PT106174A - THREE-DIMENSIONAL POROSA STRUCTURE THAT PERMITS SEGMENTARY VASCULARIZATION FOR COMPLEX TISSUE ENGINEERING AND METHODS OF PRODUCTION THEREOF - Google Patents

Abstract

The present invention relates to the development and method for the production of porous and implantable three-dimensional structure (EIS), which allows (M) to control cell growth, segmental vasovascularization and inertness . The porous three-dimensional structure (A) includes (in) the porous polymer matrix of support and a gel or hydrogel (EIS), since the gelation of the gel (in which the gelation may occur in situ BY IONIC AND PHOTO-RETICULATION). THE POROSA (S) THREE-DIMENSIONAL STRUCTURE (S) MAY BE USED AS IMPLANT. The present invention further describes the method of processing the fabric of the implantable fabric carrier structure and the various applications thereof, including the manufacture of partial or total fabric implants for textile fabrication and as textile fabrics. -OR MULTI-LAYERS FOR THE TREATMENT OF OSTEOCONDARY DEFECTS. The above described implant (s) are particularly suitable for the treatment of degenerative disorders or injuries, in which the patient's natural response does not allow the defect to be repaired.

Description

DESCRIPTION " POROUS THREE-DIMENSIONAL STRUCTURE THAT PERMITS SEGMENTARY VASCULARIZATION FOR COMPLEX TISSUE ENGINEERING AND PRODUCTION METHODS THEREOF "

TECHNICAL FIELD OF THE INVENTION The present invention relates to porous (s) and implantable (s) three-dimensional structure (s) combining a porous polymeric support matrix and the hydrogel (s) allowing integration into the locus of the defect, allowing segmental vascularization, and its use as a biodegradable implant in the regeneration of damaged complex tissues, namely, defects of the meniscus and cartilage (grade IV). The method (s) for the preparation of the porous three-dimensional structure (s) comprises an implant formed from a porous polymeric support matrix combined with a gel or hydrogel ( s) in a manner favorable to the defect site of all tissue. The structure (s) may be formed in a single or multi-sequential process. These structures exhibit controlled performances and allow for the encapsulation of therapeutic and bioactive cells and / or detection agents, further enabling the control of cell infiltration, vascularization and innervation in different parts of their porous three-dimensional (3D) structure. The present invention represents a promising approach in order to mimic the organization of the native tissue (s), with respect to the vasculature; this approach can prevent implant failure (contraction) and contributes to innervation by improving the symptoms. 1

State of the art

For in situ regeneration and tissue growth, there is a need to use temporary porous three-dimensional (3D) matrices " scaffolds ". Three-dimensional porous matrices with special design, degradability and adjustable mechanical properties, biocompatibility and ability to sustain dense cell populations have been developed. The function of the three-dimensional porous matrix is temporary, but crucial to the success of the repair / regenerative methodology. Although the choice of the type of biomaterial is of great importance, the need to develop a porous three-dimensional matrix with a suitable architecture, especially with regard to porosity, pore size and interconnectivity are also factors that will play a critical role in the formation of fabric. The surface properties of porous three-dimensional matrices will also dictate the response of the cells and eventually adjust their behavior, as well as promote cell adhesion, orientation and proliferation. However, the equilibrium between porosity / mechanical performance of porous three-dimensional matrices is an issue that has also been investigated. The strategy of implanting porous three-dimensional arrays incorporating cells is considered relevant since it allows a rapid colonization of the cells / vascularization of the tissue to be repaired. But, if rapid and random vascularization is desirable in addressing bone repair / regeneration, for example, this may be a disadvantage to other avascular or complex tissues (e.g., meniscus, intervertebral discs, and cartilage), and therefore, should be avoided. The healthy articular cartilage and the nucleus pulposus in the intervertebral disc are devoid of blood vessels, 2 whereas the formation of vessels of pathological sanquines has been shown to increase the degeneration of both tissues. The meniscus is a more complex tissue, i.e. it is partially vascularized and vascularization is observed only in the " red " and " red-to-white ". In normal tissue, the blood vessels are not present inside the meniscus (white zone).

Hydrogels have attracted much attention for their ability to swell and retain large amounts of water, to thereby provide a highly hydrated microenvironment (which resembles the extracellular matrix, MEC) necessary for cell encapsulation, culture and survival. In addition, they can be injected and gelled in situ. Through adjustments in the crosslinking it is possible to control the rate of degradation and permeability to solutes and cells, among other properties of the hydrogels. Various hydrogels have been used to prepare supports for tissue engineering. Gelatin hydrogels (HSG) loaded with fibroblast growth factor-2 (FGF-2) were studied for meniscus repair in a rabbit model (1). This study showed that HSG incorporating FGF-2 significantly stimulated proliferation and inhibited the death of meniscus cells for up to 4 weeks, thereby increasing the cell density at the meniscus and improving its repair. Another study (2) on the replacement of the meniscus by a hydrogel showed a significant degeneration of cartilage and implant failure at 12 months, and overall performance was inferior when compared to allograft transplantation. Improvements in the properties of the hydrogel material, design and surface characteristics have advanced as a means of improving the results and as a way of allowing an exact match of size. Control of innervation and angiogenesis during tissue repair may contribute to the improvement of symptoms. Treatment with a matrix metalloproteinase inhibitor has been shown to reduce joint damage, osteochondral angiogenesis and evidence of pain behavior (3). The association between osteochondral angiogenesis and pain behavior has been related to perivascular nerve growth or stimulation of subchondral nerves followed by loss of osteochondral integrity. Thus, there is scientific evidence that the targeting of angiogenesis may have utility in the treatment of pain associated with structural damage in diseased / damaged tissues. Matrigel has been used in research as a positive control for angiogenesis. Non-angiogenic hydrogels have been reported for tissue engineering applications (4, 5). Cellulose-based hydrogels and chitosan have been shown to lack angiogenic potential, whereas hydrogels encapsulating human mesenchymal stem cells (CEMhs) hydrogels increase the angiogenic response on the chorioallantoic membrane (MCA) of the assay. Albumin gel (GA) crosslinked with poly (ethylene glycol), supplemented with hyaluronic acid as the matrix for the implantation of autologous chondrocytes, was also proposed. This study showed that GA gel prevented endothelial invasion, in vitro. The MCA assay revealed that the AG gel acted as a barrier to the blood vessels. In a recent study (6), the angiogenic response of gellan gum (GG), gellan-ionic gum (iGG-MA) and photochromic gellan 4 photochromic gum (phGG-MA) gels was also investigated through an MCA assay . Analysis of macroscopic convergence of blood vessels demonstrated that gellan-based hydrogels are non-angiogenic compared to positive controls. In addition, it was observed by immunohistochemistry that the iGG-MA and phGG-MA hydrogels do not allow growth into chick endothelial cells after 4 days of implantation. Histological data also indicated that GG-based hydrogels do not cause any acute inflammatory response. The study clearly demonstrated that GG-based hydrogels were permeable to cells, whereas the iGG-MA and phGG-MA hydrogels prevented infiltration of both cells and blood vessels.

So far, no scientific papers have been reported on the development of porous three-dimensional support structures " scaffold " (s) in combination with non-angiogenic hydrogels to be used as biodegradable matrix (s) which are simultaneously capable of controlling the infiltration of segmental cells, innervation and growth of blood vessels. Several patents were granted based on the use of three-dimensional porous matrices for different applications. The following examples should be considered based on their importance in the domain (s) of the present invention:

WO 2012/021108 of February 12, 2012 relates to a biodegradable ocular implant for the controlled release of a drug comprising a first layer containing a first biodegradable polymer wherein the first layer incorporates a dispersed drug or dissolved. The multi-layer ocular implant is also described herein.

WO patent 2012/020412 of February 12, 2012 relates to a method for obtaining a fibrin matrix as a carrier for mesenchymal stem cells (MSCs) and / or umbilical tissue derived cells (UTC) at the site of administration, over an extended period of time. The scaffold preparation is made with a fibrinogen component which comprises fibrinogen, the matrix and the incorporated MSCs and / or UTCs can be used to increase vascularization and / or wound healing.

WO 2012/003370 of January 5, 2012 relates to biocompatible polysaccharide hydrogels that allow functional vascularization. The present invention shows a synergistic effect of multiple angiogenic growth factors (FG); co-encapsulation of VEGF with other FGs induces more and larger blood vessels than any individual GF. In addition, the combination of all FG increased the size and number of newly formed vessels functional.

EP 2388020 of November 23, 2011, the invention relates to a matrix, method for the preparation, wherein the matrix is a mixture of bone powder and fibrin glue.

WO 2011/119059 AI of September 29, 2011 relates to the photo-crosslinked hydrogel materials based on methacrylated gellan gum (GG-MA), suitable for tissue engineering and regenerative medicine applications or as drug delivery systems . The disclosed biomaterials allow to adjust the physico-chemical and biological properties according to the specific use and allow to control the cross-linking process while supporting the encapsulation of human and animal cells and / or drugs, as well as any combination thereof. Different types of three-dimensional porous matrices can be produced using manual or automated systems and their application as acellular or cellular system can be done by manual or automatic injection followed by crosslinking directly at the application site.

WO 2010/099463 of September 2, 2010 relates to medical compositions and devices i.e. biodegradable porous three-dimensional matrices for repair and replacement of cartilaginous meniscus. The invention includes, in certain situations, three-dimensional porous matrices that can promote tissue regeneration of a meniscus of the temporo-mandibular joint.

EP 2197504 of June 23, 2010 relates to biomaterials which allow the growth of directional tissue comprising soluble fibers having a variable cross-sectional area, which dissolve in a directional mode in situ. The direction and speed of microchannel formation within the biomaterial may be useful for directing the growth of blood vessels, nerves and other repair cells through the biomaterial in defined directions.

WO 2010/057142 of May 20, 2010 discloses compositions and methods for obtaining poly (ethylene glycol) (PEG) modified silk fibroin. Silk attached to PEG is intended to find applications as anti-adherent and anti-thrombotic material. Since it exhibits different surface properties within the matrix, the association with bioactive agents can enhance tissue growth into the structure on the side of the adherent matrix.

WO 2009/133532 of November 05, 2009 relates to a method for obtaining regenerated silk fibroin solution (s), a fibroin material and an implant which can be used for cartilage repair.

WO 2009/101518 of August 20, 2009 relates to the use of GG for regenerative medicine and for tissue engineering strategies. The present invention concentrates on the use of GG, in processing methods and in devices. In connection with this patent, the GG undergoes ionic crosslinking in the presence of a phosphate buffer, an acidic or alkaline solution.

U.S. Patent 11/285 928 of July 14, 2009 relates to a method for the in situ treatment and repair of a meniscal injury, laceration or lesion limited to the white (avascular) area. The method comprises the following steps: (i) arthroscopic surgical intervention, (ii) determination of the size and extent of meniscus injury, laceration, or injury, (iii) filling of the defect area with the hydrogel complex consisting of collagen (iv) gelation of the hydrogel in situ, and has an area of injury or laceration together during a healing period and induces cell migration and formation of the extracellular matrix, and (v) the suture of the arthroscopic and adherent incision of said hydrogel to the surrounding tissue and gluing said collagen matrix to said surrounding meniscus tissue.

WO 2008/138000 of November 13, 2008 refers to three-dimensional spongy apoptotic epithelial matrices. The supports are intended to find application in promoting the re-vascularization of the damaged tissues. Porous three-dimensional matrices are isolated or ex vivo sorted tri-cell tissues. Also provided are methods of promoting wound healing and wound re-vascularization by contacting the injured tissue with the matrices.

WO 2007/020449 of February 22, 2007 and WO 2006/030182 of March 23, 2006 disclose a cartilage tissue repair device with a biocompatible, resorbable or other fiber three-dimensional silk and a substantially porous resorbable biocompatible hydrogel of silk or other hydrogel partially or substantially filling the interstices of the fiber, with or without a means of firmly anchoring the device in the bone of a patient. The 3D silk scaffold is obtained by lyophilization and the hydrogel by gelling in the presence of PBS (ionic gelling). A mineralizing silk gel is obtained by combining the silk hydrogel with a solution of CaCl2 ·

WO 2001/087575 of November 22, 2001 describes a method for obtaining porous, polymeric and biodegradable (natural or synthetic) three-dimensional matrices with well-controlled interconnectable pores, and a method for forming porous materials. Biodegradable polymers (PLLA and PLGA) were dissolved in a solvent 9 and cast onto the negative replicate. Thereafter, the polymeric porous scaffold is obtained by dissolution / leaching of the porogen. Porous three-dimensional matrices can be used in different tissue engineering applications. Most intellectual property related to vascularization control, i.e. either increasing or inhibiting it, utilizes bioactive molecules, such as drugs, peptides, GFS or antibodies. Hydrogels when used alone in the repair of meniscal tissue have been shown to allow inward cell growth, infiltration of blood vessels thus leading to poor repair results of the defect in long-term evaluations. The stents were developed to find applications in vascular occlusion and treatment of aneurysms. Biodegradable implants have also been described for ocular applications. The present invention relates to the development and method of producing a porous three dimensional support structure comprising a matrix of biodegradable polymer and non-or anti-angiogenic hydrogel (s) for the control of segmental vascularization , innervation and growth within their cells, which may be beneficial in the regeneration of complex tissues, such as the meniscus, intervertebral disc and in the treatment of osteochondral lesions.

The present invention relates to the development and method (s) for the production of porous (s) and implantable (s) three-dimensional structure (s) which enables to control cell infiltration and proliferation, vascularization and segmental innervation. The structure (s) are characterized by containing a porous polymeric support matrix and a non-angiogenic gel (s) (in the gelling can occur in situ by means of both photocrosslinking or ionic). The porous three-dimensional structure (s) may be used as the implant (s). Said hydrogel does not allow the adhesion of endothelial cells and prevents the infiltration of blood vessels; can be sutured; the gelation of the hydrogel can occur in situ. The carrier porous polymeric matrix is preferably a polymer, which may be natural or synthetic, preferably biodegradable or bioabsorbable. The hydrogel is preferably gellan gum, more preferably gellan gellan hydrogel (meth) which may be either ionic or photopolymerized. The implantable structure (s) may be used for a variety of applications, including the manufacture of partial or complete implant (s) for meniscal tissue engineering and as porous three-dimensional - or multi-layers for the treatment of complex tissues (eg, osteochondral defects). The described implant (s) are particularly suitable for the treatment of degenerative diseases or lesions, in which the patient's natural response is not able to repair the defect.

Brief Description of the Figures

The following figures provide preferred examples to illustrate the description and should not be construed as limiting the scope of the invention. Figure 1 illustrates photographs of: (A) porous polymeric support matrix " scaffold " 12% silk fibroin, (B) micrographs obtained by scanning electron microscopy of the 12% silk fibroin carrier porous polymer matrix, and (C) implantable porous three-dimensional structure composed of silk fibroin and GG- BAD. Figure 2 shows photographs of: (A) carrier porous polymer matrix " scaffold " (1-represents the silk layer and 2 corresponds to the silk-CaP layer), and respective images of scanning electron microscopy and microcomputer tomography, and (B) structure bi-layer implantable porous three-dimensional membrane comprising the GG-MA-Silk / Silk-CaP hydrogel. Figure 3 shows the stereomicroscopy images of the CAM section corresponding to the implanted materials, after 4 days of implantation: (A) 12% Silk, (B) 12% Silk + iGG-MA, (C) Silk 12 (E) 12% + iGG-MA + VEGF, (D) Silk 12% + meniscus cells, (E) 12% + iGG-MA + meniscus cells, (F) 12% Silk + iGG-MA + meniscus cells + VEGF , (G) Filter paper and (H) Filter paper + VEGF. The implanted materials were produced with a typical diameter of 4 mm. Figure 4 shows the stereomicroscopy images of the CAM section corresponding to the implants after 4 days of implantation: (A) 12% Silk, (B) 12% Silk + iGG-MA, (C) Silk 12% (F) Silk 12% + iGG-MA + meniscus cells + VEGF, (E) 12% + iGG-MA + VEGF, (D) 12% silk + meniscus cells, (G) Filter paper and (H) Filter paper + VEGF. The implanted materials were produced with a typical diameter of 4 mm. Figure 5 graphically depicts ex-ovo quantification of convergent macroscopic blood vessels to implants after 4 days of implantation. The mean number of blood vessels results from counting a minimum of 15 disks per implant performed by three independent observers. (A) Silk 12%, (B) Silk 12% + iGG-MA, (C) Silk 12% + iGG-MA + VEGF, (D) 12% silk + meniscus cells, (E) 12% silk + iGG -M + meniscal cells, (F) Silk 12% + iGG-MA + meniscus cells + VEGF, (G) Filter paper (PF), and (H) Filter paper + VEGF. Figure 6 shows the optical microscopy images of hematoxylin and eosin (H & E) staining of excised CAM sagittal sections corresponding to implants after 4 days of implantation: (A) 12% (S) Silk, (B) Silk 12% + IGF-MA (SGG), (C) 12% Silk + IGF-MA + VEGF (SGG + VEGF)

Meniscal cells (SGGCel), (F) Silk 12% + iGG-MA + meniscus cells + VEGF (SGGCel + VEGF) Silk 12% + meniscus cells (SCel), (E) 12% , (G) Filter paper (PF), and (H) Filter paper + VEGF (PF + VEGF). The arrows indicate infiltrated cells. Figure 7 illustrates the optical microscopy images of the immunohistochemical staining of excised CAM sagittal sections corresponding to the implants after 4 days of implantation: (A) 12% (S) Silk, (B) 12% Silk + iGG-MA (SGG),

(C) Silk 12% + iGG-MA + VEGF (SGG + VEGF), (D) 12% Silk + meniscus cells (SCel), (E) 12% Silk + iGG-MA + meniscus cells (SGGCel), (F) Silk 12% + iGG-MA + meniscus cells + VEGF (SGGCel + VEGF), (G) Filter paper (PF), and (H) Filter paper + VEGF (PF + VEGF). The sections were stained with Lectin-SNA specific for endothelial cells (brown) and the images obtained by light microscopy. The arrows indicate infiltrated cells.

Detailed Description of the Invention The present invention relates to the development and methods used for the manufacture of porous and implantable three-dimensional structure (s), including a porous biodegradable carrier polymer matrix and non- angiogenic (s), and their use in the field of tissue engineering and regenerative medicine. This invention describes the method (s) for the production of new porous three-dimensional structures for application as partial or total implants, and which provide segmental vascularization and control of innervation. By this means it can: 1) promote tissue integration and regeneration; 2) avoid implant shrinkage; and 3) to allow pain management. The preparation of the implantable support structure (s) is not limited to the examples used of the polymer matrix, hydrogel, porogen (s), and the method (s) and technique (s) applied. The implantable structure (s) may also be used alone or in combination with different cell types and / or other combinations intended to restore, maintain or enhance tissue functions. This system further allows conjugation with bioactive molecules and therefore, can be used for controlled release.

The following particular examples complement the description of the present invention, and are intended to provide a better understanding thereof, although these examples are not to be considered as being of a restrictive nature.

Production of the implantable support structure (s): method 1

Step 1. Silk polymer matrix production by salt leaching / lyophilization techniques The extraction of silk fibroin from Bombyx mori was carried out by boiling the cocoons in a solution of sodium carbonate (0.02 M) for 60 minutes followed by washing with distilled water. Purified silk fibroin was dissolved in a solution of lithium bromide at 9.3 mol / L at 70 ° C for 60 minutes. The solution was then dialyzed against distilled water using a benzoylated dialysis membrane (cut molecular weight: 2000) over the 48 hour period. Then, the dialysed solution of silk fibroin was concentrated by dialysis in a solution of 20% (w / w) poly (ethylene glycol) (20,000 g / mol) for 9 hours. Finally, the dialysis membrane was carefully washed in distilled water, and the concentrated silk fibroin solution was collected into a vial. The final concentration of concentrated silk fibroin was determined by measuring the dry weight of the silk fibroin solutions. The obtained silk fibroin solution was stored at 4 ° C until further use. The concentrated silk fibroin solution was diluted to 10%, 12% and 16% (w / w), respectively. Silk fibroin structures were prepared by adding 2.0 g of sodium chloride particles (500-1000 μm) to 1.0 ml of silk fibroin solution in a 9 mm diameter silicone tube. After drying the structures in air for 2 days, the sodium chloride particles were extracted in distilled water for 2 days. The surface film of the structures was removed using a stainless steel punch. Silk polymer matrices were finally obtained (Figure 1A-B) after freezing at -80 ° C, followed by lyophilization in a lyophilizer.

The support structure (Figure 1C) was produced by combining the porous silicon polymer matrix with the ionically crosslinked gellan gellan hydrogel (iGG-MA) MA) using a suitable mold (steel or silicone), having a shape and size corresponding to the area of the tissue defect to be repaired / regenerated. The GG-MA gel was obtained by dissolving the GG-MA powder in distilled water at a final concentration of 1-2% (w / w) under vigorous stirring and at room temperature for 1-2 hours. Then, an appropriate volume of the GG-MA solution was added to the mold, and the porous silicone polymer matrix immersed in the gel. This step allows the total or partial impregnation of the polymer matrix with the gel, depending on the volume of gel that is used. The uniform dispersion of the gel can be best achieved under vacuum as it also prevents blistering. GG-MA gelation was performed by immersion in PBS (pH 7.4) for 15 minutes (minimum time). Finally, the support structure including the porous silicon polymer matrix and the iGG-MA hydrogel was removed from the mold. The structure (s) may be used and / or find subsequent application after its preparation. The carrier structure may also be frozen at -80øC and lyophilized to remove the solvent (s). Prior to gelation of the hydrogel or after lyophilization of the structures, it is possible to incorporate different types of bioactive cells or molecules into the different parts of the structure.

GG-MA hydrogel formation in the implantable structure (Photo-crosslinking, phGG-MA) - As reported for the combination of the porous polymer silicon matrix and the iGG-MA hydrogel, a similar mold was used (steel or silicone ) for the production of a structure combining the same silk polymer matrix with photoglycated gellan hydrogel gellan (phGG-MA). The phGG-MA gel was also produced from the GG-MA powder, which was dissolved in distilled water to a final concentration of 1-2% (w / w) under vigorous stirring at room temperature for period of 1-2 hours. Upon complete dissolution, the methyl benzoylformate (MBF) photoinitiator is added to a final concentration ranging from, preferably, 0.05 to 0.1% (w / w). Thereafter, after dissolution of the photoinitiator (15 minutes), a suitable volume of the blend was transferred into a mold, and the porous polymer silica matrix immersed in the gel. This process is essential for partial or complete impregnation of the silk polymer layer with the hydrogel solution. Uniform dispersion of the gel can be achieved most effectively under vacuum. The hydrogel crosslinking was performed by exposing the gel to UV light for 5-10 minutes. The structures were then stabilized by immersion in PBS (pH 7.4). Finally, the support structure including the porous silicon polymer matrix and the phGG-MA hydrogel was removed from the template. The structure (s) may be used and / or find subsequent application after its preparation. The carrier structure may also be frozen at -80øC and lyophilized to remove the solvent (s). Before gelation of the hydrogel or after lyophilization of the structures, it is possible to incorporate 17 different types of cells or bioactive molecules into the different parts of the structure.

Production of the implantable support structure (s): Method 2

Step 1. Production of Silica / Silk-CaP bi-layer polymer matrix by salt leaching / lyophilization - The silk fibroin and calcium phosphate composite was prepared using an in situ method. Initially, the concentrated silk fibroin solution (prepared as described in Method 1) was diluted to 16% (w / w). Different amounts of a calcium chloride solution (6 mol / L) were mixed with the silk fibroin solution, followed by addition of different amounts of a solution of dibasic ammonium phosphate (3.6 mol / L). The theoretical atomic ratio of calcium to phosphate was maintained at 1.67 in each group. The pH value of the system was adjusted to 8.5 by addition of ammonia (30%). The suspension was allowed to stir for 30 minutes and then allowed to stand for 24 hours at room temperature. Silica / CaP composites with a theoretical CaP content (theoretical CaP mass divided by the total mass of silk fibroin) of 4, 8, 16 and 25% (w / w) were prepared. The preparation of the sodium chloride granules was performed by screening sodium chloride using sieves in the range of 500-1000 Âμm. The silk / CaP (bottom layer) structures were prepared in a silicone tube (internal diameter: 9 mm) by the addition of sodium chloride granules (500-1000 Âμm) to the silk / CaP suspension in the proportion of 1 g of sodium chloride to 1 ml of silk fibroin solution; followed by drying the material inside the silicone tube at room temperature for 2 days. The silk structures (top layer) were obtained by the addition of 18 silk fibroin solutions on the silica / CaP / NaCl structure, followed by the incorporation of sodium chloride particles (500-1000 Âμm), in the proportion of 1 g of sodium chloride to 1 ml of silk fibroin solution. Silica / CaP bi-layer polymer matrices were air-dried for 48 hours, followed by leaching of sodium chloride in distilled water for 36 hours. The surface film of the structures was removed using a stainless steel punch, and the bi-layer polymer matrices (Figure 2) obtained after freezing at -80øC, followed by lyophilization.

Step 2. Formation of the GG-MA hydrogel in the implantable structure (cartilage-like layer) - The implantable bi-layer structure was produced by combining the silk / silk-CaP bi-layer matrix and hydrogel (s) ) of GG-MA, and using a suitable mold (steel or silicone) of a shape and size corresponding to the area of the tissue defect to be repaired / regenerated. The GG-MA gel was obtained by dissolving the GG-MA powder in distilled water at a final concentration of 1-2% (w / w) under vigorous stirring and at room temperature for the period of 1-2 hours . To produce the bi-layer structure, the porous matrix silk-fibroin layer in silk / silk bi-layer CaP was immersed in the GG-MA gel within the mold. This step allows the partial impregnation of the silk polymer layer. Thus, the uniform dispersion of the gel can be achieved most effectively under vacuum. The gelation of iGG-MA was carried out by immersion in PBS (pH 7.4) for 15 minutes (minimum time).

For the production of an implantable multilayer structure (s), another layer of GG-MA gel may be added to the silk / silk-CaP bi-layer structure 19 following the procedure described in method 1 step 3 (photo-crosslinking). Thus, bi-or multi-layer structures (s) including a porous polymer matrix and different layers of GG-MA hydrogels can be obtained. The implantable structure (s) can be used and / or found after application. The implantable structure (s) may also be frozen at -80 ° C and lyophilized to remove the solvent (s). Prior to gelation of the hydrogels or after lyophilization of the structures, it is possible to incorporate different types of bioactive cells or molecules into the different layers of the framework (s).

For in vitro cell encapsulation and in vivo use, the porous silicone polymer matrix and the GG-MA powder are pre-sterilized. The implantable structure (s) may be produced under sterile conditions prior to use, i.e., using sterile materials and reagents within a laminar flow chamber, for example.

In vivo study of the angiogenic / anti-angiogenic potential of combined cellular and cellular silk / GG-MA structures (human meniscus cells)

Step 1. Chicken egg chorioallantoic membrane (CAM) assay - The CAM assay was performed to investigate whether the combined silk / GG-MA structures are able to control the growth of the cells into their interior , segmental vascularization and innervation. Fertile chicken eggs (n = 90-120) were incubated at 37øC for 3 days. After this period a circular window was cut in the bark in order to evaluate the viability of the embryo and allow access to CAM. The opening in the shell was sealed with clear adhesive tape to prevent dehydration and the eggs were again incubated at 37 ° C until the 10th day of embryonic development.

The different sterile disks (n = 10, Table 1) produced according to method 1 (steps 1 and 2) were implanted in the CAM on the 10th day of embryonic development under sterile conditions and the assay continued until the 18th day of development embryonic. These support structures are derived from a solution of 12% (w / w) silk fibroin (silk-12) alone or in combination with iGG-MA hydrogel and / or human meniscus cells. Silk-12 + iGG-MA and silk-12 + iGG-MA + meniscal cells were also tested in the presence of VEGF (100 ng). The samples were produced with a diameter of 4 mm and a height of approximately 3 mm. Sterile filter paper discs (diameter 4 mm) were used as controls and also implanted in CAM at 10 ° embryo development (n = 10). Filter paper disks were used without (PF) or with the addition of 100 ng of VEGF (PF + VEGF). After two days, 100 ng of VEGF was added to the PF + VEGF control group under sterile conditions.

Table 1. Description of the implantable porous three-dimensional silicon-based structures produced according to method 1 (steps 1 and 2), and tested in the CAM model.

Type of biomaterial Sample name Silk-12 Acellulate S Silk-12 + iGG-MA SGG Silk-12 + iGG-MA + VEGF SGG + VEGF 21

Silica-12 + Cells of the meniscus Cellular Scel Silk-12 + iGG-MA + SGGcell Cells Silk-12 + iGG-MA + SGGcel Cells + VEGF Meniscus + VEGF Filter Paper PF Controls Filter Paper + VEGF PF + VEGF

Implant images on CAM were obtained in ovo at the end of the assay (Figure 3). Embryos and respective membranes were then fixed in ovo by addition of a small volume of 4% (w / w) paraformaldehyde followed by incubation at -80 ° C for 10 minutes. Implanted materials and portions of the CAM immediately underlying them were dissected and transferred to 12-well plates. Ex-egg images were then captured for each structure implanted in the CAM (Figure 4). The excised membranes were processed for histological and immunohistochemical analysis. Three independent CAM trials were performed. It can be seen from Figures 3 and 4 that no evidence of material absorption was observed, although an integration of all silk-based structures in the CAM was detected.

Step 2. Quantifying Convergent Blood Vessels - The macroscopic assessment of angiogenic response was performed using a semi-quantitative method, by ex-ovo analysis of blood vessel convergence for implanted discs, as described by Ribatti et al. (7). The ex-egg images obtained at the 14th day of embryonic development were processed using the WCIF ImageJ program to facilitate the counting of the total number of blood vessels converging to the implanted disk. For quantification, the magnification of the stereomicroscopic images was kept constant (7x), as well as the processed image area (800 x 800 pixels). The total number of macroscopic microscopes converging to the implant was quantified for each egg by three independent observers (Figure 5). Statistical analysis was performed by applying a variance analysis &quot; one-way &quot; followed by Tukey's multiple comparison test (p <0.05 for a 95% confidence interval). Macroscopic quantification of blood vessels (Figure 5) revealed that there are no statistically significant differences in relation to the number of macroscopic blood vessels for silk-based implants. No significant differences were also detected in the number of converging blood vessels for the filter paper controls (with and without VEGF). As previously demonstrated by Silva-Correia et al. (6), the filter paper has a positive effect on angiogenesis and, therefore, it can be considered that the porous silicon-based structures also promote the induction of angiogenesis. This effect was observed mainly in silk-based structures without iGG-MA incorporation. When the hydrogel was combined with the 12-fold structure (SGG, SGG + VEGF, SGGcel and SGGcel + VEGF structures), an inhibitory effect of angiogenesis was observed, as demonstrated by the low number of convergent blood vessels. Although the addition of iGG-MA appears to inhibit the formation of macroscopic blood vessels converging to the porous structures, the observed differences are not statistically significant. In this assay, it was also observed that the addition of VEGF to the SGG and SGGcel structures did not influence the amount of blood vessels converging to the implanted materials. Likewise, when added to the filter paper, this molecule did not significantly induce blood vessel proliferation.

Step 3. Hematoxylin-eosin staining and immunohistochemical analysis - Sections of the 3 pm thick CAM were stained with H & E. Immunohistochemical analysis was also performed on representative sections of the CAM containing the implanted material, using the primary SNA-lectin antibody specific for chicken endothelial cells. The sagittal histological sections of the samples were observed by light microscopy. Immunohistochemical analysis revealed that porous three-dimensional silk-based matrices did not give rise to any acute inflammatory response (Figure 6). It can be seen from Figures 6 and 7 that the three-dimensional silk matrices are highly susceptible to cell growth for their interior and to the formation of blood vessels. The porous nature of the structures is highly effective in facilitating cell infiltration. Silk-12 structure, when combined with the iGG-MA hydrogel (Figures 6 and 7-B, C, E and F), has a lower number of endothelial cells invading the interior. As expected, the iGG-MA hydrogel acts as a physical barrier to vascular invasion and, thus, the CAM cells are confined to the CAM / hydrogel interface and do not penetrate the tissue structure. There was no effect of VEGF incorporation on the silk-based structures (Figures 6 and 7). However, it is possible to observe an increase in the number of microscopic blood vessels in the filter paper group, promoted by the addition of VEGF.

The filter paper controls are intensely colonized by cells, as previously demonstrated (6). An increase in cell density and number of cells infiltrated at the CAM / filter paper interface was also observed, and this was much more evident in the VEGF + filter paper group, which was completely invaded by endothelial cells. Higher microscopic blood vessel density was clearly observed in the CAM tested with the groups which included filter paper compared to the other silk-based groups. Under all conditions tested, no granulation tissue formation was detected.

References 1. Narita A, Takahara M, Sato D, Ogino T, Fukushima S, Kimura Y, et al. Biodegradable gelatin hydrogels incorporating fibroblast growth factor 2 promote healing of horizontal tears in rabbit meniscus. Arthroscopy. 2012; 28 (2): 255-63. 2. Kelly BT, Robertson W, Potter HG, Deng X-H, Turner AS, Lyman S, et al. Hydrogel meniscal replacement in the sheep knee. Am. J. Sports Med. 2007; 35 (1): 43-52. 3. Mapp PI, Walsh DA, Bowyer J, Maciewicz RA. Effects of a metalloproteinase inhibitor on osteochondral angiogenesis, chondropathy and pain behavior in a rat model of osteoarthritis. Osteoarthritis and Cartilage. 2010; 18 (4): 593-600. 4. Ahmadi R, Burns AJ, of Bruijn JD. Chitosan-based hydrogels do not induce angiogenesis. J. Tissue Eng. Reg Med. 2010; 4 (4): 309-15. 5. Scholz B, Kinzelmann C, Benz K, Mollenhauer J, Wurst H, Schlosshauer B. Suppression of adverse angiogenesis in an albumin-based hydrogel for articular cartilage and 25 intervertebral disc regeneration. Eur Cell Mater. 2012; 20: 24-37. 6. Silva-Correia J, Miranda-Gonçalves V, Salgado AJ, Sousa N, Oliveira JM, Reis RM, et al. Angiogenic potential of gellan gum-based hydrogels for application in nucleus pulposus regeneration: In vivo study. Tissue Eng. Part A. 2012; 18 (11-12): 1203-12. 7. Ribatti D, Nico B, Vacca A, Presta M. The gelatin sponge-chorioallantoic membrane assay. Nat. Protoc. 2006; 1 (1): 85-91.

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Claims (16)

Porous three-dimensional structure characterized in that it comprises a porous polymeric matrix of structural support and a hydrogel material which allows controlling the spatial distribution of the blood vessels within the implant and / or implanted cells. The porous three-dimensional structure obtained according to claim 1, characterized in that it has a hydrogel material within the supporting porous polymer matrix, or surrounds the supporting porous polymer matrix, or adjacent the porous polymer support matrix, preferably within of the carrier porous polymer matrix. The porous three-dimensional structure obtained according to claim 1-2, characterized in that the biomaterial is a polymer, which may be natural or synthetic, preferably of silk fibroin, more preferably 10 to 16% (by weight). The porous polymer matrix obtained according to claim 1-3, characterized in that the matrix is monolayered silk fibroin, preferably 40-95% porosity. The carrier porous polymeric matrix obtained according to claim 1-4, characterized in that the polymer matrix is a carrier of bi- or multi-layers of silk fibroin and silk fibroin combined with calcium phosphates, preferably 1 preferably comprising a second of a different layer of calcium phosphate / fibroin fibroin layers, plus silk and silk. The carrier porous polymeric matrix obtained according to the preceding claim, characterized in that the calcium phosphate / silk fibroin layer consists of 1 to 25% (by weight) of calcium phosphate, preferably the phosphate concentration of calcium to be 16%. The hydrogel material obtained according to claim 1-2, characterized in that the material is a gellan gum or its derivatives, and the concentration is from 0.01 to 4% (w / v), preferably 1 or 2% (w / v). The hydrogel material obtained according to claims 1-2 and 7, characterized in that said hydrogel material can be polymerizable by said ionic cross-linking using mono and divalent cations, preferably sodium, calcium and magnesium. The hydrogel material obtained according to any of claims 1-8, characterized in that it further comprises a second light-curing gellan gum hydrogel. Composition of the three-dimensional polymer structure obtained according to claims 1-9, further comprising an active biological agent or a therapeutic agent, preferably sterile. Pharmaceutical composition obtained according to the preceding claim, characterized in that the active biological agent is a cell, stem cell, protein (s), a therapeutic agent, a biomolecule, platelet rich plasma, diagnostic marker and probe / label, or a mixture of these. A process for obtaining the three-dimensional polymeric structure, characterized in that it comprises the porous polymeric support matrix and the hydrogel material, and is implantable after cross-linking or crosslinking occurs in situ, according to claims 1-11. A method characterized in that it comprises administering to a subject in need thereof the three-dimensional polymer structure and composition, which allows controlling the infiltration of blood vessels and the distribution of the active agent / biological or therapeutic molecule (s), according to the invention. claims 1-12. A method characterized in that the administration is implantation, preferably using minimally invasive surgical techniques, according to the preceding claim. A method characterized by controlling the healing and infiltration of blood vessels enhances the regeneration of complex tissues or organs and prevents premature failure of the implant (s) according to claims 1-14. The use according to claim 15, characterized in that it is preferably for the treatment of meniscal, intervertebral disc, and osteochondral defects. Lisbon, March 25, 2013 4