TISSUE REPAIR SCAFFOLD AND DEVICE

Abstract

The present invention provides a tissue repair scaffold comprising a knitted body, wherein the knitted body is made of a yarn comprising polycaprolactone (PCL), and the knitted body comprises a plurality of apertures that remain open when the scaffold is stretched under load. The scaffold is particularly adapted for tendon repair. The present invention also relates to a tissue repair device comprising the scaffold, and a method of making the scaffold.

Claims

1. A tendon repair scaffold comprising a knitted body, wherein the knitted body is made of a yarn comprising polycaprolactone (PCL); the knitted body comprises a plurality of apertures; and the apertures are open at and between a first configuration in which the knitted body is unloaded and has a first length, and a second configuration in which the knitted body is loaded and has a second length which is greater than the first length.

2. The tendon repair scaffold according to claim 1 wherein the second length is at least 10% greater than the first length.

3. The tendon repair scaffold according to claim 1, wherein the yarn comprises a plurality of fibres comprising PCL, optionally wherein the fibres are made by electrospinning and/or wherein the plurality of fibres are aligned and/or wherein the yarn is formed by twisting the plurality of fibres.

4. The tendon repair scaffold according to claim 1, wherein the average diameter of the fibres is less than 1 μm, preferably between 400 nm and 850 nm.

5. The tendon repair scaffold according to claim 1, wherein the average diameter of the yarn is less than 500 μm, preferably between 225 μm and 275 μm.

6. The tendon repair scaffold according to claim 1, wherein the first length of the knitted body is between 15 mm and 25 mm.

7. The tissue repair scaffold according to claim 1, wherein the average width of the knitted body in the first configuration is between 1.5 mm and 2.5 mm.

8. The tendon repair scaffold according to claim 1, wherein the apertures individually have an area of at least 10,000 μm.sup.2 in the first configuration and an area of at least 10,000 μm.sup.2 in the second configuration.

9. The tendon repair scaffold according to claim 1, comprising a first tendril extending from a first end of the knitted body, optionally comprising a second tendril extending from a second end of the knitted body.

10. The tendon repair scaffold according to claim 9, wherein the first tendril and, if present, the second tendril are made of the same yarn as the knitted body.

11. A tendon repair device comprising a surgical cord attached to the tissue repair scaffold according to claim 1.

12. The tendon repair device according to claim 11, wherein surgical cord is a suture, optionally wherein the suture is barbed.

13. The tendon repair scaffold according to claim 1 for use in a method of treatment of the human or animal body, wherein the method comprises treating a damaged tendon.

14. A method of making a tendon repair scaffold according to claim 1, the method comprising the steps of: (A) electrospinning a plurality of fibres comprising PCL; (B) forming a yarn by aligning and twisting the plurality of fibres; and (C) knitting the yarn to form a knitted body comprising a plurality of apertures.

15. The method according to claim 14, wherein the yarn is knitted using a three-needle weft knitting process.

Description

SUMMARY OF THE FIGURES

[0088] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0089] FIG. 1 shows a typical currently clinically used technique of a Kessler suture knot to repair two cut tendon ends.

[0090] FIG. 2 shows an SEM micrograph of a known yarn formed by plaiting a plurality of primary fibre bundles.

[0091] FIG. 3 shows a schematic of the electrospinning apparatus used to make fibres, and the rotating mandrel used to align the fibres for inclusion in scaffolds of the present invention.

[0092] FIG. 4 shows an SEM micrograph of electrospun aligned fibres formed by a method of the present invention.

[0093] FIG. 5 shows an SEM micrograph of a yarn formed by twisting electrospun aligned fibres.

[0094] FIG. 6 shows a three-needle arrangement on a conventional knitting machine.

[0095] FIG. 7 shows an SEM micrograph of a knitted body formed by knitting the yarn of twisted fibres.

[0096] FIG. 8 shows an image of a tissue repair scaffold of the present invention.

[0097] FIG. 9 shows the position of the tissue repair scaffold to augment a current clinically used suture knot.

[0098] FIG. 10 shows the position of the tissue repair scaffold when used with a micro-barbed surgical cord.

[0099] FIG. 11 shows an image of a tissue repair device of the invention inserted into a human cadaver hand tendon.

[0100] FIG. 12 shows a histogram of the number of apertures across different aperture areas for a comparative plaited structure.

[0101] FIG. 13 shows a histogram for the number of apertures across different aperture areas for a knitted structure according to the present invention.

[0102] FIG. 14 shows a bar chart of the coverage of type-1 collagen and fibronectin for loaded plaited and knitted structures after 21 days in an in vitro culture.

[0103] FIG. 15 shows a bar chart of the maximum load for cell-seeded plaited and knitted structures after 28 days in an in vitro culture.

[0104] FIG. 16 shows the variance in number average molecular weight (Mn) and weight average molecular weight (Mw) over the course of a 12-week rabbit study.

[0105] FIG. 17 shows the percentage reduction of surface area compared with a control at different portions, using ultrasound in a 12-week sheep study.

[0106] FIG. 18 shows a comparison of collagen type-1 results over 12 months from histological analysis of sheep tendon.

[0107] FIG. 19 shows a comparison of decorin results over 12 months from histological analysis of sheep tendon.

[0108] FIG. 20 shows a comparison of inflammatory cell results over 12 months from histological analysis of sheep tendon.

DETAILED DESCRIPTION OF THE INVENTION

[0109] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0110] Examples of tissue repair scaffolds are described herein, comprising a three-dimensional knitted body made of yarn formed by twisting a plurality of aligned electrospun PCL fibres. These fibrous constructs are intended to mimic both the morphological anatomy and the biomechanical properties of natural human tendon. This tissue is known to be composed of a hierarchical organisation of aligned collagen fibres. In embodiments, whilst the fibres contained within the PCL yarn are not of the same size as the collagen fibres, the dimensions and morphology of the yarn closely resembles those within the natural tendon tissue.

[0111] The scaffolds described herein are biodegradable and/or bioresorbable. This suitably eliminates the need for secondary surgery. Furthermore, in embodiments, the rate of degradation matches the rate of new tissue formation. Preliminary studies suggest that the degradation rates are suitable for accommodating natural healing times for tendons of about three months.

[0112] Testing of embodiments has shown that the scaffolds are able to withstand high tensile loads and demonstrate flexibility. This latter characteristic is particularly promising given that some tendons are required to bend round bony prominences.

[0113] Described herein are studies which demonstrate how electrospinning of fibres can be used to produce the scaffolds of the present invention. Variation of the parameters associated with electrospinning can be used to control the fibre characteristics.

[0114] The electrospun fibres described herein are drawn off a mandrel and twisted onto a bobbin. This is then used to form the twisted multifilament yarn. The yarn is subsequently knitted into a structure with a pre-determined pattern, by a weft knitting process using three needles, giving rise to a slim cross-section which is broadly circular. If other knitting styles are used, then a different cross-section is generated. For example, the use of four needles gives rise to a flatter, more ribbon-like cross-section. Moreover, a four-needle process generally results in greater deformation of yarn during knitting.

(1) Fibre Formation

(a) Electrospinning and Selection of Parameters

[0115] Electrospinning provides a way of generating fibres of controlled diameter. In particular, electrospinning enables the fabrication of long, continuous fibres of controlled diameter. At its simplest, the application of a high voltage to a polymer solution within a syringe causes expulsion of a polymer jet towards an earthed collector.

[0116] It is possible to electrospin a wide range of polymers, including biocompatible polymers.

[0117] A number of parameters can be used to control the properties of the fibres formed using electrospinning. The following parameters are mentioned as particularly useful in controlling the collected fibres: [0118] 1. Solvent [0119] 2. Molecular weight of polymer [0120] 3. Concentration of polymer-solvent solution [0121] 4. Applied voltage [0122] 5. Tip to collector distance [0123] 6. Flow rate of polymer-solvent solution

[0124] These and other parameters are discussed in more detail below.

Solvent

[0125] Suitable solvents include acetone, chloroform, dichloromethane (DCM), 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and tetrahydrofuran (THF).

[0126] Solvents with a higher dielectric constant yield a greater number of fibres, due to the charge repulsion interactions which occur as the polymer jet travels towards the collector.

Molecular Weight of Polymer

[0127] A suitable molecular weight (Mn) is in the range 10,000 to 100,000, but other molecular weights can be used, as described herein.

[0128] Molecular weight can have a significant effect on the fibre morphology. PCL of higher molecular weight (Mn 80,000) is found to minimise the occurrence of “beads” along the fibres.

Concentration of Polymer-Solvent Solution

[0129] Suitable concentrations are in the range 3% w/v to 15% w/v, preferably 5% w/v to 10% w/v.

[0130] Fibre diameter can be tailored by altering the solution concentration. Generally, it is found that higher concentrations result in fibres of greater diameter.

Voltage

[0131] The voltage applied to the polymeric solution has a direct effect on fibre morphology. High voltage causes the polymer jet to be emitted with rapid acceleration, limiting the jets flight time and subsequently decreases the amount of stretching and solvent evaporation prior to collector impact [Ramakrishna et al., 2005]. The resulting fibres may be thicker and contain high levels of residual solvent. The flight time of the polymer jet is, however, dependent upon the tip to collector distance.

[0132] Keeping the applied voltage comparatively low (e.g. 15 kV) is effective in producing electrospun fibres of fine, submicron diameter. Nevertheless, it is desirable to avoid much lower voltages so as to ensure sufficient electro-static charging of the polymer solution and ejection of a polymer jet.

Needle-Tip to Collector Distance

[0133] Varying the distance between the needle-tip to the collector has an important role in determining fibre characteristics. A short deposition distance reduces the polymer jet flight time, limiting the rate of solvent evaporation and polymer stretching; often resulting in the fabrication of thick, merged fibres. Generally, a minimum distance, which allows significant drying and stretching of the jet, is required for the production of long, fine fibres [Reneker et al., 2000].

[0134] The deposition distance between the needle-tip and collector should be long enough to ensure adequate time for the polymer jet to undergo sufficient stretching and solvent evaporation prior to its impact. Suitable distances are in the range of 10 cm to 20 cm, preferably 12 cm to 15 cm.

Flow Rate of Polymer-Solvent Solution

[0135] The applied flow rate determines the quantity of polymeric solution available to the electrospinning process. Generally, high flow rates yield fibres of larger diameter. This is because the greater volume of solution pumped out may not have sufficient time for solvent evaporation and adequate stretching of the jet prior to contact with the collector.

[0136] It is desirable to use a low flow rate, as high flow rates may result in fibres of wider diameter (e.g. larger than 1 μm).

Solution Viscosity

[0137] The viscosity of solution is directly affected by the concentration of polymer present. If the polymer concentration is high, greater quantities of polymer chains are present, increasing the number of chain entanglements with solvent molecules and ultimately raising solution viscosity. A polymer's molecular weight also affects solution viscosity. A polymer of low molecular weight reduces the number of solvent/polymer entanglements because of the shorter length chains, and hence decreases solution viscosity. Fibre production is dependent on the concentration of solution being electrospun.

[0138] Generally, if the concentration is too low bead formation as opposed to fibre production is observed; and if too high, pumping of the solution will be difficult and fabricated fibres are mostly micrometer in diameter.

Solution Conductivity

[0139] The solvent chosen to dissolve the polymer has a significant role in the level of conductivity present within the solution, and this directly affects the fibre morphology generated from the electrospinning process. Solvents with high dielectric constants cause the emitted polymer jet to experience increased longitudinal force brought about by the higher accumulation of charge present within the polymeric solution [Wannatong et al., 2004]. Consequently the polymer jet experiences a greater degree of charge repulsion, leading to an increased level of stretching and elongation, resulting in fibres of finer diameter [Fong et al., 1999].

Surface Tension

[0140] The surface tension of the polymeric solution must be overcome in order for the electrospinning process to be initiated. The polymeric solutions viscosity directly affects its surface tension; high viscosity reduces the surface tension due to significant entanglement between solvent molecules and polymer chains preventing molecule clustering [Shawon et al., 2004].

Humidity

[0141] The humidity surrounding the electrospinning process can have a significant effect on fibre morphology, in terms of surface porosity: rising levels of humidity lead to an increase in pore size, number and distribution over the fibre surface [Casper et al., 2004].

Temperature

[0142] Increasing the polymeric solution temperature may result in a faster rate of fibre deposition and fibres may therefore have a decreased diameter. These effects may be attributed to the reduced viscosity of polymeric solution caused by decreased entanglements between polymer and solvent molecules as a result of polymer chain expansion [Mit-uppatham et al., 2004].

Specific Parameters

[0143] Unless otherwise stated, the electrospinning parameters employed for the fabrication of all electrospun fibre matrices described herein were as follows: voltage 15 kV (Series 120 Watt regulated high-voltage DC power supply, Glassman High Voltage, Inc.), flow rate 4.5 mL/hour (SP230IW2, World Precision Instruments), needle tip (∞ 0.8 mm, BD Microlance) to collector distance 15 cm, solution concentration 8% w/v polycaprolactone (PCL, Purasorb PC 12) (80,000 Mn) [Purac Corbion] in HFIP [Sigma Aldrich], humidity 50%, temperature 23° C.

(b) Fibre Orientation

Fibre Alignment and Collector Method

[0144] The orientation of fibres deposited from the electrospinning process is dependent upon their method of collection. Fibre alignment is determined by the angle between the fibre and the direction of alignment—the smaller the angle, the greater the alignment.

[0145] Random, non-woven arrangements of fibres are created when the collector is an earthed stationary plate.

[0146] Purposefully orientated fibres can be fabricated by electrospinning between the gap of two fixed metal plates or onto a mandrel rotating at an optimised speed. The method described herein uses a rotating mandrel. Generally, the mandrel must be rotated at sufficient speed so as to ensure that rotation speed is not too slow compared to the speed of fibre emission, in which case alignment may be inhibited. Similarly, if rotation is too fast, fibre breakage can occur [Huang et al., 2003].

[0147] Alternatively, collection of fibres between two parallel earthed plates, also known as “gap method of alignment” may be used to produce aligned fibres [Dzenis, 2004].

[0148] Fibrous yarns containing aligned individual fibres that are grouped together can also be fabricated by spinning the polymer solution directly into an earthed liquid reservoir [Smit et al., 2005]. The network of fibres collected on the liquid surface is drawn off and into the air. Fibres align and coalesce due to the effects of surface tension between the fibres and liquid during the drawing process. Three-dimensional fibrous bundles are the end product after lifting the fibres off the liquid surface.

[0149] The various methods for fibre collection resulted in different fibre orientation. The stationary plate produces fibres with least alignment. Collection on the rotating mandrel results in fibres of greatest alignment.

Optimising Rotation Speed for Creating Aligned Fibres on a Mandrel

[0150] The orientation of fibres when collected on a mandrel depends on the mandrel's rotation speed. For a narrow mandrel (width=30 mm; diameter=120 mm), low speeds of 300 RPM result in fibre deposition similar to the fibrous networks collected on stationary plates. If the speed of rotation is too fast, the fibres are predominantly random in appearance. At an optimal speed, the fibres are collected parallel to the direction of rotation. 500-600 RPM (e.g. 500 RPM) results in the greatest alignment.

[0151] For a wide mandrel (width=90 mm; diameter=65 mm), the optimum rotation speed for the alignment of fibres is different from that of a narrow mandrel. For the wide mandrel, speeds of 1200 RPM and greater are employed to generate sufficiently aligned fibres. Alignment may be further increased at rotation speeds above 1800 RPM, giving a lower angle of alignment and hence greater degree of parallelism to the longitudinal axis.

(c) Surface Characteristics

[0152] The wettability of the scaffold surface is a characteristic of the scaffold that can be adjusted to suit the function of the scaffold. Suitably, to encourage cell attachment, the exterior of the material is hydrophilic or wettable as this will permit cells to contact with the surface over a greater area to allow attachment and spreading, and for providing cells with an environment similar to their natural environment. To discourage cell attachment or protein adhesion, however, hydrophobic or non-wetting surfaces can be created. The skilled reader is familiar with appropriate surface treatments.

(2) Scaffold Formation

[0153] FIG. 3 shows a schematic of the electrospinning apparatus used to make PCL fibres, and the rotating mandrel used to align the PCL fibres for inclusion in scaffolds of the present invention. The electrospinning apparatus comprises a syringe needle (1) attached to a syringe pump (2), powered by a high-voltage DC supply (3). The mandrel (5), which has a rotating and translating element, collects aligned PCL fibres (4). An SEM micrograph of electrospun aligned PCL fibres formed by this method is shown in FIG. 4.

[0154] Yarn is then created in an automated machine process by pulling the aligned PCL fibres from the mandrel and creating a twist. The resultant yarn is collected on a bobbin, typically in a length of greater than 1 m. An SEM micrograph of yarn formed by this method is shown in FIG. 5.

[0155] Subsequently, the yarn is knitted in a weft knitting process to produce the tissue repair scaffold, using a three-needle arrangement on a conventional 10 gauge flatbed knitting machine, as shown in FIG. 6, with proprietary bobbin holder/tensioner. A bobbin containing yarn is loaded into the moving holder/tensioner. The loose end of yarn is fed through a tensioning arm and three subsequent feed holes. The loose end is passed through the knitting beds. From underneath the knitting beds, the yarn is taken and the end is clamped into a clamp attached to the knitter. The knitter is moved across the needles from right to left to cast on. The knitter is then moved across the needles from left to right to knit a first row, and from right to left to knit a second row. This is repeated until 16 rows are knitted to produce a knitted body of 20 mm in length. The two trailing lengths of yarn from either end of the knitted body are each trimmed to 100 mm in length.

[0156] The tissue repair scaffold comprises a knitted body as shown by the SEM micrograph in FIG. 7. The scaffold has a broadly circular cross-section, and comprises a number of apertures in between different segments of the yarn resulting from the knitted structure. The scaffold also has a number of significantly smaller, micropores generated by twists in a segment of yarn.

[0157] An image of a tissue repair scaffold is shown in FIG. 8, in which the scale bar represents 1 mm. The scaffold is made from a single piece of yarn. The scaffold comprises three distinct regions: a trailing length of electrospun PCL yarn (first tendril), the knitted body, followed by a second tendril.

[0158] As exhibited by the schematic in FIG. 9, the tissue repair scaffold (6) can be used to augment a current clinically used suture knot (7). The scaffold is inserted in the centre of a tendon (8), across a cut (9) in the tendon. The scaffold is attached to the suture knot such that the scaffold is alongside and approximately parallel to the suture. An alternative, using a micro-barbed surgical cord (10) in place of the suture knot, is shown in the schematic in FIG. 10. The scaffold together with the suture knot or micro-barbed surgical cord can be considered as a tissue repair device. An image of a device inserted into a human cadaver hand tendon is shown in FIG. 11 (note that the gauze-like structure in the background of the image is not part of the device).

(3) Exemplified Scaffolds

[0159] The specifications of exemplified tissue repair scaffolds formed by the method described above are shown in Table 1:

TABLE-US-00001 TABLE 1 Scaffold specifications PCL fibre diameter (nm) 700 (± 20%) Yarn diameter (μm) 225-275 Tendril lengths (mm) 100 Knitted body length (mm)  20 Knitted body width (mm)  2

[0160] One knitted body of a scaffold of these specifications was scanned with pCT. The scan was analysed using a semi-automated Avizo (voxel number based) procedure, which was repeated three times. The scan was divided into 5 different regions to calculate the final porosity, comprising microporosity and macroporosity.

[0161] Microporosity is the result of the void generated by twists within the yarn, divided by the solid material. Macroporosity is the void space percentage against the whole volume of the knitted body, imagined as if it were wrapped in an envelope (so is essentially the sum of the solid and void space). Macroporosity relates to the apertures in the knitted structure as described herein. The procedure does not account for the space between individual electrospun fibres, as the resolution of the machine does not distinguish between fibres.

[0162] It was found that measured macroporosity of the knitted body was 53.65%. The measured microporosity was 3.99%.

[0163] Three scaffolds of the above specifications were taken and placed under a microscope, and the aperture area of each knitted body (unloaded) was measured at 4× magnification using ImageJ (free-hand selection). The knitted bodies were then manually pulled to a length 10% greater than the length when unloaded, and the aperture area of each knitted body (loaded) was then measured again in the same way.

[0164] For comparison, three scaffolds having a structure of plaited yarn comprising PCL fibres were also taken (see FIG. 10 of WO 2010/067086 and the methods disclosed therein). These comparative scaffolds were placed under a microscope, and the aperture area of each plaited structure (unloaded) was measured at 4× magnification using ImageJ (free-hand selection). The plaited structure was then manually pulled to a length 10% greater than the length when unloaded, and the aperture area of each plaited structure (loaded) was then measured again in the same way.

[0165] The results are exhibited in the histograms of FIGS. 12 and 13.

[0166] FIG. 12 shows a histogram of the number of apertures across different aperture areas for all plaited structures, comparing the number of apertures when unloaded and loaded as described above. FIG. 13 shows a histogram of the number of apertures across different aperture areas for all knitted bodies, comparing the number of apertures when unloaded and loaded as described above. It can be seen that the knitted body has a significantly greater number of apertures, both when unloaded and loaded. In particular, when loaded the knitted body retains almost 40 open apertures having an area in the range of 10,000-20,000 μm.sup.2, and approximately 20 open apertures having an area greater than 20,000 μm.sup.2. By comparison, the plaited structure has a negligible number of open apertures having an area greater than 10,000 μm.sup.2 when loaded. Specifically, in this range the plaited structure has only 1 open aperture when loaded, which has an area between 10,000-12,000 μm.sup.2, and has no open apertures having an area greater than 12,000 μm.sup.2.

[0167] It can be concluded from these data that the knitted body possesses a greater aperture area than the plaited structure. Whereas the plaited structure is more two-dimensional and predominantly has apertures which are discrete from each other, the knitted body is a truer three-dimensional structure with interlocking pores. Although the aperture area does reduce upon loading for both structures, this is significantly more evident for the plaited structure. The knitted body retains an open-aperture structure on loading, which is more conducive for repeated loading of tissue without scissoring.

(4) Protein Deposition Studies

[0168] Type-1 collagen and fibronectin represent two important proteins to assess the success of tendon repairs.

[0169] Type-1 collagen is the most abundant protein in tendon extracellular matrix (ECM), as it constitutes between 80% and 90% of total tendon dry mass and represents approximately 60% of the total collagen amount. Type-1 collagen performs several functions, but its main role is to maintain tissue hierarchical structure, and to absorb and transmit the forces generated by muscles, preventing tissue mechanical failure. In particular, the hierarchical organisation of collagen in normal tendon plays a key role in tissue biomechanics, since it ensures that the tissue is able to withstand high tensile forces, and allows minor damage to be confined at the site rather than spreading to the entire tissue.

[0170] Fibronectin is another important protein found in tissue ECM, as it provides binding sites for cells, facilitating cellular adhesion to the matrix. This cell-protein interaction plays a key role in translating mechanical stimuli into cell responses, as fibronectin helps cells to organise collagen fibrils into bundles and to maintain the tissue hierarchical structure.

[0171] For these reasons, the success of tendon repair strategies can be evaluated by observing cell deposition of type-I collagen and fibronectin. Indeed, repaired tendons with high levels of these proteins more closely resemble the ECM structure of uninjured and healthy tendons, thus indicating a stronger and more functional repair.

[0172] Human mesenchymal stem cells (1×10.sup.6) were seeded onto each of the three knitted and three plaited PCL structures as described above, in an in vitro culture. The structures were each loaded so that the lengths of the structures were 10% greater than the lengths when unloaded. After 21 days in the culture, the amounts of type-I collagen (COL1A1) and fibronectin (FN) in each structure were analysed from confocal images using ImageJ and quantified. The results are provided in the bar chart in FIG. 14, showing data as mean±standard deviation. When going from the plaited structure to the knitted structure, there was an increase in coverage of collagen from 24.5% to 42.0%. Similarly, when going from the plaited structure to the knitted structure, there was an increase in coverage of fibronectin from 27.8% to 37.2%.

(5) Tensile Testing

[0173] Human mesenchymal stem cells (1×10.sup.6) were seeded onto each of six knitted and six plaited PCL structures in an in vitro culture. Three of each type of structure were loaded so that the lengths of the structures were 10% greater than the lengths when unloaded. The remaining three of each type of structure were not loaded.

[0174] After 28 days in the culture, tensile testing was conducted with the structures, in order to determine the maximum possible load. The results are provided in the bar chart in FIG. 15, showing data as mean±standard deviation. Both the plaited and knitted structures showed little difference in maximum load in terms of whether that particular structure had been loaded or not. However, when going from the plaited structure to the knitted structure, there was an increase in maximum load from approximately 1.5 N to approximately 2.8 N.

(6) Studies

(a) Cytotoxic Response

[0175] The cytotoxic response of the knitted scaffold was tested in vitro and in accordance with ISO10993-5 (Namsa, L929 cells). Epithelial cells, mesenchymal stem cells and tenocytes were cultured both in direct and indirect contact with the scaffold up to 21 days in vitro. All studies concluded that the scaffold is non-cytotoxic.

(b) Murine Study

[0176] PCL is biocompatible, non-cytotoxic and bioresorbs slowly. The biological safety of PCL yarns was demonstrated in a 12-month murine study. In this study, PCL yarns were delivered into the flexor digitorum longus (FDL) tendon in the hind paw of mice. Excised tissue was used as an autograft in the FDL tendon of the opposite hind paw. Positive cell infiltration, no significant increase in inflammation at any time and long-term PCL degradation were observed. Over the 12-month period, a positive presence of collagen was observed both within and around the PCL yarn.

(c) Rabbit Study

[0177] Biological safety was also demonstrated in a 12-week rabbit study, in which the local tissue response to implantation of PCL yarn was investigated relative to a standard suture material, monofilament polydioxanone (PDS). Each rabbit served as its own control, with either PCL (left hind) or PDS (right hind) implanted into the Achilles tendon.

[0178] Qualitative assessments of sections found few clear differences between the two materials in terms of the reaction elicited, including inflammation. However, a notable exception was the clear integration into PCL yarn of cells, with demonstrable progress from week 3 to 12 (whereas there was no evidence of cellular infiltration into PDS). Increased collagen and tenascin-C deposition were observed with PCL compared to PDS.

[0179] FIG. 16 shows the progressive reduction in number average molecular weight (Mn) and weight average molecular weight (Mw) of PCL over the course of this study. The data are shown as mean±standard deviation. Specifically, a 10% decrease in Mn and an 8% decrease in Mw were observed over the 12 weeks (84 days), illustrating the degradation of the PCL yarn.

(d) Sheep Efficacy Studies

[0180] Studies were carried out involving two groups of ten sheep (a test group and a control group). Both groups underwent a transection of the deep digital flexor tendon of the left hind limb with a two-strand Vicryl Kessler repair with additional circumferential suture.

[0181] The test group was implanted with the novel scaffold using a curved, hollow needle. Meanwhile, the control group received an equivalent intervention but no device was implanted. The full limb was cast for 14 days.

[0182] The results of a first in vivo study are shown in FIG. 17. Using ultrasound, the percentage reduction of surface area compared with the Vicryl control was measured after 4, 8 and 12 weeks, after normalisation to the native tendon. Results were obtained for the proximal, cut edge and distal portions (the definitions of which are indicated in the figure). Good surface area reduction was observed, demonstrating an improved healing response.

[0183] A second in vivo study after 21 days demonstrated cell and ECM alignment across the knitted structure.

[0184] Following termination at 12 months, the tendons were harvested for mechanical testing or histological analysis. The mechanical testing showed that the repaired test tendons were strong enough to withstand a load in excess of 350 N.

[0185] Three tendons from each group were used for histological analysis. The results are shown in FIGS. 18-20. It was observed by histology that ECM proteins collagen type-I and decorin (which also plays a role in matrix assembly) are significantly upregulated in the presence of the knitted PCL scaffold with suture, compared to the Kessler suture control. Specifically, there was a 160% increase at 3 months and a 260% increase at 12 months in collage type-I (see FIG. 18). There was also a 39% increase at 3 months and a 38% increase at 12 months in decorin (see FIG. 19). Meanwhile, there was a 41% decrease at 3 months and an 8% decrease at 12 months in inflammatory cells (see FIG. 20).

[0186] The results demonstrated that the nanofibrous architecture of the PCL knitted scaffold confers contact guidance cues to the cells and ECM, and that the cells are able to penetrate through to the core of the scaffold in vivo.

(7) Sterilisation and Packaging

[0187] Studies have shown that there is a slight, but not significant, decrease in molecular weight of the PCL after sterilisation, accompanied by no significant change in tensile strength of the scaffold. The sterilisation was a low-temperature ethylene oxide process (Anderson Ltd, ISO 13485).

[0188] The scaffold can be packaged in Dispos-a-Vent® packaging (Oliver Tolas). This has two seals: one applied after device fabrication (Tyvec/foil) and one after the sterilisation process. Both seals are strong enough to meet the minimum force recommend in BS EN 868-5:2009. The test complied with international standards ISO 11607 and ASTM F88/F88M-09. A shelf-life study showed no significant difference in peel strength over 12 months.

[0189] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0190] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0191] 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.

[0192] Any section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described.

[0193] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0194] 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%.

REFERENCES

[0195] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein. [0196] WO 2010/067086 A2. [0197] Cao Y L, Liu Y T, Liu W, Shan Q X, Buonocore S D, Cui L, (2002), “Bridging tendon defects using autologous tenocyte engineered tendon in a hen model”, Plastic and Reconstructive Surgery, 110(5), 1280-1289. [0198] Casper C L, Stephens J S, Tassi N G, Chase D B, Rabolt J F, (2004), “Controlling surface morphology of electrospun polystyrene fibres: effect of humidity and molecular weight in the electrospinning process”, Macromolecules, 37, 573-578. [0199] Curtis A S, Wilkinson C D, Crossan J, Broadley C, Darmani H, Johal K K, Jorgensen H, Monaghan W, (2005), “An in vivo microfabricated scaffold for tendon repair”, Eur Cell Mater, 9:50-57. [0200] Dzenis Y (2004), “Spinning continuous fibres for nanotechnology”, Science, 304, 1917-1919. [0201] Fong H, Chun I, Reneker D, (1999), “Beaded nanofibers formed during electrospinning”, Polymer, 40, 4584-4592. [0202] Huang Z M, Zhang Y Z, Kotaki M, Ramakrishna S, (2003), “A review on polymer nanofibers by electrospinning and their applications in nanocomposites”, Compos Sci Technol, 63, 2223-2253. [0203] Li W-J, Danielson K G, Alexander P G, Tuan R S, (2003), “Biological response of chondrocytes cultured in three-dimensional nanofibrous poly(ϵ-caprolactone) scaffolds”, J Biomed Mater Res A, 67A(4), 1105-1114. [0204] Maganaris C N, Paul J P, (1999), “In vivo human tendon mechanical properties”, Journal of Physiology, 521.1, 307-313. [0205] Magnusson S P, Hansen P, Aagaard P, Brønd J, Dyhre-Poulsen P, Bojsen-Moller J, Kjaer M, (2003), “Differential strain patterns of the human gastrocnemius aponeurosis and free tendon, in vivo”, Acta Physiologica Scandinavica, 177(2), 185-195. [0206] Mit-uppatham C, Nithitanakul M, Supaphol P, (2004), “Ultrafine electrospun polyamide-6 fibres: effect of solution conditions on morphology and average fibre diameter”, Macromol Chem Phys, 205, 2327-2338. [0207] Ramakrishna S, Fujihara K, Teo W-E, Lim T-C, Ma Z, (2005), “An introduction to Electrospinnining and Nanofibers”, World Scientific Publishing Co Ltd. [0208] Reneker D H, Yarin A L, Fong H, Koombhongse S, (2000), “Bending instability of electrically charged liquid jets of polymer solutions in electrospinning”, J Appl Phys, 87(9), 4531-4547. [0209] Shawon J, Sung C, (2004), “Electrospinning of polycarbonate nanofibres with solvent mixtures THF and DMF”, J Mater Sci, 39, 4605-4613. [0210] Smit E, Buttner U, Sanderson R D, (2005), “Continuous yarns from electrospun fibres”, Polymer, 46, 2419-2423. [0211] Smith D J (Jr), Jones C S, Hull M, Kleinert H E, (1986), “Evaluation of glutaraldehyde-treated tendon xenograft”, J Hand Surg [Am], 11(1), 97-106. [0212] Venugopal J, Ma L L, Yong T, Ramakrishna S (2005), “In vitro study of smooth muscle cells on polycaprolactone and collagen nanofibrous matrices”, Cell Biology International, 29, 861-867. [0213] Wannatong L, Sirivat A, Supaphol P, (2004), “Effects of solvents on electrospun polymeric fibres: preliminary study on polystyrene”, Polymer International, 53, 1851-1859.