C02). The aliquoted 100 mI of metabolised dye in a 96 well plate and florescence was measured with a micro plate reader (Labsystems Fluroskan Ascent Microplate Reader, UK) at the emission wavelength of 590 nm.

Alamar blue florescence at 570 nm was measured as an absorbance unit (AbU) for day 1 , 3 and 5 (see Fig. 14). Cell metabolism results indicated that for VSC (3.30 ± 0.38 AbU) and CDC (3.11± 0.13 AbU) at day 1 there was no significant difference in the absorbance in the VSC. In the Day 3 and day 5 proliferation of the cell was decreased for VSC (3.43± 0.16 AbU to 3.33± 0.20 AbU), which signifies that cells were less proliferative or viable by end of 5 days. However, in contrast for CDC it had significantly increased (3.29± 0.15 AbU to 3.99± 0.13 AbU). The results signify that cells were able to attach to the matrix and in the CDC with no antagonist effect on the cells during compression process and mechanical loading. In addition, they could metabolise and proliferate more as compared to the VSC.

Cellular alignment

Post loading constructs were fixed in 4% formalin saline for 1 hour and blocked in 0.15% triton-X in PBS for 15 minutes. Constructs were then stained in 1.5% phalloidin (Invitrogen, Paisley, UK) and 1% DAPI (Vector labs, Peterborough, UK) and mounted on glass slides with a coverslip. Images were obtained by using an upright fluorescent microscope Olympus BX61 (Olympus, Tokyo, Japan).

Cellular morphologies were studied after 24 hours in VSC and CDC and showed that in VSC (Fig. 15(A)) cells were bipolar and stellate morphology with nonaligned orientation as they were trapped in a collagen matrix. Whereas cyclic loaded CDC (Fig. 15(B)) showed improved cellular response by aligning themselves under applied uniaxial cyclic load cellular morphology. This indicates that in CDC cellular behaviour was mimicking native tissue environment in tendon than in VSC Statistical analysis

Statistical analysis was done by one-way ANOVA using SPSS 21 .0. (SPSS IBM cooperation, Chicago, USA) and Post Hoc analysis was done with Bonferroni correction. In all cases p < 0.05 was considered significant. All the data expressed as a mean ± standard deviation (SD).

References

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Claims

Claims:

1. A method of producing a biomaterial construct comprising steps of providing a vessel containing a hydrogel; and simultaneously rotating the vessel about both a first axis of rotation and a second axis of rotation to apply a centrifugal force to the hydrogel, thereby causing plastic compaction of the hydrogel to form the biomaterial construct by expulsion of at least some of a fluid phase of the hydrogel through a surface of the hydrogel.

2. The method according to claim 1 wherein the rotation about the first axis applies a radial centrifugal force to the hydrogel, and the rotation about the second axis distributes the radial centrifugal force over the fluid leaving surface of the hydrogel.

3. The method according to claim 1 or claim 2 wherein the vessel comprises a region for collection of the expelled fluid phase of the hydrogel, optionally wherein the region contains an absorbent medium for absorption of the expelled fluid phase.

4. The method according to any one of the preceding claims wherein the vessel has a substantially circular cross-section in a direction perpendicular to the second axis of rotation.

5. The method according to any one of the preceding claims wherein the first axis of rotation is offset from the centre of the vessel, and wherein the second axis of rotation passes through the centre of the vessel (i.e. the vessel spins around the second axis).

6. The method according to any one of the preceding claims wherein the centrifugal force is a variable centrifugal force.

7. The method according to claim 3 wherein the centrifugal force is varied proportionally to the weight of the biomaterial construct.

8. The method according to any one of the preceding claims wherein the rotation about the first axis of rotation is at 100-1000 RPM.

9. The method according to any one of the preceding claims wherein the ratio of RPM about the first axis of rotation to RPM about the second axis of rotation r is from 0.05 to 5.

10. The method according to any one of claims 1 to 9 wherein rotation of the vessel about the primary axis of rotation and the secondary axis of rotation is provided by a gearing system which is a planetary gearing system comprising a sun gear and one or more planetary gears, wherein the vessel is connected to a planetary gear.

11. The method according to any one of claims 1 to 9 wherein rotation of the vessel about the primary axis of rotation and the secondary axis of rotation is provided by a gearing system comprising a gear train having one or more double gears

12. The method according to claim 10 or claim 11 wherein the gearing system is powered by a motor, and wherein voltage supplied to the motor is controlled using pulse-width modulation to thereby control the speed of rotation of the gearing system.

13. The method according to any one of the preceding claims wherein the centrifugal force is applied for a time between 1 minute and 2 hours.

14. The method according to any one of the preceding claims wherein the hydrogel is plastically compacted by expulsion of 50% or more of the original fluid content of the hydrogel.

15. The method according to any one of the preceding claims wherein the gel is a collagen gel.

16. The method according to any one of the preceding claims wherein the hydrogel further comprises viable cells and the hydrogel is plastically compacted to produce a biomaterial comprising the viable cells.

17. A biomaterial construct produced according to the method of any one of claims 1 to 16.

18. The biomaterial construct according to claim 17 wherein the biomaterial construct is substantially cylindrical.

19. The biomaterial construct according to claim 17 or claim 18 wherein the construct comprises collagen fibres or fibrils.

20. The biomaterial construct according to claim 19 wherein the biomaterial construct comprises a substantially symmetrical collagen distribution.

21 . The biomaterial construct according to claim 19 or claim 20 wherein the density of the collagen increases progressively along a radius from the centre to the edge of the construct.

22. A tissue equivalent implant comprising or consisting of a biomaterial construct according to any one of claims 17 to 21.

23. The tissue equivalent implant according to claim 22 wherein the tissue equivalent implant is a tendon graft.

24. A method of treatment of a damaged tissue in an individual comprising producing a tissue equivalent implant according to claim 22 or 23 and fixing said implant to said damaged tissue to repair and/or replace said tissue.