THREE-DIMENSIONAL BODY IMPLANTS

Abstract

Three-dimensional body implants including a hydrogel, which includes cross-linked alginate and gelatin, and in particular breast implants. The hydrogel of the implants has a mechanical strength of 1 kPa to 1000 kPa, and the hydrogel of the implants may further include fibrinogen. The implants include a porous zone, and the implants are acellular, i.e., free of cells during their manufacture.

Claims

1.-31. (canceled)

32. A three-dimensional body implant which comprises a hydrogel comprising cross-linked gelatin and cross-linked alginate, wherein said hydrogel has a mechanical strength of from 1 kPa to 1000 kPa and that said implant has at least one porous zone, the porous zone comprising a plurality of pores each having a pore size, the porous zone having an overall porosity of from 100 μm to 10000 μm, the overall porosity corresponding to an average of the pore sizes measured in the porous zone.

33. The implant according to claim 32, wherein the pores of the porous zone have homogeneous pore sizes.

34. The implant according to claim 32, wherein the pores of the porous zone are homogeneously distributed.

35. The implant according to claim 32, wherein the pores of the porous zone extend along central axes having respectively homogeneous orientations.

36. The implant according to claim 32, wherein the pores of the porous zone have respectively homogeneous geometries.

37. The implant according to claim 32, wherein the pores of the porous zone are formed by the three-dimensional structure of the implant in the form of gyroid, cubic or hexagonal lattices.

38. The implant according to claim 32, comprising one porous zone or a plurality of porous zones.

39. The implant according to claim 38, wherein said plurality of porous zones comprises at least two porous zones in which the pores have different pore sizes and/or shapes.

40. The implant according to claim 39, wherein the porous zones are arranged to form a gradient of pore sizes distributed across the implant, the porous zones succeeding each other along a gradient direction in an order selected from an ascending order and a descending order of pore sizes.

41. The implant according to claim 40, wherein the implant comprises: a first porous zone forming a base representing 5% to 40% of a total volume of the implant, and having a pore size between 500 micrometers and 5000 micrometers, a second porous zone forming a core representing 20% to 70% of the total volume of the implant and having a pore size between 500 micrometers and 2500 micrometers, a third porous zone forming a shell representing 5% to 40% of the total volume of the implant, and having a pore size between 1000 micrometers to 10000 micrometers.

42. The implant according to claim 41, wherein the implant comprises: a first porous zone forming a base representing 20% to 40% of a total volume of the implant, and having a pore size between 500 micrometers and 5000 micrometers, a second porous zone forming a core representing 30% to 50% of the total volume of the implant and having a pore size between 500 micrometers and 2500 micrometers a third porous zone forming a shell representing 10% to 40% of the total volume of the implant, and having a pore size between 1000 micrometers to 10000 micrometers.

43. The implant according to claim 32, having at least one non-porous zone, the non-porous zone having a fill rate greater than 99%.

44. The implant according to claim 43, wherein said at least one non-porous zone comprises a perimeter surrounding the porous zone.

45. The implant according to claim 32, wherein said at least one porous zone covers a substantial portion of the implant.

46. The implant according to claim 32, consisting of a plurality of layers each having a mesh made up of a plurality of meshes, the layers being stacked on top of one another in such a way that the meshes form the pores.

47. The implant according to claim 32, wherein the implant has a volume in a range from 0.05 mL to 3 L.

48. The implant according to claim 32, wherein the implant is a breast implant.

49. A manufacturing process for obtaining a three-dimensional body implant comprising successively: a step of preparing a hydrogel comprising gelatin and alginate, a step of three-dimensionally shaping the hydrogel so as to form at least one porous zone, the porous zone comprising a plurality of pores each having a pore size, the porous zone having an overall porosity of between 100 μm and 10000 μm, the overall porosity corresponding to an average of the pore sizes measured in the porous zone, and a step of cross-linking the hydrogel with at least one divalent cation, and transglutaminase, said hydrogel having a mechanical strength of 1 kPa to 1000 kPa.

50. The manufacturing process according to claim 49, wherein the at least one divalent cation is calcium.

51. The manufacturing process according to claim 49, wherein, during the cross-linking step, the divalent cation and the transglutaminase are added concomitantly.

52. The manufacturing process according to claim 49, wherein the hydrogel further comprises cross-linked fibrinogen.

53. The manufacturing process according to claim 49, in which, during the three-dimensional shaping step, an additive manufacturing process is implemented.

54. The manufacturing process according to claim 49, further comprising a sterilization step.

55. A method for implementing the implant according to claim 32 in the context of reconstructive or cosmetic surgery, comprising a step of implanting the implant in the body of a subject.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0225] Further features, purposes and advantages of the invention will be apparent from the following description, which is purely illustrative and non-limiting, and which should be read in conjunction with the attached drawings in which:

[0226] FIG. 1 is a schematic representation of an implant according to the invention, of the breast type, which has a pore size gradient distributed over three zones.

[0227] FIG. 2 represents the comparison of the Young's modulus (A) and viscosity (B) of AG and FAG hydrogels that constitute the implants according to the invention.

[0228] FIG. 3 shows the comparison of the Young's modulus E0 (Pa) of an AG hydrogel in which the gelatin is cross-linked with and without transglutaminase and stored for up to 7 days at 37° C.

[0229] FIG. 4 represents the comparison of Young's modulus E0 (Pa) of an AG hydrogel and commercial hydrogels cross-linked or not with transglutaminase. *: Liquid compound at 37° C.; +: visible polymerization but insufficient gel stiffness at 37° C. for DMA measurement

[0230] FIG. 5 represents the viability and cell growth measured kinetically on FAG and AG hydrogels which constitute the implants according to the invention, and which were colonized in vitro by fibroblasts, after their manufacture.

[0231] FIG. 6 represents the viability and cell growth measured kinetically on FAG and AG hydrogels which constitute the implants according to the invention, and which have been colonized in vitro by adipose tissue stem cells, after their manufacture.

[0232] FIG. 7 represents the metabolic activity of AG implants according to the invention at different culture points following their in vitro colonization by a purified adipose tissue fraction, after their manufacture.

[0233] FIG. 8 represents the histological analyses by Hematoxylin, Phloxin, Saffron (HPS) staining of AG implants according to the invention after 2 days (4 images on the left) or 7 days (2 images on the right) of in vitro incubation with a fraction of purified adipose tissue, after their manufacture (Top: external edges of the matrices; Bottom: internal pores of the matrices; images taken in white light; magnification 100×; scale 100 μm).

[0234] FIG. 9 represents perilipin-1 immunostaining and Dapi staining of cell nuclei, on AG implants according to the invention after 2 days (top image) or 7 days (bottom image) of in vitro incubation with a purified adipose tissue fraction, after their manufacture (fluorescence imaging; magnification 200×; scale 50 μm).

[0235] FIG. 10 represents the comparison of Young's moduli of AG implants for varying durations of cross-linking at 21° C. (B) and 37° C. (A).

[0236] FIG. 11 represents the comparison of Young's moduli E0 and viscosities of AG and FAG implants after cross-linking with different concentrations of CaCl2 (A, D), TAG (B, E) and thrombin (C, F).

[0237] FIG. 12 represents the comparison of Young's modulus E0 (A-B) and viscosities (C-D) of AG and FAG implants after sequential or concomitant cross-linking with CaCl2, TAG and thrombin.

[0238] FIG. 13 represents the comparison of Young's modulus E0 (A) and viscosity (B) of AG and FAG implants after cross-linking with a solution containing calcium chloride or barium chloride.

[0239] FIG. 14A illustrates the study of the variation of dimensions (A1-A2) and pores (A3-A4) of AG and FAG implants according to the invention before and after cross-linking.

[0240] FIG. 14B illustrates the impact of sterilization on the dimensions (B1-B2) and Young's modulus (B3-B4) of these implants.

[0241] FIG. 15 illustrates the repeatability of the production of AG implants according to the invention in terms of dimensions (A), volume (B) and pore size (C).

[0242] FIG. 16 illustrates the repeatability of the shrinkage of AG implants according to the invention after consolidation.

[0243] FIG. 17 illustrates the repeatability of shrinkage of AG implants according to the invention as a function of the sterilization method.

[0244] FIG. 18A illustrates the repeatability of the extrusion diameter. FIG. 18B illustrates the repeatability of pore length (B1-B2) of AG implants according to the invention.

[0245] FIG. 19 represents images of varying pore sizes in an AG implant according to the invention.

[0246] FIG. 20 represents the surgical plan (left) of the in vivo subcutaneous implantation (right) of AG and FAG implants according to the invention.

[0247] FIG. 21 represents the histological analyses after staining with Masson's trichrome on sections of AG implants according to the invention, after subcutaneous implantation in vivo in rat back sites for 3 weeks (images at low, medium and high magnification).

[0248] FIG. 22 represents the average pore length of implants produced with different pore sizes.

[0249] FIG. 23 represents the average pore length of implants produced with an increasing pore size gradient from the base to the top.

[0250] FIG. 24 represents the apparent Young's modulus values of the different subparts of implants produced with different pore sizes.

[0251] FIG. 25 shows the compression tests on whole dentures with different architectures, stress-displacement curves.

[0252] FIG. 26 shows microscopic observation of the implant base without (left) or with (right) the addition of a perimeter.

[0253] FIG. 27A represents images of the 3D printing of a large-volume A/G implant (9 cm long, 7 cm wide, and 2.7 cm thick implant), the resulting implant after cross-linking, and the large pores obtained in the structure. FIG. 27B represents images of the 3D printing of a large volume A/G implant (12.6 cm diameter implant, and 5.3 cm thickness), the resulting implant after cross-linking and the large pores obtained in the structure.

[0254] FIG. 28 represents the macroscopic observation of the pores of implants with different filling rates.

[0255] FIG. 29 represents the average distance between the centers of the pores of implants with different filling rates.

EXAMPLES

[0256] The present invention will be better understood by reading the following examples which non-limitatively illustrate the invention.

Materials and Methods

[0257] Protocol #1 Preparation of an AG hydrogel: In order to prepare the AG hydrogel, 2 g of alginate (very low viscosity, Alpha Aesar, France), 5 g of gelatin (Sigma-Aldrich, France) are dissolved at 37° C. for 12H in 100 mL of a 0.1M NaCl solution (Labelians, France).

[0258] Protocol #2 Preparation of a FAG hydrogel: In order to prepare the FAG hydrogel, 2 g of alginate (very low viscosity, Alpha Aesar, France), 5 g of gelatin (Sigma-Aldrich, France) and 2 g of fibrinogen (Sigma-Aldrich, France) are dissolved at 37° C. for 12 H in 100 mL of a 0.1M NaCl solution (Labelians, France).

[0259] Protocol #3 Moulding of an AG and FAG hydrogel: 1.8 mL of the hydrogel prepared according to protocol #1 and #2 are deposited in the wells of a 6-well culture plate and incubated at 21° C. for 30 minutes.

[0260] Protocol #4 Cross-linking of an AG hydrogel: A cross-linking solution is prepared by dissolving 4 g of Transglutaminase (Ajinomoto, Japan), 3 g of CaCl2 (Sigma Aldrich, France) in 100mL of a 0.1M NaCl solution (Labelians, France). The cross-linking solution is then put in contact with the hydrogel for 1 H30 at 37° C. (unless otherwise specified).

[0261] Protocol #5 Cross-linking of a FAG hydrogel: A cross-linking solution is prepared by dissolving 4 g of Transglutaminase (Ajinomoto, Japan), 3 g of CaCl2 (Sigma Aldrich, France) and 400 Units of thrombin (Sigma Aldrich, France) in 100 mL of a 0.1M NaCl solution. The cross-linking solution is then put in contact with the hydrogel for 1 H30 at 37° C. (unless otherwise specified).

[0262] Protocol #6 Dynamic Mechanical Analysis (DMA) in compression: The mechanical properties of FAG and AG hydrogels are measured in triplicate with a rotational rheometer (DHR2, TA Instrument, France), a Peltier plane (TA Instrument, France) and an 8 mm notched geometry (TA Instrument, France). Three 8 mm diameter disks are cut from the molded hydrogels according to protocol #3. The disk is placed on the lower geometry at 37° C. for 60 seconds and then a 10 μm oscillatory compression procedure is performed from 0.1 to 10 Hz at 100 μm/s and 37° C. The values of Young's modulus EO (Pa) and viscosity η0 (Pa.Math.s) of the hydrogel are obtained from a visco-hyperelastic solid modeling using the E′ and E″ values acquired during the test.

[0263] Protocol #7 3D printing of hydrogels: Hydrogels prepared according to protocol #1, #2 are transferred into a 30 mL cartridge (Nordson EFD) equipped with a 410 μm diameter extrusion nozzle (Nordson EFD). The cartridge-nozzle assembly is then placed in a 3D printer (BioassemblyBot, Advanced Solution Lifescience, USA) allowing constant pressure to be applied to the cartridge while moving in all three directions of space. The printing parameters are a speed of 10 mm/sec, a pressure of 25-35 PSI and a temperature of 21° C. The different filling rates are obtained by the internal slicer of the printer control software (Tsim, Advanced Solution Lifescience, USA).

[0264] Protocol #8 In vivo implantation in rats: The in vivo implantation studies in rats were conducted on the BIOVIVO—Institut Claude Bourgelat (Lyon, France) preclinical research technical platform. The experiments were conducted in accordance with the European Directives 2010/63/EU. The 16 animals (Sprague Dawley rat, 250-300 g) were anesthetized by inhalation (oxygen and 5% isoflurane). The dorsal implantation sites were shaved and disinfected with povidone and sterile gauze, and sterile drapes were placed to delineate the surgical area. General anesthesia was maintained with isoflurane (2%) and oxygen inhalation. Pre-surgical analgesia was performed subcutaneously with meloxicam and morphine at 1 mg/kg respectively. Body temperature and pulse rate of the rats were monitored during surgery. Two skin incisions of 2-3 cm were made in the back region. A bioprosthesis was implanted in the dorsal subcutaneous region of each animal. The control group was performed with only the incision and dissection. In one animal per group, 4 surgical sites were performed, three bioprostheses and one control specimen. The surgical site was closed in layers using subcutaneous and cutaneous sutures with absorbable braided sutures (PDS® polidioxanone, 4/0 and Nylon 3/0, Ethicon J&J). Postoperatively, the animals were monitored for signs of suffering, and the surgical wounds were inspected daily for skin healing and absence of infection. Explantation took place 21 days after implantation.

[0265] Protocol #9 Histological analysis: Implants were fixed for 24 hours in a 4% formalin solution (Alphapat, France) and then dehydrated by successive baths of absolute ethanol (vwr chemicals, France) and methylcyclohexane (vwr chemicals, France) with an STP 120 dehydrator (Myr, Spain) and then embedded in kerosene (Sakura, Japan). Sections of 5 μm thickness were made with a HM 340e microtome (Microm, France). Hematoxylin Phloxine Saffron (HPS), Masson's Trichrome and DAPI staining were performed.

[0266] Protocol #10 Dynamic mechanical analysis (DMA) in compression: The mechanical properties of FAG and AG hydrogels were measured in triplicate with a rotational rheometer (DHR2, TA Instrument, France), a Peltier plane (TA Instrument, France) and a 25 mm geometry (TA Instrument, France). Punches of 25 mm diameter are cut in the implants produced according to the protocol #9. The punch is placed on the lower geometry at 37° C. for 60 seconds and then a 10 μm oscillatory compression procedure is performed from 0.1 to 10 Hz at 100 μm/s and 37° C. The values of Young's modulus EO (Pa) and viscosity η0 (Pa.Math.s) of the hydrogel are obtained from a visco-hyperelastic solid modeling using the E′ and E″ values acquired during the test.

[0267] Protocol #11 Total mechanical analysis of the implants in compression: Placement of the implants on a Lloyd tensile/compression machine with a 1 kN load cell and compression plates, a test speed of 10 mm/min is used.

Example 1—Mechanical Properties of Alginate/Gelatin (AG) and Fibrinogen/Alginate/Gelatin (FAG) Hydrogels

[0268] AG and FAG hydrogels were prepared from protocols #1 and #2, molded according to protocol #3, and then cross-linked using protocols #4 and #5 to study their DMA mechanical properties using protocol #6.

[0269] The results are shown in FIG. 2 (A-B). The measured Young's modulus and viscosity values are similar between the AG hydrogel and the FAG hydrogel following their cross-linking by the process of the invention. The Young's moduli under the specific conditions of this study are around 68000 Pa.

Example 2—Impact of Cross-Linking with Transglutaminase on the Mechanical Properties of Alginate/Gelatin Hydrogel (AG).

[0270] Molded samples of AG were prepared from protocols #1 and #3 and cross-linked from a variant of protocol #4. In this variant, the cross-linking solution is composed of a 30 mg/mL calcium chloride solution only or a 30 mg/mL calcium chloride and 40 mg/mL transglutaminase solution. Four gels of each condition were cast and tested in DMA on the same day and after 1, 4 and 7 days of storage at 37° C., respectively, in order to mimic physiological conditions.

[0271] The samples were then studied by DMA using protocol #6.

[0272] The results are shown in FIG. 3. This study shows the advantageous effect of the use of transglutaminase during cross-linking on the mechanical properties of hydrogels. This effect is even greater when the gels are converted at 37° C., justifying the particular interest of cross-linking according to the invention for hydrogels intended to be implanted.

Example 3—Impact of Cross-Linking with Transglutaminase on the Mechanical Properties of Commercial Gelatin and/or Collagen Hydrogels

[0273] Molded samples of GA were prepared from protocols #1 and #3 and cross-linked from protocol #4. The commercial hydrogel samples listed in Table 4 below were prepared according to the protocols provided by the suppliers and molded according to protocol #3.

TABLE-US-00004 TABLE 4 Hydrogel commercial name Reference Supplier Gel4Cell BIS-101 Bioink Solution Gel4Cell-VEGF BIS-103 Bioink Solution Col4Cell BIS-108 Bioink Solution GelMA VL5000000010 Cellink Lifeink 200 Type I Collagen 5278-5ML CellSystems BiogelX B5X-0000 Biogelx Rat tail Collagen A10483-01 Sigma

[0274] Hydrogels were cross-linked with a variation of protocol #4, using either a solution comprising only calcium at 30 mg/mL (no TAG), or a solution of calcium at 30 mg/mL and transglutaminase at 40 mg/mL, in order to observe the impact of TAG.

[0275] The uncross-linked and cross-linked samples with TAG were then studied by DMA using protocol #6.

[0276] The results are grouped in FIG. 4. Six of the seven commercial hydrogels studied were cross-linked with transglutaminase. Collagen-based hydrogels (Col4Cell, Rat Collagen) are not stiff enough to be analyzed by DMA but gelatin-based hydrogels (Gel4cell, Gel4cell-VEGF and GelMa) have a significantly higher Young's modulus after transglutaminase cross-linking (7.3, 9.9 and 50 kPa respectively). This study shows the effect of cross-linking with transglutaminase on the stiffness of commercial hydrogels.

Example 4—Influence of the Amount of Alginate and Gelatin in a Fibrinogen/Alginate/Gelatin (FAG) Hydrogel on Mechanical Properties

[0277] FAG hydrogels were prepared from a variant of protocol #2, molded according to protocol #3, then cross-linked thanks to protocol #5, then their mechanical properties were studied by DMA thanks to protocol #6. In this variant, we studied these mechanical properties by preparing the FAG hydrogel, with 1 or 3 or 2 g of alginate, and 10 or 7.5 or 5 g of gelatin, respectively, and 2 g of fibrinogen.

[0278] The results are grouped in Table 5 below. The Young's moduli under the specific conditions of this study range from 200 to 800 kPa.

TABLE-US-00005 TABLE 5 Young's modulus Gelatin (%) Alginate (%) Fibrinogen (%) (kPa) 10 1 2 800 7.5 3 2 600 5 2 2 200 2 2 0 70 1 1 0 35

Example 5—Evaluation of Fibrinogen/Alginate/Gelatin (FAG) and Alginate/Gelatin (AG) Hydrogels Colonization by Fibroblasts

[0279] AG and FAG hydrogels were prepared from protocols #1 and #2. Square implants of 1.5 cm side and 0.2 cm thickness were then printed using protocol #7 and cross-linked using protocols #4 or #5. The printed implants were produced with a 50% fill rate and an extrusion nozzle of 410 μm internal diameter. A negative control (empty well) is also used.

[0280] Normal human fibroblasts in passage 6 are thawed and amplified in 175 cm2 culture flasks in culture medium containing DMEM supplemented with 10% calf serum and 1% antibiotics. Each implant was seeded on its surface with a cell suspension of normal human fibroblasts at a concentration of 4000000 fibroblasts/ml. 250 μl of this suspension was drip-fed onto each implant, i.e. 1000000 fibroblasts/implant. After 1 hour of adhesion, the implants were immersed with culture medium. Implants were cultured in culture medium composed of DMEM containing 10% calf serum supplemented with vitamin C and EGF (Epidermal Growth Factor) at 37° C., 5% CO2. Implants were cultured with this same medium for 21 days, renewed every 3 days.

[0281] The metabolic activity of the fibroblasts within the implants was studied by colorimetric analysis with Alamar Blue on days 3, 5, 8, 10, 14 and 21 after seeding. The solution was made by diluting 10-fold a solution of Alamar Blue (DAL 1100, Invitrogen) in DMEM. After 19 hours of incubation at 37° C., 100 μl of the supernatants were collected and their absorbance at 570 nm and 600 nm was measured by spectrophotometer (NanoQuant® infinite M200PRO, TECAN).

[0282] Cell viability and growth were monitored over 21 days of culture using 6-point kinetics on days 3, 5, 8, 10, 14 and 21. The results are shown in FIG. 5.

[0283] The results confirmed that all implants allowed fibroblast adhesion and survival as early as day 3 of culture. Cell growth is observable for each porous implant over the 21 days of culture, on both types of hydrogels (FAG and AG) as well as for each overall porosity employed.

Example 6: Evaluation of Colonization of Fibrinogen/Alginate/Gelatin (FAG) and Alginate/Gelatin (AG) Hydrogels by Adipose Tissue Stem Cells (ASC)

[0284] AG and FAG hydrogels were prepared from protocols #1 and #2. Square implants of 1.5 cm side and 0.2 cm thickness were then printed using protocol #7 and cross-linked using protocols #4 or #5. The printed implants were produced with a 50% and 75% fill rate and an extrusion nozzle of 410 μm internal diameter. Sterilization was performed by the company IONISOS (France) by irradiating the implants with a dose of 30 kGy of Gamma ray.

[0285] Normal human adipocyte stem cells in passage 2 to 5 were thawed and amplified in 175 cm2 culture flasks in culture medium containing DMEM supplemented with 10% serum and 1% antibiotics. Each implant was seeded on its surface with a cell suspension of ASC at a concentration of 6, 12, or 24 million ASC/ml. 250 μl of these suspensions were drip-fed onto each implant, i.e. 1.5, 3 or 6 million ASC/implant. After 1 hour of adhesion, the implants were immersed with culture medium. Implants were cultured in culture medium containing DMEM supplemented with 10% serum and 1% antibiotics for 7 days and then in medium containing DMEM supplemented with 10% serum, insulin, rosiglitasone and 1% antibiotics for 14 days. The culture media are renewed every 3 days.

[0286] The metabolic activity of fibroblasts within the implants was studied by colorimetric analysis with Alamar Blue on culture days 3, 5, 7, 14 and 21 after seeding. The solution was made by diluting 10-fold a solution of Alamar Blue (DAL 1100, Invitrogen) in DMEM. After 5 hours of incubation at 37° C., 100 μl of the supernatants were collected and their absorbance at 570 nm and 600 nm was measured by spectrophotometer (NanoQuant® infinite M200PRO, TECAN).

[0287] Cell viability and growth were monitored over 21 days of culture using 6-point kinetics on days 3, 5, 7, 14 and 21. The results are shown in FIG. 6.

[0288] The results confirmed that all implants allowed adipocyte stem cells to adhere and survive from day 3 of culture. Cell growth is observable for each porous implant over the 21 days of culture, on both types of hydrogels (FAG and AG) as well as for each density of seeding.

Example 7: Evaluation of the Colonization of Alginate/Gelatin (GA) Hydrogels in Contact with a Purified Adipose Tissue Fraction

[0289] GA hydrogels were prepared from protocol #1. Cubic implants of 1.5 cm side and 0.8 cm thickness were then printed using protocol #7 and cross-linked using protocol #4. The printed implants were produced with a 50% fill rate and an extrusion nozzle of 410 μm internal diameter.

[0290] The lipoaspirate is centrifuged at 1500 RPM for 2 minutes and then rinsed with 1×PBS. The lipoaspirate was again centrifuged at 1500 RPM for 30 seconds and then the 1×PBS was removed. The lipoaspirate was considered purified.

[0291] Each implant was then immersed in 6 mL of purified lipoaspirate, and the whole set was placed in a culture insert in a 6-well plate with incubation in medium containing DMEM supplemented with 10% serum and 1% antibiotics at 37° C. 5% CO2 for 2 days or 7 days.

[0292] Following contact with lipoaspirate, the implants were grown in 6-well plates in culture medium containing DMEM supplemented with 10% serum, insulin, rosiglitasone, and 1% antibiotics, with 3 medium changes per week until 21 days.

[0293] Cellular metabolic activity within the implants was studied by colorimetric analysis with Alamar Blue on culture days 2, 7, and 21 after seeding. The solution was made by diluting 10-fold a solution of Alamar Blue (DAL 1100, Invitrogen) in DMEM. After 5 hours of incubation at 37° C., 100 μl of the supernatants were collected and their absorbance at 570 nm and 600 nm was measured with a spectrophotometer (NanoQuant® infinite M200PRO, TECAN).

[0294] Cell viability and growth were monitored over 21 days. The results are shown in FIG. 7. A much higher metabolic activity than the negative control was observed in the implants that were in contact with purified lipoaspirate.

[0295] Histological analyses were performed to complete this study according to protocol #9. The results are shown in FIG. 8. The images reveal the presence of agglomerated, polygonal, uniform, unilocular, and bulky adipocytes. These morphological characteristics are those of healthy adipocytes, which can be found in adipose tissue.

[0296] Immunostaining for perilipin-1 was also performed. Samples were included in OCT (CellPath, KMA-0100-00A) and then stored at −80° C. Sections of 16 μm thickness were made for each sample with a cryostat (Microm, HM 520). The sections were then fixed in acetone/methanol (v/v) solution for 20 minutes and rinsed 3 times in 1×PBS. A 1-hour incubation at room temperature in 4% PBS-BSA solution was performed to saturate the aspecific sites. The sections were then incubated overnight at room temperature with perilipin-1-specific primary antibody solution. The next day, the sections were rinsed three times with 1×PBS and then incubated 45 minutes with Alexa fluor 568-coupled secondary antibody solution at room temperature. Then the sections were rinsed three times with 1×PBS and mounted between slide and coverslip with Dapi fluoromount-G® mounting medium (SouthernBiotech). The images obtained are grouped together FIG. 9.

[0297] The images show adipocytes with large spherical or polygonal vacuoles depending on the clustering of the cells. The adipocytes appear as unilocular and their size is also physiological as it ranges from 50 to 200 μm.

[0298] Taken together, these results confirm the adhesion, survival, and regeneration of a human adipose tissue brought into contact with the implants. The particular structure and composition of the implants thus form a favorable environment for the regeneration of healthy adipose tissue.

Example 8—Influence of Temperature and Cross-Linking Time on the Mechanical Properties of an Alginate/Gelatin Hydrogel (AG)

[0299] Molded samples of AG were prepared from protocols #1 and protocol #3 and cross-linked from a variant of protocol #4. In this variation, the cross-linking times and temperatures were changed from 10 minutes to 14 H and from 37° C. to 21° C.

[0300] The samples were then studied by DMA using protocol #6.

[0301] The results are shown in FIG. 10 (A-B). The cross-linking times as well as the cross-linking temperature have very little influence on the final mechanics (Young's modulus) of the hydrogels. However, it seems that an optimum can be found around 1:30, whatever the temperature.

[0302] These Young's moduli are also very stable over 7 days after cross-linking at 37° C. The cross-linking of the gelatin was efficient since there was no loss of dissolved gelatin in the medium.

Example 9—Impact of Cross-Linking Solution Component Concentration on the Mechanical Properties of Alginate/Gelatin (AG) and Fibrinogen/Alginate/Gelatin (FAG) Hydrogels Once Cross-Linked

[0303] Molded samples of AG and FAG were prepared from protocols #1, #2 and #3, cross-linked from a variant of protocols #4 and #5. In this variation, the concentrations of the components of the cross-linking solution were changed (transglutaminase, calcium chloride, and thrombin).

[0304] The samples were then studied by DMA using protocol #6.

[0305] The results are grouped in FIG. 11 (A-F). Over this range of reagent concentrations, no significant variations were observed (E9 all very similar).

Example 10—Impact of Sequential or Concurrent Cross-Linking of Alginate/Gelatin (AG) and Fibrinogen/Alginate/Gelatin (FAG) Hydrogel

[0306] Molded samples of AG and FAG were prepared from protocols #1, #2, and #3, cross-linked from a variant of protocols #4 and #5. In this variant, we investigated sequential cross-linking on FAG and AG, which involves cross-linking the hydrogel in several steps.

[0307] Each step took 1 h and three rinses with 0.1M NaCl solution were performed between each step to remove residual cross-linking agents.

[0308] The samples were then studied by DMA using protocol #6.

[0309] The results are shown in FIG. 12 (A-D). The sequential cross-linking set (FAG and AG) generate hydrogels with lower Young's moduli than the single step cross-linking.

[0310] It can be observed that very soft and brittle gels are obtained if calcium is not added first, indeed TAG and thrombin are calcium-dependent their activity is therefore largely decreased without the addition of CaCl2. The gels are therefore difficult to manipulate without the calcium cross-linking. When thrombin is added first, the gels have very little mechanical strength and holes appear.

Example 11—Impact of the Nature of the Divalent Cation for the Cross-Linking of Alginate/Gelatin (AG) and Fibrinogen/Alginate/Gelatin (FAG) Hydrogels

[0311] Molded samples of AG and FAG were prepared from protocols #1, #2, and #3, cross-linked from a variant of protocols #4 and #5. In this variant, we studied a cross-linking in the presence of barium chloride 30 mg/mL.

[0312] The samples were then studied by DMA using protocol #6.

[0313] The results are shown in FIG. 13 (A-B). Cross-linking in the presence of barium results in gels with Young's moduli very similar to those obtained with CaCl2. However, since barium increases the viscosity of the gels, the formation of additional pendant chains can be assumed.

Example 12—Maintenance of the Three-Dimensional Structure and Mechanical Properties of Alginate/Gelatin (AG) and Fibrinogen/Alginate/Gelatin (FAG) Hydrogel Implants After Sterilization

[0314] The AG and FAG hydrogels were prepared from protocols #1, #2 and #3, cross-linked using protocols #4 and #5, optically observed and then studied by DMA using protocol #6. The printed shapes are half-spheres of 2 cm diameter produced with variable filling rates (30, 50 and 75%).

[0315] Sterilization was performed by the company IONISOS (France) by irradiation of the implants with a variable dose (30 kGy and 40 kGy) of Gamma ray.

[0316] The impact of the cross-linking step on the dimensions of alginate/gelatin and fibrinogen/alginate/gelatin hydrogel implants was studied. These dimensions were measured from macroscopic images.

[0317] The dimensions of the pores obtained as a function of the filling rate were also studied. These dimensions were measured from images made with a microscope (Olympus, ×4 magnification).

[0318] The results are shown in FIG. 14 (A-B). The implants shrink by an average of 10% following the cross-linking step. However, the pore size does not vary significantly (FIG. 14A (A1-A4)).

[0319] Regarding sterilization, it appears that the 40 kGy dose leads to a higher shrinkage of the constructs than the 30 kGy dose. Concerning EO, sterilization does not lead to any change in the mechanics of the material for both doses (FIG. 14B (B1-B4)).

Example 13: Production Quality of Large Alginate/Gelatin (AG) Hydrogel Implant: Repeatability of Impression Dimensions, Dimensions After Consolidation and Sterilization of the Implant by Several Methods

[0320] AG hydrogels were prepared from protocol #1. Half-sphere shaped implants of 6 cm diameter and 2 cm thickness were then printed according to protocol #7 and cross-linked using protocol #4, then optically observed and measured. The printed shapes were produced with variable filling rates (25 to 65%) and extrusion nozzles of 410 or 840 μm internal diameter. Sterilization was performed by IONISOS (France) by irradiating the implants with 2 doses (30 kGy and 40 kGy) of Beta rays or a range dose of 30 kGy.

[0321] The impact of the cross-linking and sterilization step on the dimensions of large alginate/gelatin hydrogel implants was studied. These dimensions were measured from macroscopic images.

[0322] The dimensions of the pores obtained as a function of the filling rate were also studied. These dimensions were measured from images made with a microscope (Olympus, ×4 magnification).

[0323] The results after printing are shown in FIG. 15 (A-C). These results show a high repeatability of the dimensions of the large 3D printed implants, reflecting a high production quality.

[0324] The results after consolidation of the implants are grouped FIG. 16. This graph shows a high repeatability of the shrinkage of the large implants after the consolidation stage.

[0325] The results after sterilization of the implants by 3 methods (β-rays doses 40 and 30 kGy and γ-rays 30 kGy) are grouped FIG. 17. These results show less shrinkage of the large implants with the β30 and 40 kGy rays.

[0326] Large implants were printed with 2 extrusion nozzles with internal diameters of 410 and 840 μm with fill rates of 25 to 65%. The repeatability of the extrusion diameter as well as the obtained pore length were measured. The results are shown in FIG. 18 (A-B).

[0327] FIG. 18A shows the high repeatability of the extruded bead size. FIG. 18B (B1-B2) shows the variation of pore length with the filling rate of the hydrogel.

[0328] Images of varying pore sizes were taken and are grouped in FIG. 19.

[0329] These data show the wide range of pores that can be obtained for the implants and their high repeatability and production quality.

Example 14—Studies of the Resistance of Implants In Vivo

[0330] GA and FAG hydrogels were prepared from protocols #1, #2 and #7, cross-linked through protocols #4 and #5. The printed shapes were 1 cm diameter half-spheres, produced with varying fill rates (30, 50 and 75%).

[0331] The porous half-spheres were sterilized with a dose of 30 kGy and then implanted subcutaneously in rats according to protocol #8.

[0332] Details of the implantation groups are described in the following Table 6, which refers to the surgical implantation plan described in FIG. 20.

TABLE-US-00006 TABLE 6 Group position of (hydrogel/filling rate) no animal no implant the implant FAG 30% 1 1 A 2 2 A 3 3 A 4 4 A 5 B 6 C AG 30% 5 7 A 6 8 A 7 9 A 8 10 A 11 B 12 C AG 50% 9 9 A 10 14 A 11 15 A 12 16 A 17 B 18 C AG 75% 13 19 A 14 20 A 15 21 A 16 22 A 23 B 24 C

[0333] Histological analyses were performed using protocol #9, and the results are grouped in FIG. 21. Explantation was used to validate the resistance of the implants to skin tension. Histological analyses were used to assess cell colonization, vascularization, extracellular matrix synthesis, and the presence of areas of inflammation.

Example 15—Production Quality of Large Implants with Various Pore Sizes and Study of the Impact of these Porosities on the Mechanical Properties of said Implants

[0334] Semi-anatomical breast prosthesis type implants (height: 8.83 cm; width: 6.37 cm; height: 2.86 cm) are produced using protocol #1 and a variant of protocol #7 (using a nozzle with an internal diameter of 840 μm) and then cross-linked using protocol #4. These implants are produced with different internal porosities: [0335] Implants with one pore size for their entire volume. [0336] Implants with two pore sizes distributed according to the fact that a first part at the base of the implant has one pore size and a second part at the top of the implant has another pore size. [0337] Implants with three pore sizes distributed according to whether a first portion at the base of the implant (named base) has one pore size, another second portion (named core) in the middle and above the base of the implant has another pore size, and another third portion (named shell) on the surface and above the base of the implant has another pore size. [0338] Implants with a gradient of pore sizes increasing from their bases have also been produced.

[0339] The dimensions of the obtained pores were studied. These dimensions were measured from images taken with a microscope (Olympus, ×4 magnification). The results of these measurements are shown in FIGS. 22 and 23. Pores of different sizes and very reproducible can be obtained in the different parts of the implant. A gradient of reproducible and increasing pore sizes from the base to the top can also be obtained.

[0340] The mechanical properties of the subparts of these implants were studied by DMA according to protocol #11 and the mechanical properties of the implants were studied by total mechanical analysis according to protocol #12. The results of these measurements are shown in FIGS. 24 and 25. The Young's moduli observed vary inversely with respect to the pore size. Thus, for example, the decrease in the size of the pores at the heart of the implants allows a higher modulus to be obtained, reflecting a greater mechanical resistance. Thus, variations in pore size and the distribution of pore size zones make it possible to obtain a wide range of Young's modulus and therefore to obtain implants with greater or lesser strength. Concerning compression tests on whole prostheses, we observe that each configuration of pores brings different mechanical properties to the implants. Indeed, for an effort of −35N, the prosthesis with only 1 zone of porosity deformed less contrary to the prostheses with 3 zones of porosity which deformed more. The curves also allow us to identify different breaking behaviors. The prosthesis with only one porosity zone deformed progressively before breaking, whereas for the prosthesis with two porosity zones, the breaking was progressive and then brutal at −217 N, resulting in a stress recovery. Thus, by varying the pore sizes and the distribution of pore size zones, implants with different mechanical properties are obtained. This makes it possible to adapt the mechanical properties of the implants according to the desired application.

Example 16—Addition of a Perimeter Around the Base of the Implant

[0341] Semi-anatomical sized breast prosthesis implants (Height: 8.83 cm; Width: 6.37 cm; Height: 2.86 cm) are produced from protocol #1 and a variation of protocol #7 (using an 840 μm inner diameter nozzle and adding a perimeter to the base of some implants) and then cross-linked from protocol #4. These implants all have a single pore size and a perimeter is added to some implants. This perimeter is characterized by the addition of a continuous filament surrounding the implant, tangentially to all the filaments located at the periphery of the implant. This perimeter is added on the first three layers of the implant.

[0342] The resulting architecture was studied using a microscope (Olympus, ×4 magnification). The results of these observations are shown in FIG. 26.

[0343] The addition of a perimeter to the base of the implant makes it possible to obtain a more cohesive base with fewer asperities at the periphery, thus limiting inflammatory friction in vivo.

Example 17—Production of Large Volume Porous Implants from Alginate/Gelatin Hydrogel

[0344] Implant 1: 200 mL of AG hydrogels were prepared from protocol #1 and then a 12 cm long, 10 cm wide and 5 cm thick anatomical breast-like implant was printed from a variant of protocol #7 (840 μm inner diameter extrusion nozzle and 30 mm/sec printing speed) with a single area of porosity and then cross-linked with protocol #4 (200 mL instead of 100 mL of consolidation solution for a large implant). The average pore size of the resulting implant was measured with an optical microscope and the dimensions of the implant were measured with a caliper. After cross-linking, an implant of 9 cm length, 7 cm width and 2.7 cm thickness is obtained with an average pore size of 1380+/−57 μm.

[0345] Implant 2: 500 mL of AG hydrogels were prepared from protocol #1 and then a 7 cm radius, 6 cm thick half-spherical breast-like implant was printed from a variant of protocol #7 (840 μm inner diameter extrusion nozzle and 30 mm/sec printing speed) with a single pore area, and then cross-linked with protocol #4 (700 mL instead of 100 mL of consolidation solution for a large implant). The average pore size of the resulting implant was measured with an optical microscope and the dimensions of the implant were measured with a caliper. After cross-linking, an implant with a diameter of 12.5 cm and a thickness of 5.3 cm is obtained with an average pore size of 3354 μm+/−273 μm.

[0346] Images of these implants are shown in FIGS. 27A and 27B.

Example 18—Measurement of the Distribution in Space of the Pores of the Same Zone of an Implant

[0347] Semi-anatomical sized breast prosthesis implants (Height: 8.83 cm; Width: 6.37 cm; Height: 2.86 cm) are produced from protocol #1 and protocol #7 and then cross-linked from protocol #4. These implants are produced with different filling rates (45, 50 and 55%).

[0348] The distances separating the centers of the pores (of square shape) obtained were studied. These dimensions were measured from images taken with a microscope (Olympus, ×4 magnification). The results of these measurements are shown in FIGS. 28 and 29. The distance separating the centers of the pores turns out to be reproducible for each filling rate and varies between the different rates. These observations reflect a homogeneous distribution of pores within a zone of the implant with a defined filling rate.