Method for Preparing a Three-Dimensional Scaffold for Medical Use

Abstract

A method for preparing a sterilized scaffold for medical use, the method comprising the steps of: i) Loading collagen to a fiber mesh containing fibers of polylactide polymer or copolymer (commonly denoted PLA) to obtain a PLA-collagen scaffold, ii) Drying the PLA-collagen scaffold obtained from step i), iii) Sterilizing the PLA-collagen scaffold obtained from the drying step ii) to obtain the sterilized scaffold.

The sterilized scaffold obtained has improved biomechanical properties compared with an unsterilized scaffold.

Claims

1. A method for preparing a sterilized scaffold for medical use, the method comprising the steps of: i) Loading collagen to a fiber mesh containing fibers of PLA to obtain a PLA-collagen scaffold, ii) Drying the PLA-collagen scaffold obtained from step i), and iii) Sterilizing the PLA-collagen scaffold obtained from the drying step ii) to obtain the sterilized scaffold.

2. A method according to claim 1, wherein the sterilized scaffold obtained has improved biomechanical properties compared with an unsterilized scaffold, and wherein the improved biomechanical properties are expressed as an increase in one or more biomechanical parameters or biomechanical features.

3. A method according to claim 2, wherein the one or more biomechanical parameters or biomechanical features is selected from invariant creep modulus, when tested under wet conditions, and dynamic modulus, when tested under dry conditions.

4. A method according to claim 1, wherein the sterilized scaffold obtained has improved biomechanical properties compared with an unsterilized scaffold, and wherein the improved biomechanical properties are expressed as a decrease or a change of 10% or less in one or more biomechanical parameters or biomechanical features.

5. A method according to claim 4, wherein the biomechanical parameters or biomechanical features are selected from permeability in creep and dynamic modulus, both tested under wet conditions.

6. A method according to claim 2, wherein the increase is 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more or 10% or more.

7. A method according to claim 4, wherein the change is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1%.

8. A method according to claim 1, wherein the improved biomechanical properties is stiffness.

9. A method according to claim 1, wherein the temperature of the PLA-collagen scaffold before sterilizing is essentially the same or higher than the temperature of the PLA-collagen scaffold during sterilizing.

10. A method according to claim 1, wherein the sterilization is carried out at a temperature in a range of from −200° C. to 40° C..

11. A method according to claim 1, wherein the sterilization is performed with gamma irradiation.

12. A method according to claim 11, wherein the gamma irradiation dose is at the most 25 kGy.

13. A method according to claim 1, wherein the loading in step i) is performed with a gel of collagen.

14. A method according to claim 13, wherein the concentration of the collagen in the gel is from about 0.1% to 2.0% w/w.

15. A method according to claim 1, wherein the scaffold obtained in step i) contains from 5 to 25% w/w collagen, the percentage being based on the total amount of PLA and collagen.

16. A method according to claim 1, wherein the collagen is recombinant collagen, tissue derived collagen, or combinations thereof.

17. A method according to claim 1, wherein the mesh containing fibers of PLA and used in step i) is obtained by i) providing PLA in solid form ii) subjecting PLA to a process whereby fibers of PLA are obtained, and iii) subjecting the obtained fibers to a process, whereby a mesh of fibers is obtained,

18. A method according to claim 17, wherein step ii) is performed by spinning.

19. A method according to claim 17, wherein the mesh in step iii) of claim 18 is subjected to process comprising carding or needle punching to obtain a 3D network.

20. A method according to claim 1, wherein PLA is a polylactide.

21. A method according to claim 1, wherein a further step of crosslinking is performed before sterilization.

22. A method according to claim 21, wherein the collagen in the PLA-collagen is crosslinked.

23. A scaffold obtainable by the method of claim 1.

24. A method according to claim 4, wherein the decrease is 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more or 10% or more.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0176] FIG. 1A. Obtained invariant modulus in creep tests, in wet conditions (see Example 3).

[0177] FIG. 1B. Obtained memory value in creep tests, in wet conditions (see Example 3).

[0178] FIG. 1C. Obtained permeability in creep tests, in wet conditions (see Example 3).

[0179] FIG. 2A. Dynamic slope moduli for dynamic strain sweep tests, in wet conditions (see Example 3).

[0180] FIG. 2B. Static slope moduli for dynamic strain sweep tests, in wet conditions (see Example 3).

[0181] FIG. 3. Dynamic modulus vs. test temperature in dry conditions. As seen, there are no effect of temperature on the modulus values, but there are statistically significant (p=0.00) differences between non-sterilized and sterilized samples (see Example 3).

[0182] The invention is further illustrated in the following, non-limiting examples.

EXAMPLES

Example 1

[0183] A method for preparing a sterilized scaffold for medical use with polylactide fibers and collagen, drying the structure by freeze-drying to achieve a scaffold and sterilizing the scaffolds to obtain sterilized and stabilized PLA-collagen scaffold, rhCo-PLA. In this example, it is demonstrated that the PLA component is not negatively affected by the processing method.

[0184] The scaffolds were manufactured as follows. A PLA component, medical grade poly(UD)lactide 96/4 (Corbion, Purac Biochem by, Gorinchem, The Netherlands) with an inherent viscosity in a range of from 1.8 dl/g to 2.2 dl/g and residual monomer amount less than 0.1% was used to manufacture thin fibers by melt spinning. Before melt spinning, the PLA raw material was dried in vacuum oven. The melt processing of the fibers was done with melt spinning under protective atmosphere within a temperature range of 70-240° C. The spinning equipment consists of micro-extruder and a high-speed spinning machine. The fibers were cut into staple fibers and carded into mesh. The PLA felt was manufactured by needle punching the carded PLA mesh. The PLA felt was washed and dried in a laminar hood and subsequently packed before placing into a clean room environment. Recombinant human type III collagen (FibroGen Ltd., CA, USA) fibril formation was done by increasing the pH of the collagen solution with basic buffer solution to 7. After formation of a collagen gel, the PLA felt was fully immersed with the collagen gel and placed into sample molds. These structures were then freeze-dried to achieve fully dried structures, i.e. rhCo-PLA scaffolds. The manufactured scaffolds were cross-linked with 95% ethanol solution with 14 mM EDC (N-[3-dimethylaminopropyl]-N′-ethylcarbodiimide hydrochloride, Sigma—Aldrich, Helsinki,

[0185] Finland) and 6 mM NHS (N-Hydroxysuccinimide, Sigma—Aldrich, Helsinki, Finland) at room temperature (RT). The manufactured rhCo-PLA scaffolds were then washed, freeze-dried, and packed before sending to sterilization by gamma irradiation 25 kGy and under dry ice to avoid temperature rise during sterilization.

[0186] For the manufactured rhCo-PLA, the following studies were performed. The monomer amount measurement of L-lactide was done by GC-MS technique with lower limit of 0.01 wt % (Rambol Analytics, Lahti, Finland), the inherent viscosity (i.v.) was measured with a Lauda PVS viscometer (Lauda DR. R. Wobster GmbH, KG, Konigshofen, Germany). Samples were prepared by dissolving the polymer in 1 mg/ml chloroform. An Ubbelohde capillary viscometer type Oc (Schott-Geräte, Mainz, Germany) was used to determine the viscosity.

[0187] By this manufacturing method, a sterilized scaffold for medical use with polylactide fibers and collagen, rhCo-PLA, was achieved with following features, as seen in Table 1:

[0188] The residual monomer amount of the PLA component did not remarkably change, as the monomer amount stayed under 0.1 wt %, which was the same as the raw material monomer amount. The used PLA processing temperatures and sterilization did not extensively alter the inherent viscosity of the extruded PLA: acceptable decrease in this experiment was around 50%.

TABLE-US-00001 TABLE 1 Feature of PLA component Feature of of the sterilized PLA raw material rhCo-PLA scaffold Residual monomer amount <0.1 wt % <0.1 wt % in PLA component Inherent viscosity of PLA 1.9 dl/g 1.0 dl/g component

Example 2

[0189] A sterilized scaffold for medical use with polylactide fibers and collagen, manufactured by drying the structure by freeze-drying to achieve a scaffold and sterilizing the scaffolds to obtain sterilized PLA-collagen scaffold. A rhCo-PLA scaffold as described in Example 1 was sterilized with gamma irradiation and the effect of sterilization, especially to the collagen component, was evaluated. As well, different gamma irradiation doses (under dry ice), to show the effect of sterilization doses, were used on the rhCo-PLA scaffolds.

[0190] The data analysis was made according to the procedure described in detail in the U.S. patent Ser. No. 10/379,106 B2. This analysis comprises extraction of the invariant data such as viscous stiffness and memory values in static and dynamic conditions respectively from the data of applied load (stress) and deformation (strain). In this specification, the following definitions are used:

[0191] “Invariant modulus” is an intrinsic elastic modulus value which does not depend on time or frequency, and which can be used in the prediction of the material behavior (i.e. true value).

[0192] “Dynamic invariant modulus” is a ratio of dynamic stress amplitude to dynamic true (logarithmic) strain amplitude, expressed with real (not complex) algebra (different from commonly used real (storage) and imaginary (loss) moduli definition)

[0193] “Memory value” is a time-invariant property of the specimen, having the value in the range between zero and one, representing the viscous tendency of the material, even if the material itself is not a fluid. Memory values do not have a theoretical prediction and always must be determined from the experiment. In the present invention memory values have been experimentally measured separately for static (creep) and dynamic conditions as they were found to be different.

[0194] “Fluid mobility” is a measure (coefficient) of the rate of the fluid movement inside a porous body, analogues to the diffusion coefficient (using the same units in mm.sup.2/s). Its nature however differs from the latter because as the movement of fluid is not only by diffusion but also due to convective part and momentum transfer. Fluid mobility describes how well fluid has a potential to flow inside under certain conditions.

[0195] “Apparent permeability” describes a capacity of a porous body to allow a fluid to pass through its porous network and it is measured in squared distance units (m.sup.2). It is a quantified topological capacity of a material for transportation of a fluid through its porous structure and only depends on material structure but not on fluid properties. Here it differs from commonly defined permeability by Darcy law, as the latter requires an increase of the fluid pressure gradient across the material specimen. However, as shown in U.S. Pat. No. 10,379,106 B2, the method applied also here allows measurement of permeability without a knowledge of the fluid pressure gradient, and to distinguish between these methods, “apparent permeability” term is used (expressed in millidarcy; 1 mDarcy=10.sup.−15 m.sup.2).

[0196] A first test was done for aseptically produced rhCo-PLA (rhCo-PLA-A), which was compared to a rhCo-PLA sterilized with a standard irradiation dose, where the irradiation dose was 25 kGy. In the process, the actual irradiation dose was measured to be 29 kGy (rhCo-PLA-S). The aseptically produced rhCo-PLA (rhCo-PLA-A) had a gamma irradiated PLA component (sterilized with the standard irradiation dose 25 kGy), but the addition of the collagen component was done after sterilization, in aseptic conditions, i.e. the collagen component itself was not irradiated. Therefore, the PLA fiber component should have had identical properties and contribution to overall biomechanical performance, and the differences are mainly due to effect of collagen and its treatment.

[0197] The manufactured scaffolds were subsequently tested with a biomechanical testing procedure using dynamic mechanical analysis (DMA) in a standard compression sample holder (15 mm diameter) in the dynamic mechanical analyzer DMA242E (Netzsch Gerätebau GmbH, Selb, Germany).

[0198] A part of the scaffolds was subjected to a creep test under constant force of 0.2N stress and another part to oscillating forces causing strains in the range of 5-50 μm at 1 Hz frequency (strain-sweep method). Briefly, after letting the probe to establish the contact with the specimen and taring the offset, the starting height of the specimen immersed in media was again measured and used further as the starting height for true strain calculations.

[0199] In all cases, scaffolds were tested fully immersed in water at room temperature and allowing them to equilibrate 15 min before the measurements. All specimens thus have been fully impregnated, and no air bubbles or dry areas were observed. The cross-sectional area of the tested scaffolds was 20-26 mm.sup.2. All tests were done up to 300 min (until dimensional changes were approaching constant values; displacement resolution ±0.0005 μm). These data were stored and exported as ASCII text file into data processing software (Microsoft Excel complemented with customized code). The primary data were converted into stress and true strain, and the ratio of strain to stress vs. experiment time. After that, numerical algorithm of time convolution was applied and processed data were non-locally integrated pair-wisely, row by row with a mathematical method described in detail in U.S. Pat. No. 10,379,106 B2.

[0200] These experimental data are shown in Table 2.

TABLE-US-00002 TABLE 2 rhCo-PLA-A rhCo-PLA-S Irradiation dose on PLA, kGy ≥25 29 Irradiation dose on collagen, kGy 0 29 Creep test under 0.2N at 25° C. in water Invariant modulus, kPa 71.5 55.9 Memory value 0.089 0.051 Fluid mobility, mm2/s 0.129 0.072 Apparent permeability, milliDarcy 0.504 0.359 Strain sweep at 1 Hz at 25° C. in water Dynamic invariant modulus, kPa 41.4 61.6

[0201] Table 2 indicates that the sterilization with the standard procedure of gamma irradiation with a dose 25 kGy affected the biomechanical properties of the rhCo-PLA scaffolds as follows: [0202] In the creep test, with a sterilization dose of 29 kGy the rhCo-PLA-S scaffolds have suffered from decrease of invariant modulus which drop after irradiation significantly (−22%). The memory value has also decreased (−43%), indicating the rhCo-PLA scaffolds to become more elastic after the sterilization. The decrease of effective fluid mobility and apparent permeability of sterilized rhCo-PLA scaffolds (almost twice) indicates the state of less movable fluid inside the scaffold, and thus the irradiated scaffold structure becomes less permeable to fluid. These changes are undesirable, and such inferior biomechanical properties of rhCo-PLA-S are not acceptable. For the creep tests, this sterilization method was therefore found to major affect biomechanical characteristics of the rhCo-PLA scaffold, making it impossible to ensure its use for the intended application. [0203] In the strain sweep test, the dynamic invariant modulus increased (+33%), indicating the rhCo-PLA-S scaffolds to become more rigid after the sterilization, which leads to their inferior ability to conform to dynamic strains in the surrounding tissue.

[0204] The second step was to evaluate more precise gamma irradiation dose effect on the rhCo-PLA scaffold. A study was done to compare the different, under the standard 25 kGy, doses of gamma irradiation to the rhCo-PLA scaffolds. Therefore, the sterilization with low dose of gamma irradiation for rhCo-PLA scaffolds was conducted with the following doses: 18 kGy (G18), 20 kGy (G20), 22kGy, (G22), and 25 kGy (G25). The non-sterile rhCo-PLA scaffold (GO) was used as a reference.

[0205] The manufactured scaffolds were tested with biomechanical testing procedure using dynamic mechanical analysis, as described above, using the same dynamic mechanical analyzer DMA242E (Netzsch Gerätebau GmbH, Selb, Germany). The scaffolds were subjected to creep test under 0.2N constant force, similarly to the above. In all cases, scaffolds were tested fully immersed in water at room temperature and allowing them to equilibrate 15 min before the measurements. The area of the tested scaffolds was 37-46 mm.sup.2.

[0206] The biomechanical test results for rhCo-PLA scaffolds with these different lower doses of gamma irradiations are shown in Table 3. The results indicate that these different amounts of gamma irradiation, 25 kGy gave no significant changes to the biomechanics of the rhCo-PLA scaffolds (this is seen as e.g. value of invariant modulus and memory values of GO samples are within the limits measured on sterilized samples G18-G25).

TABLE-US-00003 TABLE 3 G0 G18 G20 G22 G25 Creep test under 0.2N at 25° C. in water Invariant modulus, 22.1 21.0 20.9 27.1 25.1 kPa Memory value 0.041 0.041 0.052 0.037 0.039 Fluid mobility, 0.028 0.030 0.034 0.033 0.037 mm.sup.2/s Apparent permeability, 1.110 1.275 1.512 1.069 1.281 mDarcy

Example 3

[0207] A sterilized scaffold for medical use with polylactide fibers and collagen, manufactured by drying the structure by freeze-drying to achieve a scaffold and sterilizing the scaffolds to obtain sterilized PLA-collagen scaffold.

[0208] A rhCo-PLA scaffold as described in Example 1 was sterilized with gamma irradiation at RT (S-RT) or at lower temperature (−70° C.) (S-LT) and the effect of sterilization, was evaluated and compared to non-sterile scaffolds (NS). In this test the possible changes in biomechanical properties of the scaffolds were demonstrated by different sterilization methods under the same expected dose (25 kGy) in general and find out if the lowered temperature during sterilization has an effect on biomechanical properties. The biomechanical testing was done for dry samples as well as for wet samples as described below.

[0209] Biomechanical analysis was made in dry conditions till 60° C. and in wet immersed conditions at 25° C. in compression mode. The manufactured scaffolds were tested with a biomechanical testing procedure using dynamic mechanical analysis in a standard compression sample holder (15 mm diameter) in the dynamic mechanical analyzer DMA242E (Netzsch Gerätebau GmbH, Selb, Germany). A part of the scaffolds was subjected to a creep test under 0.2N constant force and another part to strain sweep in the amplitude range from 5 to 25 μm at 1 Hz frequency. For the latter the loading cycles were repeated 10 times, similarly to Example 2.

[0210] Scaffolds were tested as fully immersed in water at 25° C. allowing them to equilibrate 15 min before the measurements. All specimens thus have been fully impregnated, and no air bubbles or dry areas were observed. A set of dry specimens was additionally tested in dry conditions (air) under 1 Hz and 25 μm amplitude but upon heating to 60° C. with 2 K/min rate with the purpose to assess thermal stability of the mechanical properties.

[0211] The cross-sectional area of the tested scaffolds was 30-40 mm.sup.2. The data analysis was made according to the procedure described in U.S. patent Ser. No. 10/379,106 B2, aimed on the extraction of the invariant data such as viscous stiffness and memory values in static and dynamic conditions respectively, with the data shown in FIG. 1-3. It is noteworthy to mention that under repetitive dynamic loading all specimens are progressively contracting with every loading sequence cycle. Hence the integral (slope) value of the dynamic stress/strain ratio (“standard dynamic modulus”) and the associated static stress/strain ratio (“standard static modulus”) at 1 Hz have been extracted to represent the values covering all loading cycles.

[0212] As seen from FIG. 1A-C, in creep tests there are significant differences in some properties values between some sample types but not others (error bars=standard deviation in wet conditions). For example, S-RT samples have lower modulus in creep and higher permeability in creep vs. S-LT samples. Hence, there is an effect of temperature control (lowered temperature) during sterilization procedure.

TABLE-US-00004 TABLE 4 Wet conditions (mean values) NS S-RT S-LT Invariant modulus, 48 45 68 in creep, kPa Invariant memory 0.042 0.06 0.55 value, in creep Permeability, in 0.98 1.65 1.0 creep, mDarcy Dynamic slope 10.5 12 10.0 modulus, kPa Static slope 5 6 5 modulus, kPa

[0213] In dynamic wet conditions (FIG. 2A-B) at 25° C. at 1 Hz there were no significant differences between dynamic and static stress/strain ratios (slope moduli) between the samples (NS, S-RT and S-LT).

[0214] Surprisingly, in dynamic dry conditions at 1 Hz with temperature rise till 60° C., there were significant differences, p=0.00 (when confidence interval is 95%, the values p<0.05 are statistically significant for two-sided permutation t-test), between samples in dynamic and static stress/strain ratios, indicating that the S-RT and S-LT scaffolds become stiffer after the sterilization process (FIG. 3). These differences were between non-sterile (NS) and sterile samples (S-RT & S-LT), but not between those sterilized in RT (S-RT) or in lowered temperature (S-LT). The change in temperature during dynamic testing had no effect on the properties until the 60° C. Also, there seem to be no effect of the test temperature on samples stiffness (i.e. the stress/strain ratio of a material) until 60° C., so it can be concluded the differences observed are mainly due to the sterilization process, and that different temperatures; room temperature (S-RT) or lower (S-LT) does not have an effect on these samples' properties.

[0215] These results indicate that the sterilization (in RT or in lower temperature (LT)) in general stabilizes the scaffold properties, which is only clearly visible in these tests in dry state.

[0216] As seen in FIG. 3 and Table 5, there are no effect of temperature on the modulus values, but there is statistically significant (p=0.00) difference between non-sterilized and sterilized scaffolds.

TABLE-US-00005 TABLE 5 Dry conditions (mean values) NS S-RT S-LT Dynamic 10 14-16 14-16 modulus, kPa

[0217] Generally, sterilization has been thought to have inferior effects on material properties of scaffolds, but in this case the sterilization leads to an unexpected result: gamma-radiation seems to have a positive effect of stabilizing the biomechanical properties of the scaffolds.

[0218] In summary this Example 3 demonstrates the following effect of the sterilization procedure on the materials vs. non-sterile materials (the changes are thus either Poor— that change of the property was not desirable, Fair— neutral effect, no statistically significant difference, or Good— the change of the property was desirable):

TABLE-US-00006 Its increase is S-RT S-LT Properties desirable? vs. NS vs. NS Invariant creep modulus Yes No Increases (wet conditions) changes Higher values help bearing (Fair) (Good) the static load Permeability in creep (wet No Increases No conditions) changes Lower values help keeping (Poor) (Good) synovial fluid in static Dynamic modulus (wet No No changes (Fair) conditions) Lower values improve compliance Dynamic modulus (dry Yes Increases (Good) conditions) Higher values with temperature indicate stability of the material structure Dynamic modulus vs. Yes No changes (Fair) temperature (dry conditions)

[0219] As mentioned herein before, a desired improvement means i) an increase in one or more biomechanical parameters or biomechanical features, ii) a decrease in one or more biomechanical parameters or biomechanical features, or iii) no change in one or more biomechanical parameters or biomechanical features.

[0220] As seen from the table above, an increase is desirable for the following biomechanical parameters: Invariant creep modulus (wet conditions), dynamic modulus (dry conditions), and dynamic modulus vs. temperature (dry conditions), whereas a decrease or no change is desired for the following biomechanical parameters: Permeability in creep (wet conditions) and dynamic modulus (wet conditions).

[0221] As mentioned herein before, a desired improvement is when an increase (or decrease) is 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more or 10% or more; or when no change is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1%.

[0222] Therefore, it can be assumed that the sterilization procedure of gamma irradiation in RT or in lower temperature (LT) is a positive step for these kinds of scaffolds to achieve more stable structure in both, dry as well as in wet state in dynamics. As well, sterilization in lowered temperature leads to preferable results compared to sterilization in RT, shown especially in wet creep conditions.

[0223] It was also confirmed that the rhCo-PLA scaffold as described in Example 1 has unexcepted reactivity toward gamma-sterilization conditions (dose and temperature control), such as improved biomechanical stability, allowing more precise control of mechanical properties and needed optimization of the materials vs. clinical demands. This effect was unexpected as it is generally known that the irradiation weakens or even destroys many organic materials and polymers.