Particle suitable for the manufacture of an implantable soft tissue engineering material

Abstract

The particle (1) is suitable for the manufacture of an implantable soft tissue engineering material and comprises: a three-dimensionally warped and branched sheet (2) where (i) the three-dimensionally warped and branched sheet (2) is made from a biocompatible material having a Young's modulus of 1 kPa to 1 GPa; (ii) the three-dimensionally warped and branched sheet (2) has an irregular shape which is encompassed in a virtual three-dimensional envelope (3) having a volume V.sub.E; (iii) the three-dimensionally warped and branched sheet (2) has a mean sheet thickness T; iv) the three-dimensionally warped and branched sheet (2) has a volume V.sub.S; (v) the particle (1) has a Young's modulus of 100 Pa to 15 kPa; and (vi) the particle (1) further comprises a number of protrusions where the three-dimensionally warped and branched sheet (2) reaches the envelope (3); (vii) the particle (1) has a number of interconnected channel-type conduits (5) defined by the branching of the sheet (2) and/or by voids in the sheet (2); and (viii) where the conduits (5) have (a) a mean diameter D.sub.C; and (b) an anisotropicity index of 1.01 to 5.00.

Claims

1. A particle for use in manufacturing an implantable soft tissue engineering material, the particle comprising a three-dimensionally warped and branched sheet, wherein: (i) the three-dimensionally warped and branched sheet comprises a biocompatible material having a Young's modulus of 1 kPa to 1 GPa; (ii) the three-dimensionally warped and branched sheet has an irregular shape which is encompassed in a virtual three-dimensional envelope having a volume V.sub.E; (iii) the three-dimensionally warped and branched sheet has a mean sheet thickness T; (iv) the three-dimensionally warped and branched sheet has a volume V.sub.S; (v) the particle has a Young's modulus of 100 Pa to 15 kPa; and (vi) the particle has an irregular shape and comprises a number of protrusions where the three-dimensionally warped and branched sheet reaches the virtual three-dimensional envelope; (vii) the particle has a number of interconnected channel-type conduits defined by the branching of the sheet and/or by voids in the sheet; and (viii) the conduits have (a) a mean diameter D.sub.C; and (b) an anisotropicity index of 1.01 to 5.00, and (ix) wherein the ratio of D.sub.C/T is larger than 1.

2. The particle according to claim 1, wherein the biocompatible material of the sheet is selected from the group consisting of poly-ethyleneglycol (PEG), poly-acrylamide, poly-(hydroxyethyl)methacrylate, and polysaccharides.

3. The particle according to claim 1, wherein the biocompatible material of the sheet is a material selected from the group consisting of carbohydrates, collagens, peptides, and extracellular matrices.

4. The particle according to claim 1, wherein the biocompatible material of the sheet is a synthetic polymer selected from the group consisting of (i) silicones; (ii) polyurethanes; (iii) polyolefins; (iv) acrylates; and (v) polyamides.

5. The particle according to claim 1, wherein the mean diameter D.sub.C of the conduits is larger than 1 micrometer.

6. The particle according to claim 1, wherein the protrusions have a mean relative maximum protrusion depth in the range between 0.05 and 1.0.

7. The particle according to claim 1, wherein the shape of the three-dimensionally warped and branched sheet is flexible and reversibly expandable upon absorption or removal of a liquid by the biocompatible material.

8. The particle according to claim 1, wherein the contact angle between water and the biocompatible material of the sheet is in the range of 0° to 90°.

9. The particle according to claim 1, wherein the sheet is reversibly compressible.

10. The particle according to claim 1, wherein the particle is hydrated and comprises at least 0.05 weight-% of the biocompatible material based on the total weight of the hydrated particle.

11. The particle according to claim 1, wherein the particle comprises a plurality of three-dimensionally warped and branched sheets.

12. A composition comprising: a) a multitude of particles according to claim 1; and b) a physiologically acceptable fluid.

13. The composition according to claim 12, wherein the amount of fluid is such that the particles are only partially hydrated.

14. An implantable soft tissue engineering material comprising a multitude of particles according to claim 1.

15. The implantable soft tissue engineering material according to claim 14, wherein the multitude of particles is admixed with one or more substances to form a malleable paste, and wherein the one or more substances are selected from the group consisting of: water, aqueous solution, blood, serum, pharmaceutically active agents, lidocaine, adrenaline, cell suspensions, biological tissues, stem cells, virus, bacteria, fungi, transfecting agents, antibodies, genetically modified cells, extracellular matrices, co-cultures of cells, growth factors, platelet rich plasma, cell differentiation factors, lipids, and high-density lipoprotein (HDL).

16. The implantable soft tissue engineering material according to claim 14, wherein the implantable soft tissue engineering material is reversibly compressible after injection into a patient by uptaking liquid from surrounding tissues.

17. A method for manufacturing particles according to claim 1, comprising: a) pre-cooling a mixture comprising a polymerizable biocompatible material in an aqueous solution at a temperature below 10° C.; b) cross-linking the pre-cooled mixture at a temperature below 0° C. such that the cross-linking is not based on a radical polymerization; and c) fractioning the cross-linked biocompatible material obtained.

18. The method according to claim 17, wherein adipic dihydrazide is used as a cross-linker.

19. The particle according to claim 1, wherein the biocompatible material of the sheet is a hydrogel.

20. The particle according to claim 1, wherein the biocompatible material of the sheet is selected from the group consisting of cellulose, alginate, chitosan, agarose, polysucrose, and dextran.

21. The particle according to claim 1, wherein in use, when a neighboring second particle is present, the two particles interlock.

Description

A BRIEF DESCRIPTION OF THE DRAWINGS

(1) A special embodiment of the invention will be described in the following by way of example and with reference to the accompanying drawings in which:

(2) FIG. 1 illustrates a three-dimensional view of an embodiment of the particle according to the invention and its virtual envelope;

(3) FIG. 2 shows the same view as FIG. 1 highlighting the protrusions at the periphery of the particle by means of broken-line circles;

(4) FIG. 3 shows the same view as FIG. 1 in which the thickness T of the sheet at various locations is indicated by arrows.

(5) FIG. 4 shows the same view as FIG. 1 in which the thickness diameter DC of some of the conduits is indicated by arrows.

(6) FIG. 5 shows the same view as FIG. 1 in which the protrusion depth is indicated by the length of a normal vector Nv from a point of the particle envelope to the closest intersection point with a sheet of the particle.

(7) FIG. 6 shows 3-D projections of a material having a Young's modulus to low (A) and of a material according to the invention (B).

(8) FIG. 7 is a graphical representation of the percentage of cells retained in the soft tissue engineering material according to the invention when seed in vitro with and without deployment effect.

(9) FIG. 8 is a graphical representation of the percentage of cellularization of the soft tissue engineering material according to the invention.

(10) FIG. 9 shows macroscopic pictures of the implants site over time.

(11) FIG. 10 represents photographs regarding histology and macroscopic observation of bio-integration of comparative examples.

(12) FIG. 11 is a graph showing cellular invasion and vascularization of comparative examples.

(13) FIG. 12 is a graph showing the vessels density (number of vessels/mm.sup.2) observed on histology pictures after the implantation of a soft tissue engineering material having a mean diameter of the conduits of 16 micrometers and a soft tissue engineering material having a mean diameter of the conduits of 127 micrometers.

DETAILED DESCRIPTION OF THE INVENTION

(14) The following examples clarify the invention further in more detail.

(15) A) Manufacture of the Particles

Example 1

(16) Carboxymethyl-cellulose (with a MW of 700 kDa) was dissolved in deionized water to the concentration of 2%, and crosslinking initiated after precooling to 4° C. by means of addition of adipic acid dihydrazide AAD (0.07%) and a small excess of the carbodiimide EDC (0.4%) and buffered to a pH-value of 5.5. by means of an excess of 2-(N-morpholino)ethanesulfonic acid (MES) buffer (50 mM).

(17) The reaction mixture was placed at −20° C. in a mold. After 1 day, the scaffolds were thawed and washed in de-ionized water (DI).

(18) The next step consisted in fractioning the scaffold. For this, a bulk scaffold or a bulk scaffold piece was placed in a plastic bag and compressed and sheared manually to create the particles according to the invention. In another embodiment, the bulk scaffold was extruded through a thin tubular element by applying a known pressure to obtain a fragmented material.

(19) The particle size was controlled by the pressure applied on the piston of the syringe and by the size of the extruding cannula. Typically, a pressure of 15 bars and a cannula of 14 G was used.

Example 2a

(20) The same procedure as in example 1 was followed but prior to freezing, the reaction mixture was distributed into a silicone mold using a pipette of 10 mL. The silicone mold contained microstructured star-shaped cavities measuring 100 micrometers in diameter and 20 micrometers in depth. The silicone mold was covered with a flat polypropylene counterpart, squeezing excess liquid from the mold. The assembly was then placed into a freezer at −20° C.

(21) Alternative Methods for the Manufacture of Particles:

(22) Particles were manufactured by placing the scaffolds into a mixer and mixing them.

(23) Particles were manufactured by ink-jet printing, 3D printing, and additive manufacturing.

(24) Particles were manufactured by mixing the reaction mixture with a photosensitizer (typically acrylamide monomer and N,N′methylenebis(acrylamide), freezing at −20° C. and photopolymerizing using a UV lamp or a visible lamp.

(25) Particles were manufactured by grinding a preliminary manufactured scaffold during at least 30 s, for example using a mixing robot (for example Kenwood Major Titanium KMM060).

(26) Particles were manufactured by cutting and/or slicing a preliminary manufactured scaffold using cutting blades, possibly organized in networks.

(27) It is important to note that a classical emulsion polymerization method would give nearly perfectly round particles and therefore would not lead to the desired structure of the particles according to the invention with significant protrusions.

Example 2b

(28) A solution of 5% of hyaluronic acid monomers with a molecular weight of 90 kDa, MES buffer pH6, adipic acid dihydrazide (2 mg/mL) was mixed with EDC (4 mg/mL) and poured onto a consolidated paraffin microspheres scaffold. The paraffin beads were prepared according to “Microspheres leaching for scaffold porosity control”, Draghi et al, Journal of Material sciences: Materials in medicine, 16 (2005) 1993-1997. The mixture was incubated at room temperature during 24 hours after which the paraffin beads were dissolved by an excess of hexane. The obtained scaffold was then rinsed with isopropanol, and a mix of isopropanol and water (40%:60%) and followed by a rinsing step with water.

(29) The obtained scaffold was then fragmented by applying an extrusion force on the scaffold through a narrow tubular element.

(30) B) Manufacture of an Implantable Soft Tissue Engineering Material Comprising a Multitude of Particles According to the Invention

Example 3

(31) Carboxymethyl-cellulose (with a MW of 1500 kDa) was dissolved in deionized water to the concentration of 2,2%, and crosslinking initiated after precooling to 3° C. by means of addition of adipic acid dihydrazide AAD (0.08%) and a small excess of the carbodiimide EDC (0.5%) and buffered to a pH-value of 5.6 by means of an excess of MES buffer (54 mM). 20 mL of the reaction mixture was placed at −15° C. in a glass mold measuring 1 mm in depth and 16 cm diameter. After 20 hours, the scaffold was thawed and washed in 50 mL of DI water. The next step consisted in fracturing the scaffold. For this the bulk scaffold was stuffed into a 50 mL syringe and extruded through a 20 G needle by applying a pressure of 15 bars. The fractioned material obtained was further washed with 50 mL of a saline solution containing 0.45 g of NaCl. After the washing step, the material was autoclaved in a bottle of glass containing 90 mL of DI water using a temperature of 118° C. during 24 minutes. The content was then put onto a filter device with a pore size of 0.22 um and fluid withdrawn by briefly applying a suction pressure of 750 mbar such as to obtain a final volume of 10 mL The material was then transferred into a syringe with luer lock for injection.

Example 4

(32) The fractioned material obtained in example 1 was further washed with phosphate buffered saline (PBS). The washing step was performed by thawing the fractioned material in a bath of saline solution. 10 mL of the fractioned material obtained in example 1 consisting of 0.6 g of dry polymer and of 9.4 g of water was washed with 50 mL of a saline solution containing 0.45 g of NaCl. After the washing step, the material was autoclaved in a bottle of glass containing 90 mL of DI water using a temperature of 121° C. during 20 minutes. The content of the bottle was then centrifuged using an acceleration of 4 g during 2 minutes; 50 mL of water was removed using a Becher and a pipette to obtain the final consistency. The consistency was adjusted by addition or withdrawal of fluid on a filter device; the final volume was about half of the original fabrication volume.

(33) C) Comparative Tests

Example 5.1

(34) The Young's modulus of the soft tissue engineering material according to the invention, in conjunction with particle geometry and hydration level, enables the deployment of the branched sheets of the particles and consequently the 3D projection of the volume created (see FIG. 6). When the Young's modulus is too low, or the hydration too large, the material does not project in 3D but spreads (picture A of FIG. 6). When the Young's modulus and hydration level are correct, the material creates a 3D implant, stable over time (picture B of FIG. 6).

(35) The effect of the mechanical properties of the soft tissue engineering material was further evaluated quantitatively by evaluation of the short-term (3 weeks) implantation behavior as a function of the mechanical properties of the implant. For this purpose, soft tissue engineering material fabricated according to 6 different recipes and characterized by their deployment pressure and Young's modulus of the soft tissue engineering material. The materials were injected subcutaneously in mice, and the implant evaluated with regard to undesired spreading from the injection site, evolution of volume for the first hour and then at three weeks, as well as regarding stability of shape and creation of a 3D projection. The results are summarized in table 1:

(36) TABLE-US-00001 TABLE 1 Young's modulus (soft tissue Deployment engineering In-vivo 3D In-vivo shape Recipe pressure material) deployment projection maintenance #1 4 Pa 40 Pa No No Flows #2 19 Pa 0.13 kPa No No Flows #3 32 Pa 0.28 kPa Inconsistent Inconsistent Inconsistent #4 95 Pa 0.74 kPa Yes Inconsistent Inconsistent #5 163 Pa 1.5 kPa Yes Yes Yes #6 274 Pa 3.3 kPa Yes Yes Yes, but too hard to the touch

(37) They indicate for that for the implantation site and procedure chosen, a minimum of about 100 Pa of deployment pressure is needed to obtain a desired consistent (yet slight) volume swelling upon implantation, and that a Young's modulus of at least 1.5 kPa is required for stable 3D projection (not surprisingly, this approximately matches the known Young's modulus of 2 kPa for adipose tissue). Only slightly higher Young's moduli (3.3 kPa) are perceived as unnaturally hard to the touch from the outside. The Young moduli indicated are drained moduli; the undrained values are about 2.5× higher. The Poisson ratio under drained conditions was near zero, whereas it was near 0.5 for undrained conditions.

(38) Uniaxial compression used for Young modulus determination was essentially perfectly reversible to high strains (at least 30%), both from geometric observation and return to baseline force within a few percent of the maximum force in particular for the drained conditions.

(39) To further characterize the mechanics of the soft tissue engineering material, we analyzed samples obtained with recipe #5 of Table 1 in oscillatory rheology, and in uniaxial creep tests. For rheology, we used a HaakeRS100 RheoStress device, FL16 vane geometry with factory settings, stress sweep from 1 Pa to 100 Pa at constant 1 Hz frequency. At low stress (<10 Pa), the sample behaves like an elastic solid with minor viscous contribution (elastic modulus G′ on the order of 5 kPa, viscous modulus G″ about 0.9 kPa), whereas at higher stresses (20-30 Pa of shear stress in the FL16 vane geometry), a yield point is observed and the sample starts to flow with G′ approaching G″; however, as soon as the movement is stopped, the samples recover their original G′ and G″ values at low frequency and stress (essentially perfect repeatability of the experiment without need for a setting period). This reversible, but nonlinear viscoelastic behavior contributes to injectability of the material (at shear stresses beyond yielding), and simultaneously its propensity to rapidly regain its stable solid-like properties once movement ceases.

(40) Creep addresses how a material behaves under a constant load. We assessed creep during uniaxial compression (samples of an about 5 mm height under a chuck of 5 cm diameter), and found an uncommon behavior: For all pressures safely accessible to the uniaxial compression machine used (<2.5 kPa), chuck movement would completely stop at a finite sample height, indicating that for slow compression, the samples can withstand very substantial pressures equal to their Young modulus or higher. Local densification due to particle compressibility as well as efficient particle interlocking in the engineering material according to the invention are at the origin of this particular behavior. Surprisingly the effect is protective for the shape achieved in-vivo under slowly applied pressures (for instance, an individual lying down on an injected site). Deployment at low pressures enables the gentle aspiration of tissues or cell suspensions for co-grafting applications (mixing with adipose tissues and injection of the mixture to create a living volume). FIG. 7 shows the percentage of cells retained in the material obtained after seeding fibroblast cells using the deployment effect or using only a passive seeding (no deployment effect).

(41) To achieve cell adhesion for the experiment described in relation to FIG. 7, material as prepared in example 3 was coated with collagen I (10% of the mass of CMC) in an sodium acetate buffer pH 4, followed DI rinsing and covalent attachment of the adsorbed collagen by use of EDC (10 mg/mL, in 100 mM MES buffer at pH 5.5), followed by inactivation of remaining EDC in basic pH and readjustment to physiological pH with PBS buffer. All steps made use of the deployment effect to enhance fluid exchange; the coating protocol is a result of optimization with respect to total collagen adsorption efficiency, homogeneity, collagen density lining the pores, absence of fibril formation and cell adhesion.

(42) For measuring the effect of the deployment advantage of the particles according to the invention, two different materials were injected in mice: one which was partially hydrated, and once injected, deployment of the particles took place by taking up interstitial fluids; and another material the particles of which were fully hydrated, and once injected, would not deploy itself because the channel-like conduits were already “full” of fluid and therefore was not capable to deploy more.

(43) In both cases the percentage of the implant area occupied by cells and collagen or other proteins (“cellularization”) was evaluated as shown in FIG. 8.

(44) Further experiments were conducted with the two materials in order to confirm working hypothesis that the deployment of the partially hydrated particles by means of their peripheral protrusions was producing a zipper effect leading to stability of the shape of the injected material (implant) and of the volume created and preventing migration of the particles in the body.

(45) The results obtained with the material with deployment ability clearly showed its superiority as represented in Table 2:

(46) TABLE-US-00002 TABLE 2 Height of Height of the implant the implant measured 3 days after the Standard after the Standard injection deviation injection deviation Material (mm) (mm) (mm) (mm) With 3 1 3 1 deployment ability Without 2 1 0.5 1 deployment ability

(47) Surprisingly it seems that the material with deployment ability is frictioning with the surrounding tissues enabling the material to stay in place (anchoring effect).

Example 5.2

(48) Since isotropicity of the conduits seems to play a major role in the deployment capability of the material further experiments were conducted in this regard. Indeed particles with high channel anisotropicity have long, highly oriented, parallel channels, and will collapse easily in the direction perpendicular to the channel orientation and therefore be unable to deploy correctly. In cross-sections of the particles, this anisotropy is visible by the occurrence of channels with very large ratios of longer to smaller diameter.

(49) In order to verify these assumptions particles were manufactured with non-isotropic conduits and used for the manufacture of an implantable soft tissue engineering material comprising a multitude of such particles.

(50) This material was compared to the material according to the invention by measuring the height of the implanted material immediately after the injection into the body and after 3 days. The results are shown in the below table. It was observed that the 3D deployment was reduced in the non-isotropic like conduits as shown in table 3:

(51) TABLE-US-00003 TABLE 3 Height of Height of the implant the implant measured measured 3 days after the Standard after the Standard injection deviation injection deviation Material (mm) (mm) (mm) (mm) With isotropic 4 1 3 1 conduits With non- 2 1 0.5 1 isotropic conduits

(52) D) Role of the Mean Diameter of the Conduits on the Vessels Ingrowth

(53) Since mean diameter of the conduits plays a major role in the vascularization of the material once implanted in vivo, further experiments were conducted in this regard. The graph in FIG. 12 shows the vessels density (number of vessels/mm2) observed on histology pictures after the implantation of a soft tissue engineering material having a mean diameter of the conduits of 16 micrometers and a soft tissue engineering material having a mean diameter of the conduits of 127 micrometers. We observe that the vessels density is significantly lower in the case of the smallest mean diameter of the conduits.

(54) E) Clinical Use of the Implantable Soft Tissue Engineering Material According to the Invention

Example 6.1

(55) Prior the intervention, the surgeon using the soft tissue engineering material defines the areas where new volumes are needed. For this, he/she evaluates visually the volume defects and traces lines using a marker defining the future injection lines.

(56) 10 mL of the soft tissue engineering material was placed in a plastic syringe equipped with a Luer-lock connector tip (corresponding to 0.6 g of dry mass of polymer). A cannula was connected to the tip and inserted in the target area of the patient through a thin skin incision. Once positioned in the target site, for example between the subcutaneous adipose layer and the pectoral muscle in a woman breast, the cannula is withdrawn at a speed of 0.5 cm/s while 10 mL of the material is injected by the surgeon by applying a pressure on the piston of the syringe of 4000 N/m.sup.2. The injection can be repeated in a neighboring area, enabling to increase the total volume injected.

Example 6.2

(57) The injection of 10 mL of the soft tissue engineering material is repeated by using the same incision point as in example 6.1. but by modifying the direction and the angle of the cannula between each injection.

(58) The surgeon performed one incision through the skin of the patient close to the area needing volume enhancement. The Luer-lock syringe containing the soft tissue engineering material was screed to a cannula (14 G for example) and the cannula was inserted in the patient's tissues. The cannula was inserted into the tissues up to reaching the target and the injection of 10 mL of the soft tissue engineering material was started by applying a pressure of 4 kPa to the piston of the syringe while withdrawing the cannula in the direction of the incision point. Then, without taking the cannula out of the patient's body, the empty syringe was unscrewed and a new filled syringe containing the soft tissue engineering material was screwed on and a new injection was performed in a new direction of interest, predefined by marked lines on the patient's skin.

Example 6.3

(59) In another embodiment, the soft tissue reconstruction material is first mixed with adipose tissues from the patient using two syringes and a connector before being injected as a mixture into the target area using a cannula.

Example 6.4

(60) In one embodiment, the soft tissue engineering material is combined with the graft of adipose tissues preliminary harvested from the patient. For example, adipose tissues are extracted by liposuction using a harvesting cannula connected to a 10 mL Luer-lock syringe. Tissues are let sediment for 5 minutes allowing to remove the blood and oil floating above the adipose tissues. In one embodiment, the user injects one spaghetti of adipose tissues of 2 mL to 10 mL and then he/she injects a spaghetti of the soft tissue engineering material. In another embodiment, the adipose tissues are mixed with the soft tissue engineering material by connecting two syringes (one containing the adipose tissues, the other one containing the soft tissue engineering material) using a Luer-to Luer connector and by pushing sequentially on the two pistons of the two syringes until obtaining a homogeneous mixture. The mixture obtained is then injected using the injection method described before.

Example 6.5

(61) In another embodiment, the material is injected in the target area and the shape of the implant is shaped manually by the surgeon from the outside of the patient in order to create the shape required.

Example 6.6

(62) In one embodiment, the implantable soft tissue engineering material is sterile and contained in a syringe. It is delivered in the target area of the patient using a tubular element such as a sterile Luer-lock infiltration Coleman cannula of 14 Gauge. Typically, the material is injected into subcutaneous tissues, into adipose tissues, into muscular tissues, between two layers of the above-mentioned tissues. For the delivery, the user performs first a small incision (1 mm to 4 mm in length) located at least at 2 cm of the targeted injection site. The user inserts the cannula through the incision up to reaching the targeted point, located at 2 cm to 15 cm from the insertion point. He/she then injects retro-gradually 5 mL of the soft tissue engineering material by pushing gradually on the piston of the syringe while withdrawing the cannula from the targeted point to the incision point. So doing, the user injects a spaghetti like volume having a diameter comprised between 1 mm and 8 mm, enabling the integration of the soft tissue engineering material within the surrounding tissues. The procedure can be repeated several times from the same injection point in order to create a 3D arrangement of spaghettis. The localization of the spaghettis is controlled manually by the user, who is able to evaluate the depth of the injection and the localization in the different planes of the patient's tissues.

(63) Other Variations of Examples 6.1. to 6.6. are Described Below

(64) In one embodiment, the user uses his/her hands to press on the skin of the patient while inserting the cannula and injecting the material in order to maintain the patient's tissues from the outside and to define the localization of the material.

(65) In one embodiment, the user injects the material using the same device described previously but injects the material in a bolus shape, which is expanding the surrounding tissues of the injection site.

(66) In one embodiment, the soft tissue engineering material is combined with the graft of adipose tissues preliminary harvested from the patient. For example, adipose tissues are extracted using a harvesting cannula by liposuction. Tissues are let sediment for 5 minutes allowing to remove the blood and oil floating above the adipose tissues. The adipose tissues are distributed in 10 mL syringes. In one embodiment, the user injects one spaghetti of adipose tissues of 2 mL to 10 mL using the Coleman method and then he/she injects a spaghetti of the soft tissue engineering material. In another embodiment, the adipose tissues are mixed with the soft tissue engineering material by connecting two syringes (one containing the adipose tissues, the other one containing the soft tissue engineering material) using a Luer-to-Luer connector and by pushing sequentially on the two pistons of the two syringes. The mixture obtained is then injected using the method described before.

(67) In another embodiment, the soft tissue engineering material is manually distributed in a body cavity (such as a breast cavity after silicone implant removal) using a sterile spatula in order to create a layer of the soft tissue engineering material.

(68) In another embodiment, the soft tissue engineering material is sutured to surrounding tissues (in the case of large particles).

(69) F) Clinical Results Obtained and Comparative Studies with Prior Art Materials

Example 7

(70) A comparison of the stability and migration of 4 different materials, including the soft tissue engineering material according to the invention was performed. The materials were the following:

(71) “HA 1” is a commercially available hyaluronic acid based filler (“Juvederm Ultra 2” from Allergan.

(72) “HA 2” is a commercially available, strongly crosslinked hyaluronic acid based filler (“Macrolane” from Q-med AB).

(73) “Matrix” is a commercially available, collagen based, flowable matrix used for wound repair (from Integra LifeSciences corporation).

(74) “Material developed” is the material obtained in examples 1 to 4.

(75) A defined volume of the different tested items (200 microliters) was injected subcutaneously in CD1 female mice in the back area of the animal. Two samples of each tested item were injected, namely one on each side of the spinal cord of the animal. In the case of the silicone item, the samples were implanted by first performing an incision in the skin of the animal and by inserting manually with tweezers the layer of silicone. The volumes of the items were monitored over time using external measurements with a Caliper and using MRI scanning and MRI images analysis. After 3 and 6 months, the animals were euthanized and histology of the different implanted materials was performed. Bio-integration (percentage of the material occupied by cells and tissues, vascularization) was quantified. The results are represented in FIG. 9, which shows macroscopic pictures of the implants site in mice for the different tested items, at different time points (t=0 is just after the injection step). In the drawings on the left side of FIG. 9, the dashed lines represent the implant localization just after the injection. The grey surface represents the implant localization 3 months after the injection.

(76) The macroscopic observation of the histology samples (see FIG. 10) enabled to show that the material developed lead to the growth of vessels and tissues, whereas the HA-based materials tested remained clear and transparent, showing neither tissular nor vascular ingrowth. The Matrix 1 material was populated with cells but it degraded before the 6 months timepoint. These results are represented graphically in FIG. 11.

(77) The presence of a capsule surrounding the implants compared was investigated on histological sections stained with Masson trichrome. An additional material was included in this comparative study, namely “Silicone” which a silicone layer sample cut from a silicone tissue expander used in breast (Natrelle 133) Tissue expander from Allergan. The implanted samples were squares of 6 mm side and measured 1.5 mm in thickness.

(78) The thickness of the capsule was measured for each material tested. The results are presented in Table 4 below:

(79) TABLE-US-00004 TABLE 4 Material Material implanted developed HA1 HA2 Matrix 1 Silicone Thickness of the No capsule No capsule 92 +/− 17 No capsule 104 +/− 20 capsule (3 months micrometers micrometers after the implantation

(80) It was observed that the soft tissue engineering material according to the invention was stable over time. It did not migrate or increase in volume. On the contrary, HA 1 was increasing in volume and the two implants merged together (they moved from the initial position). HA 2 was also stable but the histological analysis showed the presence of a foreign body reaction (thin capsule around the implant, presence of giant cells), which could explain the stability of the position. The material was isolated from the body and did not migrate. The matrix 1 material was rapidly resorbed and did not produce a durable volume.

(81) Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the scope of the appended claims.

(82) It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.