Process for obtaining a neuroprotective conduit with controlled release of bioactive molecules for nerve regeneration, neuroprotective conduit and uses thereof

Abstract

The present invention relates to a process for obtaining a biocompatible neuroprotective conduit with controlled release of bioactive molecules for nerve regeneration, comprising the steps of: 3D printing an NGC and guide tube from PCL biomaterial; sterilising the NGC; preparing GelMA functionalised with the growth factor FGF-2; inserting the guide tube into the NGC; applying GelMA functionalised with the growth factor FGF-2; photocrosslinking with a UV light head; and removing the guide tube. Furthermore, the present invention also relates to a biocompatible neuroprotective conduit with controlled release of bioactive molecules for nerve regeneration and to uses thereof.

Description

[0001] “PROCESS FOR OBTAINING A NEUROPROTECTIVE CONDUIT WITH CONTROLLED RELEASE OF BIOACTIVE MOLECULES FOR NERVE REGENERATION, NEUROPROTECTIVE CONDUIT AND USES” FIELD OF THE INVENTION [1] The present invention relates to a process for obtaining a biofunctional, biocompatible and biodegradable neuroprotective conduit that releases fibroblast growth factor-2 (FGF-2), a thermostable recombinant human protein, in a controlled manner. [2] The present invention falls within the fields of biotechnology, materials engineering and regenerative medicine. More specifically, it refers to neuroprotective conduits for the recovery of damaged nerves, obtained through the combination of the synthetic biomaterial polycaprolactone (PCL), natural methacrylated gelatin (GelMA) and FGF-2. BACKGROUND OF THE INVENTION [3] In the treatment of nerve injuries, the state-of-the-art alternatives encompass various surgical and therapeutic approaches.Neurorrhaphy and the use of autografts are commonly employed for nerve lacerations with short defects. This involves approximating the nerve stumps with sutures, but when tension-free neurorrhaphy is not feasible, alternative techniques such as tubulization or nerve grafts are used to provide support until axon reconnection. However, this approach has limitations, including the need for a donor nerve, long surgical times, and the lack of donor nerve tissue in some cases. [4] Another option is nerve conduits with bio-tissues, which use fresh tissues to create a bandage, reduce scar formation, and improve nerve regeneration in nerve crush injuries. However, the efficacy and safety of these bandages have not been fully proven by clinical studies, and the limited availability of these tissues may be a barrier.[5] Nerve conduits with natural and / or synthetic biomaterials are used in lesions with longer intervals. These conduits are designed as hollow tubes made of materials such as polyglycolic acid, polyvinyl alcohol, or type I collagen. Despite their flexibility and permeability, these conduits generally do not have bioactive properties and have long biodegradation times, which may require additional surgical interventions for biomaterial removal. [6] Although several alternatives exist for the treatment of nerve injuries, all of them face significant challenges, such as low therapeutic efficacy, the need for donor tissues, and limitations in customization according to lesion size. [7] Exemplary examples of prior art documents highlight the aforementioned deficiencies, such as: [8] Document CN116808308 discloses a drug-loaded composite nerve conduit, as well as a method for its preparation and application.The drug-laden nerve conduit is prepared by combining a biodegradable polymer with graphene oxide and melatonin, in order to solve the technical problems of existing technology, such as relatively low biocompatibility of graphene-derived biomaterials, difficulty of degradation and poor repair effect on nerve tissue damage. [9] In this sense, unlike the present invention, the nerve conduit of document CN116808308 is obtained by an electrospinning solution by mixing an organic solution of a biodegradable polymer, graphene oxide and melatonin, having a continuous release.

[0010] Document US2020376167A refers to a method for preparing a nerve conduit using bioprinting technology and a nerve conduit prepared by the same, being able to easily prepare a nerve conduit simulating a nerve bundle and nerve tissue, and the like, by three dimensional printing bioinks comprising a neuronal regeneration material on one side of a porous polymer structure.

[0011] However, compared to the present invention, document US2020376167A presents greater complexity and time involved in its manufacturing process, which is lengthy and requires multiple steps, resulting in high product variability. Furthermore, this technology depends on the use of processed biological material, both for the hydrogel and for the neuronal cells, which increases the costs and complexity of the process.

[0012] The article entitled “3D printing of functional nerve guide conduits” presents a review of 3D printing technologies for the fabrication of functional NGCs, including inkjet printing, extrusion printing, stereolithography-based printing, and indirect printing. However, the technologies mentioned in this article are limited in the following ways: (a) Inkjet printing: low mechanical strength for multiple layers, limited resolution, and inability to precisely control the directionality and size of the droplets; (b) Extrusion: low resolution, open systems without a sterile cabinet; (c) Stereolithography (SLA): high cost, possible cytotoxicity from photoinitiator and uncured resin residues. Furthermore, the impossibility of combining thermoplastic polymers (PCL) with hydrogels (GelMA) limits it to a few light-sensitive biomaterials; (d) Indirect: customization and limited resolution.Additionally, unlike this article, the present invention does not use live cells and commercially available thermosensitive neurotrophic factors, as these have limitations related to variability in therapeutic efficacy, production time, cost, immune reactions, and lack of stability with temperature variations.

[0013] Thus, it is clear that, although different proposals for neuroprotective conduits have been discussed in the prior art, none of them allows the combination of thermoplastic polymers with hydrogels to deliver FGF-2 to damaged nerve tissues.BRIEF DESCRIPTION OF THE INVENTION

[0014] The present invention relates to a process for obtaining a biocompatible neuroprotective conduit with controlled release of bioactive molecules for nerve regeneration, comprising the steps of: 3D printing of a sterile NGC and guide tube from PCL biomaterial; preparation of GelMA incorporated with sterile FGF-2 growth factor; insertion of the guide tube into the NGC; application of the GelMA functionalized with FGF-2 growth factor; photocrosslinking with a UV light head; and removal of the guide tube. Furthermore, the present invention also relates to a biocompatible neuroprotective conduit with controlled release of bioactive molecules for nerve regeneration and its use. BRIEF DESCRIPTION OF THE FIGURES

[0015] Figure 1 illustrates the detection of the functionalization (DoF – degree of functionalization) of proteins through the loss of free amines via a ninhydrin colorimetric assay.(A) Methacrylic anhydride (MAA) monomers react with lysine and hydroxylysine groups of gelatin. The anhydride reaction occurs primarily with free amines. (B) Scheme of the ninhydrin assay reaction. Ninhydrin reacts with primary free amines to generate the Ruhemann purple product. (C) Image of the gelatin standard curve. The solutions produced a visible color change that corresponded to the free amine content in the solution. (D) Image of the plate after the ninhydrin test in the GelMA solution. The samples produced color changes that inversely correlate with their respective DoF values.

[0016] Figure 2 illustrates the synthesis and purification of the GelMA biomaterial. (A) Chemical illustration of the GelMA synthesis. (B) Illustrates the procedure for diluting type A porcine skin gelatin solution (300 bloom) in DPBS and adding MAA at a rate of 0.5 mL / min with vigorous stirring, forming a dense solution.The GelMA precursor solution was centrifuged, forming a non-reactive MAA pellet. Then, in the purification step, the solution was dialyzed for one week and filtered through 22 µm polystyrene (PES) filters. Finally, the precursor solution was subjected to flash freezing in liquid nitrogen and transferred to the freeze dryer. The freeze-drying product consisted of a white, highly porous, and lightweight foam.

[0017] Figure 3 presents a graph for determining the DoF% of GelMA samples. The standard curve shows linear absorbance of gelatin between 2-8 mg / mL (R = 0.9585; p < 0.0001). The GelMA samples prepared at 10 mg / mL (n=3) exhibited a DoF of 74.06%, 74.34%, and 72.28% (colored dots). Values ​​are represented as mean ± SEM.

[0018] Figure 4 illustrates a 365 nm UV photopolymerization test of GelMA 5% and 10% (wt / vol). (A) Irradiation with a 365 nm UV light source.(B) Samples of GelMA + photoinitiator IC2959 were added to molds and light-cured for 40, 100, 200, 240, and 300 seconds. (C) Back view of the 10% GelMA constructs and (D) back view of the 5% GelMA constructs. Thin edges and structure fidelity are observed from 200s with 10% GelMA. The 5% GelMA constructs subjected to UV light for 40s and 100s were not shown because they did not show physical stability.

[0019] Figure 5 illustrates a 365 nm UV light-curing test of 5% and 10% (wt / vol) GelMA. (A) Front (A) and side (B) views of the GelMA constructs after light-curing with UV light for 40, 100, 200, 240, and 300 seconds. Better fidelity and preservation of the structure was observed from 200s in GelMA 10% and 300s in GelMA 5% in the dorsal and lateral views, respectively. (C) After the 45° tilt. o Adhesion and preservation of the structure were observed from >200s in GelMA 10%. (D) After tilting at 45° oFlow, lack of adhesion, and loss of structure were observed in samples <240s in GelMA 5%.

[0020] Figure 6 illustrates the pH evaluation in GelMA 5% and 10% photocrosslinked samples over 5 days. (A) GelMA constructs were immersed in DMEM basal culture medium and incubated for 120 hours at 37 o C and 5% CO2. (B) Macroscopically, the GelMA constructs showed a reddish coloration indicating absorption of phenol red from the culture medium. (C) Graph showing the pH values ​​measured 24, 48, 72 and 120 hours after incubation. There were no significant changes in pH values ​​between the analyzed groups (p > 0.05).

[0021] Figure 7 illustrates the graphs of the rheological properties of three hydrogel compositions: GelMA 5%, GelMA 10% and GelMA 10% / gelatin 1.25%. (A) Viscosity as a function of shear rate at 22 oC. (B) Viscosity as a function of temperature. (C) Effect of temperature on storage modulus (G') and loss modulus (G”). Values ​​are represented as mean ± SEM. Black arrows indicate the gel-point of each concentration. The circle indicates the proximity of the gel-point of the GelMA 10% and GelMA 10% / gelatin 1.25% concentrations.

[0022] Figure 8 illustrates the rheological analysis of crosslinked GelMA. (A) Storage modulus (G') and loss modulus (G”) of angular frequency (ω). (B) Damping factor (tan (δ)) of angular frequency (ω) (n=2). Values ​​are represented as mean ± SEM.

[0023] Figure 9 illustrates the immunophenotyping of dental pulp-derived stem cells (DPMSCs) for biological assays. (A) DPMSCs with elongated morphology and ~40% confluence were observed 4 days after thawing. (B) Greater ~90% confluence was observed during the 8 days after thawing of the DPMSCs.(C) Gate of human DPMSCs (cell size versus granularity). (BF) Histograms and percentage of expression (red). (D) CD90: 99.57%; (E) CD105: 98.74%; (F) CD73: 99.75%; (G) CD44: 99.10%; (H) Negative cocktail with CD45-, CD34-, CD11b-, CD19- and HLA-DR (0.43%). Scale bar (20µm).

[0024] Figure 10 illustrates the Live / Dead assay of DPMSCs cultured in GelMA 5% and 10% (wt / v). (A) Representative image of 5% GelMA encapsulated with live cells stained green (calcein AM) and dead cells stained red (ethidium homodimer) 2 days after photopolymerization with UV light (240s, ~50 mW / cm. 2 (B) Representative image of 5% GelMA encapsulated with live cells stained green (calcein AM) and dead cells stained red (etidium homodimer) 7 days after photopolymerization with UV light (240s, ~50 mW / cm²). 2(C) Representative image of 5% GelMA encapsulated with live cells stained green (calcein AM) and dead cells stained red (etidium homodimer) 15 days after photopolymerization with UV light (240s, ~50 mW / cm²). 2(D) Cell viability at 2, 7, and 15 days in GelMA 5% (n = 3) (p > 0.05). (EG) Representative image of GelMA 10% encapsulated with cells 2, 7, and 15 days after photopolymerization with UV light (240s, ~50 mW / cm2). (H) Cell viability at 2, 7, and 15 days in GelMA 10% (n = 3). Values ​​are represented as mean ± SEM. p < 0.05*; p < 0.01**; p < 0.001***.

[0025] Figure 11 illustrates fluorescence images from the Live / Dead assay of DPMSCs cultured in GelMA 5% and 10% (wt / v). (A) Representative image of 5% GelMA constructs encapsulated with live cells 2 days after UV light photopolymerization (240s, ~50 mW / cm2). (B) Representative image of 5% GelMA constructs encapsulated with live cells 15 days after UV light photopolymerization (240s, ~50 mW / cm2). The dashed line delimits the surface of the construct. The white arrows indicate the presence of dead cells on day 2. (C) Control constructs in the absence of cells.(D) Representative image of 10% GelMA constructs encapsulated with live cells 2 days after UV light photopolymerization (240s, ~50 mW / cm2). (E) Representative image of 10% GelMA constructs encapsulated with live cells 15 days after UV light photopolymerization (240s, ~50 mW / cm2). The dashed line delimits the surface of the construct and the white arrows indicate the presence of dead cells on day 2. (F) Control construct without cells.

[0026] Figure 12 shows green fluorescence microscopy images of DPMSCs immunolabeled for human mitochondria in 5% and 10% GelMA (wt / v). (A) Representative images of 5% GelMA constructs with immunolabeled cells after 2 days. (B) Representative images of 5% GelMA constructs with immunolabeled cells after 7 days. (C) Representative images of 5% GelMA constructs with immunolabeled cells after 15 days. (D) Representative images of 10% GelMA constructs with immunolabeled cells after 2 days.(E) Representative images of 10% GelMA constructs with immunolabeled cells after 7 days. (F) Representative images of 10% GelMA constructs with immunolabeled cells after 15 days. Positive labeling of human mitochondria is observed, characterized by the presence of filiform structures in the cytoplasm of the cells around the nucleus. Scale bar: 20µm.

[0027] Figure 13 shows the relative gene expression after 3 days of stimulation of DPMSCs with FGF-2 STAB. (A) Relative expression of VEGF. (B) Relative expression of BDNF. Samples were tested for the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (n=3). Data are represented as mean ± SEM. p < 0.05*; p < 0.01**; p < 0.001***.

[0028] Figure 14 shows the quantification of controlled FGF-2 release over 30 days using ELISA. (A) Daily release of FGF-2 over 30 days. Insert in (A): Analysis of daily FGF-2 release in the first 7 days.(B) Cumulative release of FGF-2 within 30 days (n=2). Data were represented as mean ± SEM. Statistical differences were represented by: p < 0.05*; p < 0.01**; p < 0.001***.

[0029] Figure 15 shows the filament fusion test (fidelity of 3D bioprinting) at GelMA concentrations of 5% and 10%. (A) Pneumatic pressures were varied between 18-26 kPa, keeping the temperature constant (22. o(C) Speed ​​(5 mm / s) and bed temperature (ambient temperature) during the bioprinting process. (B) Diffusion rate as it increases the pore size of the scaffold after bioprinting with GelMA hydrogel. (C) Printability value as it increases the pore size of the scaffold after bioprinting with GelMA hydrogel.

[0030] Figure 16 illustrates the scanning electron microscopy (SEM) characterization of GelMA constructs after UV photocrosslinking and lyophilization. (A) GelMA 5% wt / vol 24 hours after lyophilization at 130x magnification. (B) GelMA 10% wt / vol 24 hours after lyophilization at 130x magnification. (C) GelMA 10% wt / vol 24 hours after lyophilization at 130x magnification. (D) GelMA 10% wt / vol 24 hours after lyophilization at 700x magnification. Scale bar 20 and 100 ^m.

[0031] Figure 17 shows the characterization and printability (Pr) evaluation of PCL bandages manufactured by 3D printing.(A) Diagram of the dimensions and dorsal view of the PCL bandage surface by scanning electron microscopy (SEM). (B) Diagram of the dimensions and lateral view of the PCL bandages by SEM. (C) Geometric parameters and Pr of the construct (n = 3).

[0032] Figure 18 presents the characterization and evaluation of the surface of the PCL NGCs. (A) Dorsal view of the external surface of the NGC. (B) Dorsal view of the base and first layer of the NGCs. (C) Dorsal view of the last layer or apex of the NGCs. (D) Cross-section at the apex level of the NGCs showing the internal diameter (ID), external diameter (ED) and wall thickness. (E) Dorsal view of the fractured nerve construct showing the internal surface and filaments. (F) Detail of the spiral filaments on the internal surface of the NGCs. (G) Dimensions of the internal diameter (ID), external diameter (ED) and wall thickness of the conduit (n=3).

[0033] Figure 19 illustrates the biofabrication process of NGCs from the GelMA+FGF-2 hybrid.(A) Experimental design of the NGC biofabrication process. (B) Macroscopic aspect of the NGC and the guide conduit manufactured by 3D printing using a multilayer spiral process. (C) Macroscopic appearance of the NGC after functionalization of the inner wall of the tube with 10% GelMA. (D) Side view showing in detail the preservation of the NGC lumen after removal of the guide tube. (E) Dorsal view of the tubular structure of the PCL+GelMA nerve guidance conduits (NGCs), showing the outer PCL wall and an inner layer of whitish GelMA.

[0034] Figure 20 illustrates the characterization and evaluation of the surface of the PCL+GelMA NGCs. (A) Transverse view of the tubular structure. (B) Enlarged view of the contact between the PCL and the GelMA. (C) Enlarged transverse view showing the microporosity and lumen of the NGC. (D) Dorsal section showing the inner GelMA wall with the presence of microporosity.

[0035] Figure 21 shows the implantation of the PCL biomaterial for biocompatibility assessment.(A) Diagram of the surgical procedure in which the lateral wall of the vertebral bone is removed to access the vertebral canal. (B) Image showing the hemilaminectomy window. (C) Images showing the implanted and fixed PCL membrane with 3 simple sutures. (D) Macroscopic appearance of the surgical field after implantation.

[0036] Figure 22 presents a macroscopic analysis of the vertebrae after biomaterial implantation. (A) Cross-section of a specimen from the control group showing the height of the hemilaminectomy (dotted line) and scar tissue entering the spinal canal slightly (arrows). (B) Cross-section of a specimen from the PCL group showing the height of the hemilaminectomy (dotted line) and the PCL membrane integrated into the adjacent musculature. (C) Lateral view of a specimen from the PCL group showing scar tissue around the PCL membrane without changes related to inflammation. Scale bar; 1 mm.

[0037] Figure 23 presents the quantitative analysis of the vertebrae after biomaterial implantation. (A) Parameters evaluated (B) Comparison of hemilaminectomy height in the control and PCL groups. (C) Comparison of height and length of fibrous tissue adjacent to the hemilaminectomy in the control and PCL groups. (D) Comparison of vertebral canal height and length in the control and PCL groups. Data are represented as mean ± SEM. Statistical differences are represented by: p < 0.05*; p < 0.01**; p < 0.001***.

[0038] Figure 24 illustrates radiographic images of the vertebral column 15 and 30 days after PCL membrane implantation. (A) Ventrodorsal (VD) view at the level of the T13 vertebrae of the control group rats (n=3) 15 days later. (B) Ventrodorsal (VD) view at the level of the L1 vertebrae of the control group rats (n=3) 15 days later. (C) Ventrodorsal (VD) view at the level of the L2 vertebrae of the control group rats (n=3) 15 days later.(D) Ventrodorsal (VD) view at the level of the T13 vertebrae of the control group rats 30 days later. (E) Ventrodorsal (VD) view at the level of the L1 vertebrae of the control group rats 30 days later. (F) Ventrodorsal (VD) view at the level of the L2 vertebrae of the control group rats 30 days later. (G) Ventrodorsal (VD) view at the level of the T13 vertebrae of the PCL group rats (n=3) 15 days later. (H) Ventrodorsal (VD) view at the level of the L1 vertebrae of the PCL group rats (n=3). (I) Ventrodorsal (VD) view at the level of the L2 vertebrae of the PCL group rats (n=3) 15 days later. (J) Ventrodorsal (VD) view at the level of the T13 vertebrae of the PCL group rats 30 days after. (K) Ventrodorsal (VD) view at the level of the L1 vertebrae of the PCL group rats 30 days after. (L) Ventrodorsal (VD) view at the level of the L2 vertebrae of the control group rats 30 days after. LE – left side.

[0039] Figure 25 illustrates radiographic images of the vertebral column 15 and 30 days after PCL membrane implantation.(A) Latero-lateral (LL) view at the level of the T13 vertebrae of the control group rats (n=3) 15 days later. (B) Latero-lateral (LL) view at the level of the L1 vertebrae of the control group rats (n=3) 15 days later. (C) Latero-lateral (LL) view at the level of the L2 vertebrae of the control group rats (n=3) 15 days later. (D) Latero-lateral (LL) view at the level of the T13 vertebrae of the control group rats 30 days later. (E) Latero-lateral (LL) view at the level of the L1 vertebrae of the control group rats 30 days later. (F) Latero-lateral (LL) view at the level of the L2 vertebrae of the control group rats 30 days later. (H) Latero-lateral (LL) view at the level of the T13 vertebrae of the PCL group rats (n=3) 15 days later. (I) Latero-lateral (LL) view at the level of the L1 vertebrae of the PCL group rats (n=3). (J) Latero-lateral (LL) view at the level of the L2 vertebrae of the PCL group rats (n=3) 15 days later. (K) Latero-lateral (LL) view at the level of the T13 vertebrae of the PCL group rats 30 days later.(L) Latero-lateral view (LL) at the level of the L1 vertebrae of the PCL group rats 30 days later. (M) Latero-lateral view (LL) at the level of the L2 vertebrae of the control group rats 30 days later. LE – left side.

[0040] Figure 26 shows an experimental lesion of the sciatic nerve in the Autograft, NGCs and NGCs+FGF2 groups. (A) In the Autograft group, sciatic nerve neurotmesis was performed (critical defect 8 mm), repositioned and repaired with perineural sutures. (B) In the NGCs groups and (C) in the NGCs+FGF2 groups, a critical defect of the sciatic nerve was performed and then the nerve stumps were introduced 1 mm into the NGCs and fixed with perineural sutures. Scale bar 10 mm.

[0041] Figure 27 illustrates a functional nerve index over 12 weeks in the Autograft, NGCs, and NGCs+FGF2 groups. Values ​​were obtained weekly over 12 weeks and are represented as mean ± SEM. p < 0.05*; p < 0.01**; p < 0.001***.

[0042] Figure 28 presents an assessment of the contact area over 12 weeks in the Autograft, NGCs, and NGCs+FGF2 groups. Values ​​were obtained weekly over 12 weeks and are represented as mean ± SEM. p < 0.05*; p < 0.01**; p < 0.001***.

[0043] Figure 29 presents an assessment of base of support over 12 weeks in the Autograft, NGCs, and NGCs+FGF2 groups. Values ​​were obtained weekly over 12 weeks and are represented as mean ± SEM. p < 0.05*; p < 0.01**; p < 0.001***.

[0044] Figure 30 presents an assessment of the regularity index over 12 weeks in the Autograft, NGCs, and NGCs+FGF2 groups. The values ​​were obtained weekly for 12 weeks and were represented as mean ± SEM. p < 0.05*; p < 0.01**; p < 0.001***.

[0045] Figure 31 represents the intensity of hyperalgesia in the experimental groups.Data on the withdrawal threshold (Δ withdrawal threshold) after mechanical pressure on the plantar region show that treatment with autograft and NGCs+FGF2 regulated hyperalgesia in the injured limb predominantly from week 7. The values ​​obtained are represented as mean ± SEM. p < 0.05*; p < 0.01**; p < 0.001**.

[0046] Figure 32 presents an evaluation of total muscle mass, cranial tibial muscle and gastrocnemius. The values ​​obtained are represented as mean ± SEM. P < 0.05*; p < 0.01**; p < 0.001**.

[0047] Figure 33 presents an evaluation of total muscle mass, cranial tibial muscle and gastrocnemius. The values ​​obtained are represented as mean ± SEM. P < 0.05*; p < 0.01**; p < 0.001**.

[0048] Figure 34 represents the immunostaining of the sciatic nerve in the Contralateral, Autograft, NGCs, and NGCs + FGF2 groups. (A) Immunostaining of Schwann cells by S100 protein for 4 weeks in the Contralateral group.(B) Immunostaining of Schwann cells with S100 protein for 4 weeks in the Autograft group. (C) Immunostaining of Schwann cells with S100 protein for 4 weeks in the NGCs group. (D) Immunostaining of Schwann cells with S100 protein for 4 weeks in the NGCs + FGF2 group. (E) Immunostaining of the neurofilament (NF) cytoskeletal protein for 4 weeks in the Contralateral group. (F) Immunostaining of the neurofilament (NF) cytoskeletal protein for 4 weeks in the Autograft group. (G) Immunostaining of the neurofilament (NF) cytoskeletal protein for 4 weeks in the NGCs group. (H) Immunostaining of the neurofilament (NF) cytoskeletal protein for 4 weeks in the NGCs + FGF2 group.

[0049] Figure 35 represents an analysis of pixel intensity after immunostaining with S100 (A) and neurofilament (B) in the Contralateral, Autograft, NGCs and NGCs + FGF2 groups. The values ​​obtained are represented as mean ± SEM. P < 0.05*; p < 0.01**; p < 0.001**.

[0050] Figure 36 represents the immunostaining of the sciatic nerve in the Contralateral, Autograft, NGCs, and NGCs + FGF2 groups. (A) Immunostaining of macrophages using the IBA-2 protein for 4 weeks in the Contralateral group. (B) Immunostaining of macrophages using the IBA-2 protein for 4 weeks in the Autograft group. (C) Immunostaining of macrophages using the IBA-2 protein for 4 weeks in the NGCs group. (D) Immunostaining of macrophages using the IBA-2 protein for 4 weeks in the NGCs + FGF2 group. (E) Immunostaining of the P75NTR neurotrophic factor receptor for 4 weeks in the Contralateral group. (F) Immunostaining of the P75NTR neurotrophic factor receptor for 4 weeks in the Autograft group. (G) Immunostaining of the P75NTR neurotrophic factor receptor for 4 weeks in the NGCs group. (H) Immunostaining of the P75NTR neurotrophic factor receptor for 4 weeks in the NGCs + FGF2 group.

[0051] Figure 37 is an analysis of pixel intensity after immunostaining with IBA-1 and P75. NTRin the Autograft, NGCs, and NGCs + FGF2 groups. The values ​​obtained are represented as mean ± SEM. P < 0.05*; p < 0.01**; p < 0.001**.

[0052] Figure 38 represents a qualitative evaluation of the selective labeling of myelin sheaths using fluoromyelin labeling in the Contralateral, Autograft, NGCs, and NGCs + FGF2 groups. (A) The triple labeling of IBA-1 (green), DAPI (blue), and fluoromyelin (red) is observed at 10x magnification, indicating complete reinnervation of the proximal and distal nerve stumps after repair in the Contralateral group. (B) Triple labeling with IBA-1 (green), DAPI (blue), and fluoromyelin (red) is observed at 10x magnification, indicating complete reinnervation of the proximal and distal nerve stumps after repair in the Autograft group. (C) Triple labeling with IBA-1 (green), DAPI (blue), and fluoromyelin (red) is observed at 10x magnification, indicating complete reinnervation of the proximal and distal nerve stumps after repair in the NGCs group.(D) Triple labeling of IBA-1 (green), DAPI (blue), and fluoromyelin (red) is observed at 10x magnification, indicating complete reinnervation of proximal and distal nerve stumps after repair in the NGCs + FGF-2 group. (E) Triple labeling of IBA-1 (green), DAPI (blue), and fluoromyelin (red) is observed at 10x magnification, indicating complete reinnervation of proximal and distal nerve stumps after repair in the Contralateral group. (F) Triple labeling of IBA-1 (green), DAPI (blue), and fluoromyelin (red) is observed at 40x magnification, indicating complete reinnervation of proximal and distal nerve stumps after repair in the Autograft group. (G) Triple labeling of IBA-1 (green), DAPI (blue), and fluoromyelin (red) is observed at 40x magnification, indicating complete reinnervation of proximal and distal nerve stumps after repair in the NGCs group.(H) Triple labeling of IBA-1 (green), DAPI (blue), and fluoromyelin (red) is observed at 40x magnification, indicating complete reinnervation of proximal and distal nerve stumps after repair in the NGCs + FGF-2 group. Greater organization of nerve fibers with greater myelination is observed in the Autograft and NGCs groups with controlled release of FGF2.

[0053] Figure 39 presents an analysis of the regenerated nerve morphology in the Contralateral, Autograft, NGCs, and NGCs+FGF2 groups. (A) Ultrastructure of the organization and distribution of myelinated axons in the Contralateral experimental group, scale bar 5^m. (B) Ultrastructure of the organization and distribution of myelinated axons in the Autograft experimental group, scale bar 5^m. (C) Ultrastructure of the organization and distribution of myelinated axons in the NGCs experimental group, scale bar 5^m. (D) ultrastructure of the organization and distribution of myelinated axons in the NGCs + FGF2 experimental group, scale bar 5^m.(E) Ultrastructure of the thickness of the myelin sheath and endoneural collagen in the Contralateral experimental group, scale bar 1^m. (F) Ultrastructure of the thickness of the myelin sheath and endoneural collagen in the Autograft experimental group, scale bar 1^m. (G) Ultrastructure of the thickness of the myelin sheath and endoneural collagen in the NGC experimental group, scale bar 1^m. (H) Ultrastructure of the thickness of the myelin sheath and endoneural collagen in the NGCs + FGF2 experimental group, scale bar 1^m. DETAILED DESCRIPTION OF THE INVENTION

[0054] Unless otherwise specified, the terms used throughout this descriptive report have their common meanings in the art, within the context of the disclosure and in the specific context in which each term is used.Certain terms used to describe the disclosure are discussed below, or elsewhere in this descriptive report, to provide additional guidance to the technician regarding the description of the disclosure. Publications cited herein are specifically incorporated by reference in their entirety.

[0055] It will be appreciated that the same thing can be said in different ways. Consequently, alternative language and synonyms may be used for any or more of the terms discussed herein. No special significance should be placed on whether a term is elaborated upon or discussed herein. Synonyms for certain terms are provided, but the exemplification of some synonyms does not preclude the potential use of others not listed herein.

[0056] In general, the present invention consists of a process for obtaining a neuroprotective conduit; a neuroprotective conduit; and the use of the neuroprotective conduit for applications such as traumatic injuries due to complete laceration of the nerves of the upper and lower extremities and face, generating short-gap defects (<2.5 cm) or long-gap defects (>2.5 cm); nerve injuries due to external crushing from trauma; nerve injuries subjected to decompression (carpal tunnel syndrome or cubital tunnel syndrome); adjuvants in nerve injuries subjected to end-to-end neurorrhaphy; adjuvants in nerve injuries subjected to autograft; compressive injuries of the nerve roots of the cauda equina; chronic paralysis resulting from spinal cord injury (SCI).EXAMPLES OF CONCRETIZATION Synthesis and purification of methacrylated gelatin (GelMA) biomaterial

[0057] The synthesis of the GelMA precursor solution was carried out by diluting 10% (w / v) gelatin in DPBS (pH = 7.4) under stirring, at 50°C. Subsequently, 6% (v / v) methacrylic anhydride (MAA) was added at a rate of 0.5 mL / min under stirring conditions, allowing it to react for 60 min at 50°C as previously described (J. Nichol, ST Koshy, H. Bae, CM Hwang, S. Yamanlar, A. Khademhosseini, Biomaterials 2010, 31, 5536; Y. Hu, Y. Wu, Z. Gou, J. Tao, J. Zhang, Q. Liu, T. Kang, S. Jiang, S. Huang, J. He, S. Chen, Y. Du, M. Gou, Sci. Rep.). MAA monomers react with gelatin-free amines, as shown in Figure 1A.The solution was diluted in a 1:1 ratio with ultrapure deionized water (40°C), dialyzed against distilled water using dialysis tubes with a cutoff of 14-16 kDa (Spectrum Labs, Repligen, Rancho Dominguez, CA, USA) for one week at 40°C under agitation. After the purification step, the pH was calibrated to 7.4, and the solution was filtered, frozen in liquid nitrogen, and lyophilized (Lio101, Liobras, São Carlos, SP, Brazil) for one week. Finally, the resulting GelMA foam was stored at -80°C, protected from light and moisture. Table 1. Variables modified during GelMA synthesis. Vol. Rate Time Pressure of giving MAA addition reaction Dilution Time of T° of Lyophil Lot Solven (v / vo of the “freeze drying” lyophilization) MAA (min) (s:n) (h) z.(°C) (uHg) 0.5 DPBS, mL / Mi -65, - 1 pH 7.46% n 60 1:1 5-6 67 75-107 0.5 DPBS, mL / Mi -65, - 2 pH 7.46% n 60 1:1 4-5 67 75-107 0.5 DPBS, mL / Mi -65, - 3 pH 7.46% n 60 1:1 4-6 67 75-107 0.5 DPBS, mL / Mi 4 pH 7.46% n 60 1:2 4 -65 91 Biochemical characterization of GelMA

[0058] The ninhydrin assay consists of detecting free primary amines that did not react with methacrylic acid (MA) during the gelatin functionalization process, as presented in Figure 1 in (A) and (B).

[0059] A standard curve of 0 to 10 mg / mL (1 mg / mL intervals) of unmodified gelatin (n = 3) was constructed and GelMA samples were plated in duplicate without dilution (n=2). After the addition of the ninhydrin solution in ethanol (2.2 mg / mL) and incubation, a linear pattern of color development was observed (30 min), as shown in Figure 1 in (C) and (D). Absorbance was measured at 570 nm (JM Zatorski, AN Montalbine, JE Ortiz-Cárdenas, RR Pompano, Anal. Bioanal. Chem.2020, 412, 6211) and, for each functionalized sample, the available amine fraction was determined by equation 1:

[0060] where the apparent concentration was obtained by comparison with the standard curve, and the nominal concentration was defined as the concentration at which the protein sample solution was prepared. The percentage (%) of DoF was. determined by equation 2: Photocrosslinking assay with ultraviolet (UV) light of GelMA

[0061] Samples of GelMA at concentrations of 5 and 10% (wt / vol) were used, maintaining a constant concentration of 0.5% (wt / vol) of the photoinitiator IC2959. The GelMA / IC2959 solution was diluted at 37ºC in an oven for 3 hours until it became transparent. After sample preparation (GelMA 5% / IC2959 0.5% and GelMA 10% / IC2959 0.5%), these were deposited in transparent rectangular silicone molds (7mm x 14mm L x 4mm) (Sigma-Aldrich, Burlington, MA, USA) and subsequently exposed to UV light using a 365nm lamp (E19UV, Wuben, Guangdong, China) at a height of 45 mm, for the formation of GelMA constructs. Five UV light exposure time points were defined: 40s, 100s, 200s, 240s, and 300s.

[0062] Images of the prismatic-shaped GelMA constructs were obtained, and stability in the longitudinal plane was observed at rest and after inclination of approximately 45º.pH Assay of Photocrosslinked GelMA

[0063] The pH of 5% and 10% photocrosslinked GelMA samples was evaluated for 200s. GelMA constructs were incubated in triplicate in sterile 24-well polystyrene (PES) plates (Nest Biotech, Wuxi, China) and supplemented with low-glucose, pyruvate-free DMEM, glutamine, and phenol red (Gibco, São Paulo, SP, Brazil) for 24, 48, 72, and 120 hours. The control consisted of DMEM alone. After the defined periods, the pH was semi-quantitatively evaluated using the colorimetric method with reagent strips (pH 0-14, Kasvi, Guangzhou, China). Filament Fusion and Printability Test of GelMA and PCL Biomaterials

[0064] The 3D printing capability was evaluated at GelMA 5% and GelMA 10% concentrations and compared with the GelMA 10% / Gelatin 1.25% system.The pressure parameters (kPa) were tested between 18-26 kPa, maintaining constant temperature, printing speed, and nozzle diameter (described in Table 3 below). The fabricated scaffold followed a two-dimensional (2D) two-layer pattern from 0°–90° with increasing filament distance (FD) from 1 to 5 mm in 1 mm increments. The theoretical dimensions of the construct were obtained and compared with the actual dimensions of the construct, following the previously described protocol (A. Habib, V. Sathish, S. Mallik, B. Khoda, Materials (Basel). 2018, 11, DOI 10.3390 / ma11030454). Therefore, considering the filament diameter (df), the scan width (Rw) was defined as Rw = FD – df.

[0065] Images of the scaffolds were obtained immediately after fabrication and photocrosslinking to avoid material spreading. Two factors were determined: (1) the percentage of diffusion rate (material spreading rate) (Dfr) and printability (Pr) (A. Habib, V. Sathish, S.Mallik, B. Khoda, Materials (Basel). 2018, 11, DOI 10.3390 / ma11030454; L. Ouyang, R. Yao, Y. Zhao, W. Sun, Biofabrication 2016, 8, 035020) como mostrado nas. Equations (3) and (4), respectively:

[0066] At defines the theoretical area and Aa signifies the actual pore area, respectively. The Dfr of a pore without any material spreading is 0 (i.e., At = Aa). In the case of Pr, A is defined as the area and L is the pore perimeter. For a perfect square, the standard printability would be 1, while Pr > 1 indicates overgelation and Pr < 1 indicates undergelation. The calculation of Pr can help to semi-quantify printability and guide the optimization of bioprinting parameters. Values ​​between 0.9–1.1 are acceptable and show morphology and mechanical stability using hydrogels (L. Ouyang, R. Yao, Y. Zhao, W. Sun, Biofabrication 2016, 8, 035020).

[0067] Printability characterization was performed separately on GelMA and PCL in order to determine the best printing parameters for the manufacture of NGCs. In order to increase the viscosity of GelMA, 10% gelatin was added (1.25% wt / v) and compared with GelMA 5% and 10%, as previously described (R. Seyedmahmoud, B. Çelebi-Saltik, N. Barros, R. Nasiri, E. Banton, A. Shamloo, N. Ashammakhi, MR Dokmeci, S. Ahadian, Micromachines 2019, 10, DOI 10.3390 / mi10100679).

[0068] The models for evaluating the printability of the GelMA formulations were designed in CAD (computer-aided design), sliced ​​in PrusaSlicer to generate the G-code, and imported into the DNA studio software of the Bio X system (Cellink-BICO, Gothenburg, Sweden). Next, after bioprinting at a temperature of 22ºC, the constructs were crosslinked with UV light using the 365nm photopolymerization module of the bioprinter. Table 2. 3D printing parameters tested for GelMA hydrogels.Diameter Pressure Temperature Nozzle Speed ​​Composition Sample (Kpa) (Tº) (mm / s) (µm) GelMA 10% 1 18 22 5 400 2 20 22 5 400 3 22 22 5 400 4 24 22 5 400 5 26 22 5 400 GelMA 5% 1 18 22 5 400 2 20 22 5 400 3 22 22 5 400 4 24 22 5 400 5 26 22 5 400 GelMA 10 / gelatin 1.25% 1 18 22 5 400 2 20 22 5 400 3 22 22 5 400 4 24 22 5 400 5 26 22 5 400

[0069] During the analysis of the GelMA formulations, photomicrographs were captured using a photographic studio (Pop Up 45, Photo Studio, Brazil) and the parameters were obtained from the measurement of all macropores of each scaffold of GelMA 10%, GelMA 5% and GelMA10% / gelatin 1.25% (n=5 / sample). The measurements were performed using image analysis software (Image J, National Institutes of Health (NIH), MA, USA).

[0070] During the analysis of the PCL biopolymer, printability (equation 4) was determined from scanning electron microscopy (SEM) photomicrographs (n=8 / sample) obtained from the PCL constructs (n=3).Furthermore, the pore height, pore length, and filament diameter of the PCL constructs (n=3) were evaluated.

[0071] The models were designed using a computer with Tinkercad software (Autodesk, San Francisco, CA, USA) and exported in STL format. The designs were 10 mm x 10 mm x 0.5 mm mesh structures. PCL pellets (Mn 80,000, Sigma-Aldrich, São Paulo, Brazil) were deposited in the thermoplastic printhead and, after 30 minutes of preheating and filament formation, several parameters were tested (Table 3), following the manufacturer's protocol. Table 3. 3D printing parameters tested for PCL.Size Pressure of the nozzle (kPa) Temperature Speed ​​(mm / s) Material (µm) Polycapropacton printing window 210- a (PCL) o 230 210-220 3-3.5 410 Parameter used 220 210 3.3 410

[0072] After obtaining the printability of the PCL, 3D printing of the nerve conduits (NGCs) characterized by 3 mm diameter x 10 mm height was performed. The models were designed in Tinkercad software (Autodesk, San Francisco, CA, USA) and exported in STL format.

[0073] During the ultrastructural characterization of the NGCs, the constructs were coated with gold in the sputter coater (BAL-TEC, SCD-050) and imaged on SEM (JSM 5800LV JEOL, Tokyo, Japan). The images were captured using the image software (SemAfore 5.21, JEOL, Tokyo, Japan). The surface and porosity were analyzed using ImageJ software (NIH, Bethesda, MD, USA) from the SEM images.The dimensions of the NGCs were evaluated and the values ​​of wall thickness, internal diameter (ID) and external diameter (ED) were obtained from 3 random measurements of the NGCs per sample (n=3).

[0074] The design of the biofabricated NGCs was based on previously reported parameters to maintain nutrient diffusion without affecting mechanical properties, with porosity between 125-550 μm and a wall thickness of ~600 μm (AM Moore, R. Kasukurthi, CK Magill, HF Farhadi, GH Borschel, SE Mackinnon, HAND 2009, 4, 180; S. Vijayavenkataraman, S. Zhang, S. Thaharah, G. Sriram, WF Lu, JYH Fuh, Polymers (Basel). 2018, 10, 1; P. Konofaos, JP Ver Halen, J. Reconstr. Microsurg. 2013, 29, 149). The internal diameter was adapted to the nerve diameter (< 200 μm) to avoid compression and support molecule diffusion. Furthermore, the length was consistent with the size of the lesion (AM Moore, R. Kasukurthi, CK Magill, HF Farhadi, GH Borschel, SEMackinnon, HAND 2009, 4, 180). Rheological analysis of GelMA hydrogel

[0075] Rheological measurements were performed on a compact modular rheometer (Anton Paar MCR-102), with a cone-plate geometry of 50 mm diameter, cone angle of 0.9815° and truncation of 0.97 ^^^^. All samples in GelMA 5%, GelMA 10% and GelMA 5% / gelatin 1.5% hydrogel were deposited on the plate to completely fill the gap (1 mm in size) between the plate and the cone.

[0076] Viscosity and shear stress measurements were performed by varying the shear rate from 1 to 500 s. -1 with a rotational test at 22ºC.

[0077] Viscosity was measured on a temperature ramp between 10 and 37ºC at a rate of 1ºC / min, and the shear rate was 1 s -1The storage modulus (G') and loss modulus (G") were measured as a function of temperature at a constant frequency rate of 1 Hz and a constant voltage of 0.1%. The hydrogel samples were equilibrated at 38°C and then cooled at a rate of 1°C per minute from 38°C to 3°C. Duplicate samples (n=2) of the GelMA 5%, GelMA 10%, and GelMA 10% / Gelatin 1.25% concentrations were analyzed. Pure gelatin at a concentration of 10% was used as a control in the temperature modulus test. Mechanical properties

[0078] The mechanical properties of the GelMA 5%, GelMA 10%, GelMA 10% / Gelatin 1.25% hydrogels and pure gelatin 10% were characterized by oscillatory shear stress sweeps in the range of 10 -1 10 3Pa to determine the yield stress, using the rheometer (Anton Paar, MCR-102, Austria). Frequency sweep tests were performed within the linear viscoelasticity region (σ0 = 1%) in the frequency range of 1 to 25 s⁻¹. -1The bioinks were photocrosslinked in rectangles (7 mm on each side and 2 mm high) by exposure to UV light (365nm) for 240 seconds and positioned between the two plates of the rheometer. Duplicates (n=2) were analyzed using samples from two different batches. Samples of pure gelatin at a concentration of 10% were used as a control. Evaluation of the interaction, morphology and metabolic activity of DPMSCs by labeling human mitochondria

[0079] Human dental pulp-derived mesenchymal stem cells (DPMSCs) (Poietics™, Lonza, MD, USA) were thawed, plated and seeded in the presence of culture medium containing 90% low-glucose DMEM, 10% fetal bovine serum (FBS), 1% penicillin (10000 IU / mL) streptomycin (10mg / mL). The metabolic activity and interaction of DPMSCs were evaluated by means of mitochondrial labeling.

[0080] 1 x 10 were used 6Cells were mixed in GelMA. The DPMSC pellet was resuspended in the GelMA solution and transferred to transparent rectangular silicone molds (7mm W x 14mm L x 4mm D) (Sigma-Aldrich, Burlington, MA, USA). The GelMA / DPMSC suspension was then light-cured with UV light (365nm), 45 mm height, for 240s (~50 mW / cm²). 2The constructs were then incubated at 37°C / 5% CO2 in DMEM / SFB 10% for 2, 7, and 14 days in triplicate. The culture medium was changed every 3 days.

[0081] After the defined times, the GelMA constructs containing the DPMSCs were fixed in 4% paraformaldehyde (PFA) for 30 min in orbital shaking and immersed in 10 and 20% sucrose in PBS (pH 7.4). They were then embedded in Tissue-tek OCT and frozen. Gelatinized slides were prepared with 16 µm sections. During the immunofluorescence reaction, the slides were washed with 0.01M PB 3x and blocked (3% BSA in 0.1M PB) for 45 min. The cells were incubated with the primary anti-human mitochondrial antibody (ab92824, Abcam, Cambridge, UK) for 2 hours. After washing in 0.01M PB, the secondary antibody (488 anti-mouse, Jackson Immunoresearch, West Grove PA, USA) was applied and staining was performed with DAPI (4',6-diamidino-2-phenylindole, 1:1000 in 0.1 M) for 10 minutes.The sections were washed with 0.01 M PB and mounted in glycerol / PB (3:1). The samples were observed under a fluorescence microscope (Leica DMB5500, Wetzlar, Germany) and documented with a digital camera (Leica DFC 345 FX) with specific filters. Gene expression of neurotrophic factors

[0082] For gene expression assessment, Taqman assays (primer + hydrolysis probes) were used. DPMSCs were stimulated at decreasing concentrations of FGF-2; 2 µg / mL, 0.2 µg / mL and 0.02 µg / mL to observe the effect on gene expression of DPMSCs, as described below.

[0083] DPMSCs were cultured in triplicate with 2x105 cells / cm. 2In 24-well plates (Costar®, 24 wells, TC-treated, Corning, NY, USA), unstimulated cells were used as controls. After 72 hours, cells were collected in RLT buffer (Qiagen, São Paulo, Brazil) and stored at -80°C. Ribonucleic acid (RNA) was extracted using the Mini RNAeasy™ kit (Qiagen, São Paulo, Brazil) and quantified by spectrophotometry. Complementary DNA (cDNA) synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies Corporation, Carlsbad, CA, USA) following the manufacturer's instructions.

[0084] Triplicate reactions were performed using the produced cDNA, TaqMan® Gene Expression Master Mix (2x) (Life Technologies Corporation, Carlsbad, CA, USA), RNA-free water, and the TaqMan assays (Life Technologies Corporation, Carlsbad, CA, USA): Hs00900055_m1- VEGF; Hs03805856_g1 – BDNF and Hs01055329_m1 – GDNF. Samples were tested with the GAPDH reference gene.Quantitative qRT-PCR procedures were performed on the MX3005P instrumentation platform (GE). Biocompatibility and degradation of PCL in vivo

[0085] To evaluate the biocompatibility and biodegradability of PCL, implantation was performed near the nervous system. Hemilaminectomy allows latero-ventral access to the vertebral canal and is commonly used in spinal cord injuries. PCL membranes were sterilized by immersion in 70% ethanol for 15s, followed by washing with distilled water, drying at room temperature and subjecting to UV irradiation of 200-280 nm for 2 hours for subsequent in vivo implantation. The experiments were conducted following the norms and principles of the Ethics Committee on Animal Experimentation (CEUA / UNICAMP, protocol no. 6148-1-2022).

[0086] The animals were divided into 2 groups: Group 1: experimental animals underwent hemilaminectomy (control) (n = 5).Group 2: The animals underwent hemilaminectomy and PCL scaffold implantation (n = 5). Under general anesthesia, hemilaminectomy was performed on vertebral segments L1 and L2 at the level of the lumbar intumescence. Subsequently, the PCL scaffold (6 mm x 4 mm x 0.2 mm) was implanted over the window and fixed with simple sutures to the adjacent musculature. An analgesic agent (tramadol hydrochloride, 5 mg / kg / SC / SID / 5 days) was administered. In the control group, hemilaminectomy was performed without modifications.

[0087] The experimental animals were observed weekly for 4 weeks to detect neurological and clinical changes or changes at the lesion site. In addition, radiographic images of the thoracolumbar vertebral segments were obtained every 15 days for 1 month using an in vivo image analyzer (FXPRO, Bruker, TX, USA). Finally, they were euthanized 30 days after the surgical procedures.Specimens from the implantation region were collected for macroscopic evaluation. The parameters measured were: (1) hemilaminectomy height, (2) width and height of fibrous tissue (hemilaminectomy fibrosis), (3) width and height of the vertebral canal (vertebral canal stenosis) (n=5). RESULTS Step 1 – In vitro Acquisition of material and synthesis of methacrylated gelatin (GelMA)

[0088] The synthesis, purification, characterization and storage of the GelMA biomaterial from 4 different batches of the biomaterial were carried out under laboratory conditions.

[0089] The final GelMA product was obtained, which consisted of a white, highly porous and sterile foam according to the results previously described (J. Nichol, ST Koshy, H. Bae, CM Hwang, S. Yamanlar, A. Khademhosseini, Biomaterials 2010, 31, 5536; Y. Hu, Y. Wu, Z. Gou, J. Tao, J. Zhang, Q. Liu, T. Kang, S. Jiang, S. Huang, J. He, S. Chen, Y. Du, M. Gou, Sci. Rep. 2016, 6, 1).Some modifications were applied without affecting the final yield of the lyophilized GelMA, as shown in Table 1.

[0090] After obtaining the dry, sterile, lyophilized GelMA samples (n = 4 / batch), they were weighed to obtain the amount of dry mass per 50 mL Falcon tube. The mass of biomaterial obtained per tube ranged from 2.1 to 2.57 g, as shown in Table 4. The findings indicate that each batch yielded approximately 8 g. The process takes 2 weeks per batch, and the quantities obtained were sufficient to carry out all the characterization and biofabrication processes of the NGCs. The greatest advantage was the immediate availability, in a sterile form at low cost and with a high degree of functionalization, as shown later. Table 4. Yield of the GelMA batches produced. GelMA Yield (g) Batch 1 2.1 ± 0.1 Batch 2 2.47 ± 0.1 Batch 3 2.02 ± 0.2 Batch 4 2.57 ± 0.1

[0091] GelMA is prepared by means of the reaction between MAA gelatin.This reaction introduces methacryloyl groups into reactive amines and hydroxyl groups into amino acid residues, as shown in Figure 2. After dissolving the gelatin in DPBS (pH 7.4) at 50 °C, MAA is added with vigorous stirring. After 1 hour of reaction, the solution is diluted and dialyzed for 5 days against distilled water at 40 °C. Then, the product is lyophilized to obtain a white solid foam (K. Yue, G. Trujillo-de Santiago, MM Alvarez, A. Tamayol, N. Annabi, A. Khademhosseini, Biomaterials 2015, 73, 254). The protocol proved versatile for obtaining the product on a laboratory scale.

[0092] Gelatin is a soluble polypeptide mixture of denatured and partially hydrolyzed collagens derived from animal tissues such as tendon, skin and bone (K. Yue, G. Trujillo-de Santiago, MM Alvarez, A. Tamayol, N. Annabi, A. Khademhosseini, Biomaterials 2015, 73, 254).We used porcine gelatin for the manufacture of GelMA, which is water-soluble and forms a thermoreversible hydrogel through physical cross-linking between collagen molecules (AI Van Den Bulcke, B. Bogdanov, N. De Rooze, EH Schacht, M. Cornelissen, H. Berghmans, Biomacromolecules 2000, 1, 31). During preparation, it was not necessary to use reactors or complex equipment for temperature control during dilution and methacrylation (G. Ninan, J. Joseph, ZA Aliyamveettil, J. Food Sci. Technol. 2014, 51, 2085) as shown in Figure 2.

[0093] Different DoF of methacryloyl can be achieved in GelMA by adjusting the amount of MAA added, allowing the production of GelMA with different mechanical properties. We used a 6% volume of MAA at an addition rate of 0.5 mL / min. The slow addition of MAA to the gelatin solution induces methacryloyl substitution in the reactive amine and hydroxyl groups of amino acid residues (AI Van Den Bulcke, B.Bogdanov, N. De Rooze, E. H. Schacht, M. Cornelissen, H. Berghmans, Biomacromolecules 2000, 1, 31). Evaluation of the degree of functionalization (DoF), photocrosslinking analysis and pH

[0094] The mechanical and biological properties of GelMA-based hydrogels are largely dependent on the degree of functionalization (DoF), polymer concentrations, photoinitiator and thermal gelation state of the GelMA precursor solution, as well as UV exposure (intensity and time) during polymer photocrosslinking (AI Van Den Bulcke, B. Bogdanov, N. De Rooze, EH Schacht, M. Cornelissen, H. Berghmans, Biomacromolecules 2000, 1, 31, W. Schuurman, PA Levett, MW Pot, PR van Weeren, WJA Dhert, DW Hutmacher, FPW Melchels, TJ Klein, J. Malda, Macromol. Biosci. 2013, 13, 551). The degree of functionalization was evaluated using the ninhydrin assay. During the assay, a linear absorbance curve was obtained from 2 to 8 mg / mL (R2 = 0.9585, p < 0.001).The GelMA samples developed little purple color, indicating a low amount of free amines present. To determine the fraction of remaining amines in a GelMA sample, the “apparent” gelatin concentration was determined from the standard curve and normalized to the nominal value of the sample concentration (Equation 1). The DoF was defined as the difference of this value from unity (Equation 2). For example, GelMA samples prepared at 10 mg / mL reacted with ninhydrin and produced an apparent concentration of 2.59 mg / mL, 2.56 mg / mL, and 2.77 mg / mL, with calculated DoFs of 74.06%, 74.34%, and 72.28%, respectively (n = 3), as shown in the graph in Figure 3.

[0095] An estimated DoF of 50% is the minimum to generate 5% (wt / vol) GelMA-based hydrogels. The results are comparable to previous studies that achieved a high degree of functionalization (75-80%) using 6% MAA (W. Schuurman, PA Levett, MW Pot, PR van Weeren, WJA Dhert, DW Hutmacher, FPW).Melchels, TJ Klein, J. Malda, Macromol. Biosci. 2013, 13, 551; D. Loessner, C. Meinert, E. Kaemmerer, LC Martine, K. Yue, PA Levett, TJ Klein, FPW Melchels, A. Khademhosseini, DW Hutmacher, Nat. Protoc. 2016, 11, 727). The ninhydrin assay allowed the preparation of the protein standard curve and is an accurate, practical, and cost-effective strategy for determining DoF (JM Zatorski, AN Montalbine, JE Ortiz-Cárdenas, RR Pompano, Anal. Bioanal. Chem. 2020, 412, 6211).

[0096] The photocrosslinking assay was performed by exposing the samples to 365 nm UV light, as can be seen in Figure 4 in items (A) and (B). The samples prepared with 10% GelMA and 0.5% photoinitiator IC2959 were fully crosslinked in 40s, as per item (C) of Figure 4. The 10% concentration showed greater preservation of structure, vertical stability and fine edges from 200 seconds onwards compared to the shorter times in the dorsal view when compared to 5% GelMA (D).Samples subjected to 40s and 100s showed thickening with visible flow at the base of the structure as shown in items (A) and (B) of Figure 5, which represent the front and side views, respectively. Samples >200 s, when subjected to inclination, did not show flow as shown in Figure 5 item (D), when compared with GelMA 5% (C) of Figure 5.

[0097] Samples prepared with GelMA 5% and the photoinitiator IC2959 were crosslinked at 240s, as shown in Figure 4 item (D). At this concentration, it was detected that samples >200s partially maintained the structure with low stability, edges and body with thickening in the dorsal, front and side views (as can be observed in Figure 4 item (D) and Figure 5 items (A) and (B). There was greater fidelity of the structure at 300s of exposure to UV light.Additionally, samples with <200s exposure to UV light (365nm) did not undergo gelation and did not maintain a vertical structure with flow after tilting (Figure 5 item (C)).

[0098] The results indicate that the GelMA 10% samples showed rapid crosslinking and stability. Photosensitive biomaterials such as GelMA allow the construction of various 3D architectures using bioprinting while preserving cell viability (W. Schuurman, PA Levett, MW Pot, PR van Weeren, WJA Dhert, DW Hutmacher, FPW Melchels, TJ Klein, J. Malda, Macromol. Biosci. 2013, 13, 551; T. Billiet, E. Gevaert, T. ;De Schryver, M. Cornelissen, P. Dubruel, Biomaterials 2014, 35, 49; W. Liu, Z. Zhong, N. Hu, Y. Zhou, L. Maggio, AK Miri, A. Fragasso, X. Jin, A. Khademhosseini, YS Zhang, Biofabrication 2018, 10, 024102).

[0099] Adjusting pH to physiological conditions is essential to maintain conditions for cellular homeostasis.After dialysis of the GelMA, the pH was adjusted close to physiological. It was noted that the 10% and 5% GelMA constructs absorbed red from the culture medium and remained stable 5 days later, as shown in Figure 6, items (A) and (B). In this context, no significant differences were observed in the pH measurement between the 10% and 5% GelMA concentrations, 1, 2, 3 and 5 days after incubation when compared to the control (p > 0.05), as shown in the graph of Figure 6 item (C). Rheological analysis of the hydrogel

[0100] The rheological properties of the biomaterial were evaluated at three GelMA concentrations (5% GelMA, 10% GelMA and GelMA / 1.25% Gel) and a constant gelatin concentration. A pseudoplastic behavior (shear thinning) was observed at all concentrations.However, the higher concentration of GelMA (10%) showed greater pseudoplastic behavior compared to the lower concentration (5%), as can be seen in graph (A) of Figure 7, where it can be observed that the viscosity was lower at the 5% concentration when compared to the higher concentration of 10% at 22. o C. Temperature-dependent viscosities are shown in graph (B) of Figure 7, with a reduction in viscosity (psudoplasticity) observed with increasing temperature. Viscosity increased at higher GelMA concentrations, and there was a slight increase after the addition of 1.25% (w / v) gelatin, with a good thermoresponsive response observed in the range between 20 and 25 oC of all concentrations.

[0101] Graph (C) in Figure 7 shows the variation in storage modulus (G') and loss (G”) of hydrogels according to temperature change. The GelMA 10% and GelMA 10% / gelatin 1.25% bioinks showed a gelation point (G' = G”) very close to 23.77 and 24.26 °C, respectively. In contrast, the GelMA 5% concentration showed a gelation point (G' = G) of 12.7 °C, which is considered low when compared to higher concentrations. All concentrations showed a lower gelation point compared to pure, unmodified gelatin (27.72 °C). During cooling, the G' value increased rapidly and crossed over at G”, indicating the presence of gel-like structures. Below the gelation point (G” > G'), all concentrations showed near-liquid behavior.Based on the rheological results, a 10% GelMA concentration with a starting temperature of 23 ºC was selected as the bioprinting temperature for the analysis of 3D bioprinting capability. Mechanical properties

[0102] Based on the previous results, the viscoelastic properties of the GelMA samples (5% and 10% + Gelatin 1.25%) crosslinked at 365nm UV light were evaluated. The viscoelastic properties of biomaterials are crucial to understanding the ability to maintain physical structures similar to native tissue. The analysis of linear viscoelastic properties is shown in graph (A) of Figure 8. In all samples analyzed, a predominance of elastic properties G' > G'' was observed in all frequency ranges. These results indicate that the photocrosslinked GelMA exhibits gel behavior with strong chemical bonds, without crossing of storage (G') and loss (G'') modules.Furthermore, the modulus and storage (G') were superior in the 10% GelMA and 10% GelMA + 1.25% Gelatin samples, indicating that exposure to UV light (365 nm) increased the mechanical properties when compared to 5% GelMA and pure gelatin.

[0103] Graph (B) in Figure 8 shows the damping factor ^^^^^^^^ = G” / G′ as a function of frequency. The damping factor indicates the strength of a gel, and solid behavior was dominant (^^^^^^ ^^ = < 1). Tangent values ​​of 0.1 indicate a weak gel. Therefore, at all frequencies studied, strong gel behavior was obtained after crosslinking. Culture of DPMSCs for bioactivity assays

[0104] During the evaluation of cell interaction and viability / cytotoxicity, DPMSCs were cultured and characterized following the minimum criteria established for the identification of cell lines of mesodermal origin (M. Dominici, K. Le Blanc, I. Mueller, I. Slaper-Cortenbach, FC Marini, DS Krause, RJ Deans, A. Keating, DJProckop, EM Horwitz, Cytotherapy 2006, DOI 10.1080 / 14653240600855905). The DPMSCs showed monolayer growth with fibroblast-like morphology and adherence to the culture plastic, as can be observed in items (A) and (B) of Figure 9. Flow cytometry analysis showed the expression of positive markers CD90+, CD105+, CD73+, CD44+ and absence of expression of CD45-, CD34-, CD11b-, CD19- and the MHC class II cell surface receptor (HLA-DR), as can be observed in graphs (D) to (H) of Figure 9. The results indicate that the cells presented a pure mesodermal phenotype with good characteristics in culture for the biomaterial's biological assays. Cell adhesion, proliferation and viability on the surface

[0105] Biofabrication of biomaterial-based constructs requires high cytocompatibility to facilitate the migration, proliferation and differentiation of endogenous tissue cells or cells embedded in the biomaterial (J. Malda, J. Visser, FPMelchels, T. Jüngst, WE Hennink, WJA Dhert, J. Groll, DW Hutmacher, Adv. Mater. 2013, 25, 5011). Gelatin is a natural, biocompatible, biodegradable, non-immunogenic polymer that can promote cell adhesion, differentiation, and proliferation.

[0106] In this context, human DPMSCs encapsulated in 5% and 10% GelMA hydrogel showed high viability of 83% in 5% GelMA, as can be seen in items (A) to (C) of Figure 10, and 85% in 10% GelMA, as can be seen in items (E) to (G) of Figure 10, during 2, 7, and 15 days after encapsulation using the LIVE / DEAD assay. Furthermore, no significant differences were observed between the groups evaluated on days 2, 7, and 15 (n = 3) (p > 0.05), according to graphs (D) and (H) of Figure 10. The trend of decreasing viability in GelMA 5% on day 2 and in GelMA 5% (graph (D) of Figure 10) may be related to UV light exposure or the effects of the encapsulation itself (S. Duchi, C. Onofrillo, CDO'Connell, R. Blanchard, C. Augustine, AF Quigley, RMI Kapsa, P. Pivonka, G. Wallace, C. Di Bella, PFM Choong, Sci. Rep. 2017, 7, 1). The results suggest that the minor effects on cell viability in the first few days generated by the cross-linking and / or encapsulation process are not permanent. These results are comparable to previous studies reporting high biocompatibility of GelMA (J. Nichol, ST Koshy, H. Bae, CM Hwang, S. Yamanlar, A. Khademhosseini, Biomaterials 2010, 31, 5536; CD O'Connell, B. Zhang, C. Onofrillo, S. Duchi, R. Blanchard, A. Quigley, J. Bourke, S. Gambhir, R. Kapsa, C. Di Bella, P. Choong, GG Wallace, Soft Matter 2018, 14, 2142).

[0107] In mesenchymal stem cells (MSCs), high cell viability >95% was observed in the first 7 days after encapsulation (CD O'Connell, C. Di Bella, F. Thompson, C. Augustine, S. Beirne, R. Cornock, CJ Richards, J. Chung, S. Gambhir, Z. Yue, J. Bourke, B. Zhang, A. Taylor, A.Quigley, R. Kapsa, P. Choong, GG Wallace, Biofabrication 2016, 8, 015019). O'Connell et al (2018), evaluated three UV light intensity and time regimes in human MSCs: 700 mW / cm. 2 37s (high), 100 mW / cm 2 100s (average) and 10 mW / cm 2(low) 316s under similar conditions (GelMA 10% + 0.5% CI 2959). The study showed 91% viability with medium intensity in the first 24 hours. However, high intensity could decrease metabolic activity in the first week compared to medium or low intensity (CD O'Connell, B. Zhang, C. Onofrillo, S. Duchi, R. Blanchard, A. Quigley, J. Bourke, S. Gambhir, R. Kapsa, C. Di Bella, P. Choong, GG Wallace, Soft Matter 2018, 14, 2142). Other studies have evaluated the viability of MSCs in GelMA matrices and observed comparable results with high viability between 75 and 90% (J. Yin, M. Yan, Y. Wang, J. Fu, H. Suo, ACS Appl. Mater. Interfaces 2018, 10, 6849; W. Liu, MA Heinrich, Y. Zhou, A. Akpek, N. Hu, X. Liu, X. Guan, Z. Zhong, X. Jin, A. Khademhosseini, YS Zhang, Adv. Healthc. On the other hand, it is suggested that DNA absorbs wavelengths of 260 nm, therefore, a frequency of 365 nm may reduce direct DNA damage (M. Liu, X. Zeng, H. Ma, Y.Yi, L. Tang, L. Zhong, W. Qiu, S. Wen, G. Jia, M. Wu, K. Ye, ACS Appl. Mater. Interfaces 2017, 9, 43449). One study observed that high percentages of GelMA reactive groups for the photoinitiator could have protective effects for cells without affecting viability after UV light-mediated photopolymerization (M. Bartnikowski, NJ Bartnikowski, MA Woodruff, K. Schrobback, TJ Klein, Acta Biomater. 2015, 27, 66).

[0108] It is suggested that concentrations <5% (wt / v) of GelMA are more suitable for maintaining cell viability. Meanwhile, the geometric fidelity of the frameworks may be compromised (W. Liu, MA Heinrich, Y. Zhou, A. Akpek, N. Hu, X. Liu, X. Guan, Z. Zhong, X. Jin, A. Khademhosseini, YS Zhang, Adv. Healthc. Mater. 2017, 6, 1; SR Shin, PA Bae, JY Cha, HR Mun, JS Kim, MR Dokmeci, A. Khademhosseini, Pequeno 2015, 11, 2096). Concentrations > 15% may increase viscosity and geometric fidelity.However, cell viability may be reduced (J. Yin, M. Yan, Y. Wang, J. Fu, H. Suo, ACS Appl. Mater. Interfaces 2018, 10, 6849; C. Zeng, D. Yang, J. Xia, S. Wang, Q. Li, X. Kong, J. Sun, Y. Xie, L. Zhang, Biomaterials 2020, 233, 119744). Therefore, it is crucial to balance physical bioprinting capability and biological functionality to achieve superior results in 3D bioprinting using GelMA.

[0109] In the qualitative assessment in the Live / Dead cell assay, mild cytotoxicity was observed on the surface, indicating susceptibility to the effects of UV light, as can be seen in items (A) to (D) of Figure 11 compared with the control represented by items (C) and (F) of Figure 11. Light-mediated polymerization could cause cell damage depending on the time and intensity of exposure (CM Madl, SC Heilshorn, Tissue Eng. Part A 2018, 24, 1475).Despite this, the cells inside the GelMA block were viable 15 days after photocrosslinking in both 5% and 10% GelMA and were not affected by irradiation, as can be seen in items (B) and (E) of Figure 11.

[0110] The interaction and metabolic activity was characterized by the preservation of mitochondrial activity. Mitochondria play an important role in homeostasis, bioenergetic functions and stem cell differentiation (H. Ning, X. Gong, FW Wua, G. Gao, JL Gong, L. Zhang, Mater. Sci. Eng. C 2018, 87, 78). Immunostaining showed thread-like structures in the cytoplasm of cells, predominantly around the nucleus, in GelMA 5% and 10% at 2, 7, and 15 days after photopolymerization, indicating mitochondrial activity, as can be observed in items (A) to (F) of Figure 12.Studies have tracked and detected MSCs from various sources, including human umbilical cord (UCMSCs), Wharton's jelly (WJMSCs), gingiva (GMSCs), and DPMSCs encapsulated in biomimetic scaffolds for musculoskeletal regeneration, retinal ganglion cells, and dental pulp, respectively (JM Kanczler, RL Oreffo, Nat. Mater. 2008, 7, 724; EL Kai, LW Yong, LY Yu, BT Hin, SC Boon, RMA Tan, AN Ho, ANL Jia, ANB Wan, Tissue Eng. Part A 2010, 16, 2719; 2016, 10, 598). In this way, immunolabeling for mitochondria allows for the specific identification of viable and metabolically active human cells (PC Alves, RM Carvalho, PC Alves, RM Carvalho, Biotechnol. Adv. 2019, 37, 107451; X. Wang, Z. Ao, X. Lu, H. Wang, Q. Yang, H. Tian, ​​J. Bioact. Compat. Polym. 2006, 21, 131).Gene expression of neurotrophic factors (NFs)

[0111] RNA measurements in the spectrophotometer indicated that the samples presented absorbance ratio values ​​of 260 / 280 and 260 / 230 within the appropriate parameters. The results show good quality RNA and absence of PCR-inhibiting substances.

[0112] Gene expression of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF) in DPMSCs was evaluated 3 days after direct stimulation with FGF-2 STAB. FGF-2 STAB concentrations of 2 µg / mL, 0.2 µg / mL, and 0.02 µg / mL were used.

[0113] VEGF transcripts were significantly increased after stimulation with FGF-2 STAB compared with the unstimulated control at concentrations of 2 µg / mL (p < 0.05), 0.2 µg / mL (p < 0.05), and 0.02 µg / mL (p < 0.01), respectively, as can be seen in item (A) of Figure 13.In the comparison between the tested FGF-2 STAB concentrations, no statistical differences were detected (p > 0.05). The results indicate an increase in VEGF transcript expression independent of the FGF-2 concentrations used. On the other hand, BDNF transcripts did not show statistical differences when compared to the unstimulated control at concentrations of 2 µg / mL, 0.2 µg / mL and 0.02 µg / mL (p < 0.05) of FGF-2 STAB, respectively, as shown in item (B) of Figure 13. The results indicate that FGF-2 did not change the gene expression profile of BDNF in the cells.

[0114] The results of GDNF expression were not included due to the impossibility of obtaining the Ct of the control calibrator group due to technical limitations during thermocycling. FGF-2 release in vitro

[0115] The amount of FGF-2 released from the constructs was quantified daily and cumulatively over 30 days.Daily analysis showed high release at both GelMA concentrations (5% and 10%) in the first week, as represented in graph (A) of Figure 14, with the 5% GelMA concentration being significantly higher on days 1, 3, 4, and 5 compared to the 10% concentration (p < 0.05). Overall, high FGF-2 release was observed at both concentrations in the first 7 days, then slowly decreased until stabilizing after 11 days and remaining stable for 30 days. From day 11 onwards, concentrations of ~1 ng / mL were released until 30 days. The cumulative release, shown in graph (B) of Figure 14, revealed a tendency for the amount of FGF-2 released from the construct to slowly decrease, remaining stable over time. The 5% and 10% GelMA formulations released 93.64 ng / mL and 90.25 ng / mL after 30 days, respectively.In terms of percentage, an inverse trend is observed in the amount of FGF-2 incorporated, with a release of 12% from GelMA 5% compared to GelMA 10% (11.58%). Significant amounts of FGF-2 remain in the conduits when 2 μg / mL were incorporated into the biomaterial.

[0116] The amount of FGF-2, between the two GelMA concentrations, was similar and the results are comparable to the reported neurotrophic factor release values, demonstrating that there was absorption / binding on the surface of the biomaterial allowing FGF-2 to remain available at a stable concentration (W. Liao, D. Xu, S. Wu, S. Li, Tissue Eng. Regen. Med. 2018, 15, 103; SL Tan, CS Ahmad, PF Ahmad, MME Lee, MMH Kamarul, J. Orthop. Surg. 2017, 25, 230949901771889; M. Sharifi, F. Hesaraki, M. Amerinatanzi, M. Fathi, H. Yazdanpanah, J. Mater. Sci. Mater. Med. 2019, 30, 49).We observed a peak in FGF-2 release in the first 3 days followed by a gradual decrease over a prolonged period of 30 days. In this regard, it is important to highlight that, for neurotrophic factors such as GDNF and NGF, release ranges between 1–10 ng / mL have been suggested as optimal concentrations for nerve regeneration (K. Zhu, H. Chen, M. Guo, L. Zhang, G. Tang, X. Wu, H. Wei, H. Ma, J. Lu, X. Liu, Mater. Sci. Eng. C 2021, 119, 111628). Filament fusion test, printability and structural characterization of GelMA

[0117] Printability and diffusion rate consist of a systematic quantitative assessment to evaluate the shape fidelity of the 3D bioprinting structure.To evaluate the effect of filament fusion and pore closure for each hydrogel concentration (GelMA 5%, GelMA 10%, and GelMA 10% + 1.25% gelatin), the area and pore perimeter of the designed scaffold of various sizes (1x1, 2x2, 3x3, 4x4, and 5x5) were measured, as can be seen in item (A) of Figure 15, and the diffusion rate and printability were determined, as previously described (A. Habib, V. Sathish, S. Mallik, B. Khoda, Materials (Basel). 2018, 11, DOI 10.3390 / ma11030454). Based on the rheological analysis, a temperature of 22 was set. oC with constant printing parameters (speed 5 mm / s, nozzle size 400µm) and a comparison was made across various pneumatic pressure ranges, as shown in Table 2. Qualitatively, the 10% GelMA showed better pore size and geometry when compared to the 5% GelMA. It was observed that the diffusion rate (fidelity) shows a decreasing trend and the printability shows an increasing trend with increasing pore size for each material concentration, as can be seen in graphs (B) and (C) of Figure 15.

[0118] The 10% GelMA showed that pressures between 20-22 kPa showed minimal material spreading compared to higher pressures of 24-26 kPa. However, the minimum extrusion pressure (18kPa) generated material shrinkage with loss of scaffold geometry. In contrast, GelMA 5% showed minimal material spreading at a pressure of 18-20 kPa compared to a pressure of 22-24 kPa.The highest pressure of 26 kPa in the 5% GelMA did not allow the scaffold to be generated due to excessive material spreading and complete pore closure, as observed in graph (B) of Figure 15. Therefore, the 10% GelMA showed minimal material spreading as the printing pressure increased, and the 5% GelMA showed greater spreading as the printing pressure increased.

[0119] The printability of GelMA 10% showed a tendency to increase (Pr, >0.90) as the pore size increased from 2x2 mm to 5x5 mm in all pressure ranges between 20-26 kPa, demonstrating an almost square pore geometry, as observed in graph (C) of Figure 15.

[0120] The printability of GelMA 5% showed a tendency to increase (Pr, >0.90) as the pore size increased from 2x2 mm to 5x5 mm, only with the use of lower pressures of 18-20 kPa, demonstrating an almost square pore geometry.On the other hand, at higher pressures of 22 to 24 kPa, low printability <0.90 was shown, demonstrating rounded pores.

[0121] In SEM characterization, the 5% and 10% GelMA samples analyzed showed high microporosity, indicating that photocrosslinking maintains the permeability of the GelMA construct, a crucial factor for the controlled release of biomolecules. In the evaluation of the cross-sections of the biomaterial, the presence of larger diameter pores was noted at lower concentrations of 5% GelMA, as observed in items (A) and (B) of Figure 17, and smaller diameter pores at the 10% concentration in items (C) and (D). A previous study showed pore sizes after lyophilization of 50, 30, and 24 µm for 49.8%, 64.8%, and 73.2% DoF, respectively (SM Richardson, CJ Hoyland, JM Mobasheri, AJ Csaki, RCM Hoyland, JA Mobasheri, Arthritis Res. Ther. 2008, 10, R85).Printability and structural characterization of PCL

[0122] During the bioprinting process of PCL, different pressure, temperature, and printing speed ranges were explored to obtain constructs with suitable characteristics. The final optimized parameters are shown in Table 4. The characterization of PCL was performed by SEM.

[0123] PCL filaments (diameter 441.2 ± 11.01 μm) were continuously deposited in a square geometry along the horizontal direction for the first layer and the vertical direction for the second layer, resulting in the formation of a bilayer membrane with a thickness of 690.9 ± 4.71 μm, as illustrated in items (A) and (B) of Figure 17. The spaces between the filaments (polymer-free areas) formed pores with a height of 143.3 ± 39.10 μm and a width of 177.5 ± 16.40 μm, as illustrated in graph (C) of Figure 17. Evaluation of the outer surface showed a smooth and even architecture.The cross-sectional view of the membranes confirmed the bilayer shape with the presence of rounded filaments and no fusion of the two layers, being constructs with excellent morphology and dimensions, as shown in item (B) of Figure 17. These findings were consistent with the semi-quantitative evaluation of printability (Pr = 1.02). It was previously determined that the Pr with adequate morphology and stability should be between 0.9-1.1 (L. Castano-Izquierdo, MEP Alvarez-Barreto, EML Diaz, MC Hernandez, CD Criado, L. Becerra, N. Fernandez-Gutierrez, AR Lopez-Lopez, JMS Cruzado, SAY De La Torre, Materials (Basel). 2018, 11, DOI 10.3390 / ma11112300). The results indicate that PCL allowed the formation of filaments with the dimensions and microstructure suitable for the subsequent bioprinting of NGCs. 3D printing of NGCs

[0124] The previously defined PCL parameters were used as the basis for the subsequent 3D printing of the NGCs.The optimized parameters consisted of a 0.4 mm metal nozzle, 200 kPa pressure; 180. o C cylinder head temperature and 10 oC temperature of the printing bed.

[0125] PCL NGCs were fabricated by continuous spiral deposition of the biomaterial, having an inner diameter (ID) of 1845 ± 37.65 μm and an outer diameter (ED) of 2307 ± 44.47 μm with a wall thickness of 461.3 ± 9.60 μm. In the SEM, the tubular structure and surface with uniform filaments were observed, as seen in item (A) of Figure 18. The base of the structure was slightly thicker, and the first basal layer showed flattening and slight irregularity, as seen in item (B) of Figure 18. On the other hand, the vertex showed rounded, regular filaments and no flattening, as seen in item (C) of Figure 18.

[0126] In the top view in the cross-section, circularity of the tubular structure is observed with regularity in the internal diameter and in the wall, as seen in item (D) of Figure 18.However, the formation of a slight lateral scar with increased focal thickness and accumulation of biomaterial was observed (item (E) of Figure 18). These findings are related to biomaterial flow due to movement in the Z-axis of the head. In the top view of the dorsal section of the tubular structure, continuous fused filaments were observed in the lateral region with preservation of filament circularity and reproducibility between layers (item (F) of Figure 18).

[0127] The 3D printed NGCs were based on parameters reported for the preservation of bioactivity (S. Bhardwaj, AK Roy, J. Nanosci. Nanotechnol. 2011, 11, 1519). NGCs have been manufactured with thicknesses between 100-300 µm. Thicknesses of 200 µm allow nutrient diffusion. On the other hand, however, 600 μm is associated with poor diffusion (CW Patrick, J. Tissue Eng. Regen. Med. 2008, 2, 296). The ID was adapted to the nerve size (<2mm) to avoid compression and maintain regenerative support (AM Moore, R.Kasukurthi, CK Magill, HF Farhadi, GH Borschel, SE Mackinnon, HAND 2009, 4, 180; P. Konofaos, JP Ver Halen, J. Reconstr. Microsurg. 2013, 29, 149). The ID was comparable to commercially available polyethylene tubing (BD Intramedic™) with an inner diameter of 1.57 mm, an outer diameter of 2.8 mm, and a wall thickness of 510 μm. Furthermore, the NGCs were designed based on the size of the experimental lesion in rats (8 mm gap) to facilitate tension-free implantation. Biofabrication of FGF-2 functionalized NGCs

[0128] A 3D bioprinting process of a mechanically stable NGC was performed using two different biomaterials: PCL and GelMA 10%. A hybrid system was developed for the fabrication of FGF-2 functionalized NGCs. Initially, 3D printing of the NGCs (3 x 10 mm) and guide tubes (2 x 10 mm) was performed using PCL biomaterial, a process represented in item (A) of Figure 19.The NGCs were sterilized by immersion in 70% ethanol for 15 seconds, followed by washing with DPBS, drying at room temperature, and subjecting to UV irradiation in the 200-280 nm range for 30 minutes for subsequent in vivo implantation. The guide tube was inserted into the NGCs to obtain an internal channel with a diameter of 1 mm. Subsequently, 50 μL of sterile 10% GelMA hydrogel containing 50 µL of FGF-2 at a concentration of 2 µg / mL was applied and immediately photocrosslinked with a UV light head for 240 seconds (365 nm), allowing for the crosslinking and encapsulation of FGF-2. The micrographs show a hybrid tubular structure composed of two concentric PCL cylinders, which is presented in more detail in item (B) of Figure 19. After incorporation of GelMA, the NGC filled with the GelMA+FGF-2 hydrogel is observed in the lumen of the tubular structure with a transparent appearance, preserving the lumen of the tubular structure, as shown in items (C) and (D) of Figure 19.After lyophilization of the NGCs containing GelMA, it was observed that the GelMA remained adhered to the inner wall of the construct, maintaining the tubular lumen. This approach is a practical and reproducible method with the possibility of controlling the desired geometry. The guide tube was easily removed by traction without altering the GelMA structure. In turn, item (E) of Figure 20 shows a dorsal view of the tubular structure of the lyophilized PCL+GelMA NGCs, showing the outer PCL wall and an inner layer of whitish GelMA. Conventionally manufactured NGCs are simple cylindrical structures, and when complex structures are designed, the manufacturing processes are difficult and slower (AH Teo, WL Tan, AD Shrestha, LS Ngiam, MH Yeo, SK Lim, JYY Hui, KL Tan, S. Ramakrishna, Nanomedicine 2011, 6, 963; S.-J. Lee, M. Nowicki, B. Harris, LG Zhang, Tissue Eng. Part A 2017, 23, 491).

[0129] The biofabricated NGCs were qualitatively characterized by SEM. In this context, they were kept in PBS (pH 7.4) for 1 week before the lyophilization process. Figure 20 shows micrographs that reveal a hybrid tubular structure consisting of an outer layer composed of PCL and an inner tubular structure of GelMA, as can be seen in item (A). The PCL has a smooth and regular surface, and the GelMA shows irregularity with a rough surface and adhesion points to the inner wall of the PCL, as can be seen in more detail in item (B) of Figure 20. The GelMA incorporated into the inner surface showed preservation of microporosity in the inner and outer walls, as illustrated in items (C) and (D) of Figure 20.In vivo degradability and compatibility of PCL constructs

[0130] The interaction of the PCL biomaterial in the form of PCL membranes (6 x 2 mm) was evaluated after implantation in rats subjected to hemilaminectomy (n=5), a procedure illustrated in item (A) of Figure 21. Hemilaminectomy allows latero-ventral access to the spinal canal and is commonly used in spinal cord injuries (JA Villanova Junior, MAB Magalhães, TD Cereja de Souza, MKB Battisti, JR Engracia Filho, CT Pimpão, MO Müeller, P. de Arruda, LGA Capriglione, Neuroscience Letters 2021, 760, DOI 10.1016 / j.neulet.2021.135973). The PCL biomaterial was implanted in the hemilaminectomy window as shown in Figure 21, items (A) to (D), in order to evaluate its degradability and biocompatibility in a region that has direct contact with the nervous system. Item (B) presents an image showing the hemilaminectomy window.Item (C) presents an image showing the implanted and fixed PCL membrane with 3 simple sutures. Item (D) Macroscopic appearance of the surgical field after implantation.

[0131] The specimens evaluated did not show PCL biodegradation 30 days after implantation. In the macroscopic analysis, in the control group, scar tissue formation was observed in the surgical area slightly invading the vertebral canal, but without spinal cord displacement, as shown in item (A) of Figure 23. In the PCL group, the presence of the PCL membrane at the implantation site in contact with the surgical window is observed, as shown in item (B) of Figure 23. There is no evidence of excess scar tissue, with good integration with the local musculature, as shown in item (C) of Figure 23. Furthermore, no areas of edema or erythematosus associated with inflammation or local reaction were observed.

[0132] The quantitative parameters of the specimens evaluated were: hemilaminectomy window height, height and length of scar tissue (hemilaminectomy fibrosis), and height and width of the vertebral canal (vertebral canal stenosis), as highlighted in item (A) of Figure 23. Significant differences were observed in hemilaminectomy height between the control and PCL groups (p = 0.04) (graph (B) of Figure 23), indicating a decrease in the size of the surgical window in animals where PCL was implanted. The results may be related to the protective barrier effect. In the evaluation of fibrosis in the surgical area and vertebral canal stenosis, no significant differences were observed between the control and PCL groups (p > 0.05), as detailed in graphs (C) and (D) of Figure 23). These results indicate that the implantation of PCL membranes does not show excessive scarring reaction outside and inside the vertebral canal.These results indicate good biocompatibility and integration after implantation of the PCL biomaterial.

[0133] The assessment of possible bone inflammatory reaction was complemented by radiographic imaging at the level of the vertebrae subjected to laminectomy (L1-L2). Figure 24 shows radiographic images of the vertebral column of rats subjected to hemilaminectomy 15 and 30 days after the surgical procedure. The PCL biomaterial on the right side of the vertebra showed radiolucent characteristics and was not observed in the radiographic projections. In the control group, VD projections showed the presence of radiopaque bone remodeling (white arrows) and absence of periosteal reaction on day 15, as per items (A) to (C) of Figure 25 and 30 after hemilaminectomy, as per items (G) to (I).In the PCL group, VD projections showed the presence of radiopaque bone remodeling with irregular vertebral contours (white arrows) on the right side, as per items (D) to (F), compared to the control, as per items (J) to (L). PCL implantation did not show periosteal reaction or edema after implantation. Bone tissue formation was observed at the PCL membrane implantation site and maintenance of vertebral alignment without displacement.

[0134] During the evaluation of LL projections in the control group, it was observed, through Figure 25, which presents radiographic images of the vertebral column of rats subjected to hemilaminectomy for the in vivo biocompatibility test, the persistence of the hemilaminectomy window with regular vertebral contours and correct vertebral alignment on day 15 (items (A) to (C)) and day 30 (items (H) to (J)).In the PCL group, a radiopaque region was observed at the hemilaminectomy site, indicating the presence of bone remodeling. Furthermore, there was an absence of periosteal reaction with preserved vertebral contours and correct vertebral alignment on days 15 (as per items (D) to (F) of Figure 25) and 30 (as per items (K) to (M) of Figure 25). Radiographic results show excellent integration of the PCL into the soft and hard tissues of the implantation site, with biocompatibility demonstrated by the absence of changes compatible with inflammation or local rejection. Results of the in vivo study Description of the experiments Traumatic injury to the sciatic nerve

[0135] Seven-week-old female Lewis rats (Rattus norvegicus) were adapted and maintained under controlled humidity and temperature conditions with normal light / dark cycles and unrestricted water and food.All procedures were performed in accordance with the ethical principles established by the National Council for Animal Experimentation (CONCEA) and with the approval of the Ethics Committee on the Use of Animals (CEUA) / UNICAMP (protocol no. 6180-1 / 2023).

[0136] The experimental lesion of the sciatic nerve was performed under isoflurane anesthesia (Isoforine®, Cristalia, Itapira, SP, Brazil) using a microsurgical microscope (DF Vasconcelos, Valença, RJ, Brazil). Rats underwent critical lesion (8 mm defect) of the sciatic nerve. In the Autograft group, the nerves were sectioned in the proximal and distal regions, maintaining the 8 mm defect, and then perineural sutures were placed (10 / 0 Ethicon, Cincinnati, OH, USA). In the NGCs and NGC+FGF-2 groups, the nerves were lesioned in the proximal and distal regions, forming an 8 mm defect, and then the nerve stumps were fixed within the NGCs with perineural sutures (10 / 0 Ethicon, Cincinnati, OH, USA), as shown in Figure 26.The surgical planes were sutured (5 / 0, Vycril, Ethicon, Cincinnati, OH, USA) and tramadol was administered at a dose of 20 mg / kg / SC intraoperatively, followed by 5 mg / kg / day / SC for 5 days. Table 5. Experimental groups, number of animals and parameters evaluated. Autograft, autograft group; NGCs, NGCs group of PCL / GelMA in absence of FGF2; NGCs+FGF2, NGCs group of PCL / GelMA incorporated with FGF2. ENMG; electroneuromyography, TEM, transmission electron microscopy.Paradigm Groups No. Experimental Technique treated (n=5 / group) Catwalk and Autograft 7 / group Short-term NGCs (4 weeks) sensory evaluation NGCs+FGF2 Long-term (12 weeks) ENMG Autograft 7 / group Long-term NGCs (12 weeks) NGCs+FGF2 Muscle mass and Autograft 7 / group Long-term MET NGCs (12 weeks) NGCs+FGF2 Immunohistochemistry Autograft 3 / group Short-term NGCs (4 weeks) NGCs+FGF2 Gait Assessment – ​​Catwalk

[0137] The analysis of the animals' motor recovery was performed using the automated catwalk system (Noldus, Wageningen, Netherlands), weekly for 12 weeks in the Autograft, NGCs, and NGCs+FGF2 groups. Four runs were obtained and classified for each animal, with the following parameters evaluated: Peroneal nerve functionality index, contact area (cm. 2), maximum contact intensity, base of support (cm), and step sequence. Sensory Assessment

[0138] Nociceptive recovery was performed weekly for 12 weeks in the Autograft, NGCs, and NGCs+FGF2 groups. Mechanical sensitivity was assessed using the Von Frey test. Increasing pressure was applied to the plantar area of ​​the injured limb with a 0.5 mm polypropylene tip. 2coupled to a portable pressure force transducer (Anesthesiometer EFF 301, Insight, Ribeirão Preto, SP, Brazil) to obtain the pressure intensity for the withdrawal reflex. The intensity of hyperalgesia (Δ withdrawal threshold, g) was calculated by subtracting the value of the measurements after the treatments from the first measurement before the treatment. Muscle and body mass

[0139] Muscle mass was assessed at week 12 in the Autograft, NGCs, and NGCs+FGF2 groups. The tibialis cranialis and gastrocnemius muscles were collected, weighed to obtain the percentage of total muscle mass (g), and compared with the contralateral normal muscle. Body mass was measured using a precision scale weekly for 12 weeks in the Autograft, NGCs, and NGCs+FGF2 groups. Sciatic nerve immunostaining

[0140] Immunostaining analysis was performed post-mortem 4 weeks after in the experimental groups: Autograft (n=3), NGCs (n=3) and NGCs+FGF2 (n=3).The activation of Schwann cells (s100), cytoskeleton organization (neurofilament), and expression of neurotrophic factor receptors (P75) were determined. NTR), macrophage activation (IBA-1) and myelination (fluoromyelin). After transcardiac perfusion, specimens were collected and divided into 3 segments: proximal, central, and distal. The central segment (nervous tissue regenerated within the NGCs) was fixed in 4% formaldehyde in PB (0.1M, pH 7.4) for 12 hours at 4°C, immersed in 10%, 20%, and 30% sucrose solutions (0.1M PB, pH 7.4 for 12 hours), and embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, USA). Longitudinal cryosections (12 μm) were obtained. During immunostaining, the slides were incubated in the blocking solution (3%, BSA, 0.1M PB, pH 7.4) for 1 hour; Incubation solution containing the primary antibodies for 4 hours (Table 6); incubation with the conjugated secondary antibody for 45 minutes. Representative images were obtained using a fluorescence microscope (BX51, Olympus Corporation, Tokyo, Japan) and quantified using ImageJ software (version 1.33u, NIH, Bethesda, MD, USA). Table 6.Primary antibodies selected for immunolabeling in the sciatic nerve. Marker Company Host Code Dilution S100 Abcam Rabbit AB868 1:500 NF-H Millipore Rabbit AB1989 1:2000 P75 Santa Cruz Goat sc-6188 1:250 IBA-1 Wako Rabbit 019-19741 1:750 Fluoromyelin Invitrogen - F34651 1:300 Morphological analysis

[0141] Morphological analysis by transmission electron microscopy (TEM) was performed at week 12 in animals from the experimental groups Autograft, NGCs and NGCs+FGF2. Perfusion and fixation with Karnovsky's solution (2% glutaraldehyde and 1% paraformaldehyde in 0.2M PB, pH 7.34) were performed. The specimens were collected and divided into 3 segments: proximal, central, and distal. The central segment analyzed was divided into two segments: proximal and distal. Subsequently, post-fixation was performed with 1% osmium tetroxide diluted in 0.2M PB.The fragments were embedded in epoxy resin (Durcupan, Fluka - Sigma Aldrich), ultrathin sections (90 nm; Ultracut, Leica, Vienna, Germany) were obtained, and collected on copper meshes (mesh 200, EMS, Philadelphia, PA). After contrasting with uranyl acetate and lead citrate, the specimens were observed using a Tecnai G2 Spirit BioTwin transmission electron microscope (FEI, Eindhoven, Netherlands) operating at 80 kV. Results

[0142] Implantation of NGCs via a posterior approach to the sciatic nerve in rats was minimally invasive and rapid, as shown in Figure 1. The application of the perineural suture showed low complexity and ease due to the transparency of the canal. Additionally, the canal dimensions were adequate for the size of the nerve. No wound dehiscence or signs of inflammation were observed 3 months after NGC implantation in the experimental animals.Gait Assessment – ​​Catwalk

[0143] During the assessment of the peroneal nerve functional index, significant differences were observed between the NGCs+FGF2 group when compared to the Autograft (p < 0.05*) and NGCs (p < 0.01**) groups at week 1. Between weeks 2 and 7, no significant differences were detected between the Autograft, NGCs, and NGCs+FGF2 groups (p > 0.05). However, significant differences were observed between the Autograft group when compared to the NGCs group (p < 0.05*) and between the NGCs+FGF2 group when compared to the NGCs group (p < 0.01**) in weeks 11 and 12. Furthermore, in weeks 11 and 12, there were no differences between the Autograft and NGCs+FGF2 groups, as shown in Figure 27. The findings indicate increased functional motor recovery in animals subjected to autograft and treated with NGCs with controlled release of FGF2. In addition, the degree of functional recovery was similar between the groups treated with autograft and NGCs+FGF2.

[0144] During the contact area analysis, no significant differences were observed between the groups (p > 0.05). However, the maximum contact area showed significant differences between Autograft and NGC+FGF2 when compared to the NGCs group (p < 0.05*), in weeks 11 and 12, as shown in Figure 28. This indicates an increase in the contact surface of the injured limb in both groups treated with autograft and NGCs with controlled release of FGF2, with the degree of contact being equivalent.

[0145] During the analysis of thoracic limb base support, significant differences were observed between the Autograft group when compared to the NGCs and NGCs+FGF2 groups (p < 0.05*) at week 2. At week 11, a significant difference was detected between Autograft and NGCs (p < 0.05*), represented by Figure 29. The findings indicate a lower tendency for thoracic limb support in the Autograft group in the first weeks.However, from week 7 onwards, there was a tendency towards decreased support of the thoracic limbs in all experimental groups, indicating a shift in the base of support towards the pelvic limbs.

[0146] During the analysis of pelvic limb base support, significant differences were observed between the Autograft group compared to the NGCs group (p < 0.05*) at weeks 9, 11, and 12. No differences were observed in the comparison between NGCs and NGCs+FGF2 (p > 0.05) at weeks 9, 11, and 12, as shown in Figure 29. The findings indicate increased support in the injured limb in the autograft-treated group and a tendency towards increased support in the NGCs+FGF2 group. In contrast, the NGCs group without controlled release showed a tendency towards decreased support of the injured limb.

[0147] During the assessment of gait regularity, no significant differences were observed between the experimental groups (p > 0.05), as illustrated in Figure 30, at all evaluated times.The findings indicate regular gait patterns during locomotion. Sensory Assessment

[0148] During the assessment of hyperalgesia after mechanical stimulation, significant differences were observed between the Autograft and NGCs groups at weeks 10, 11, and 12 (p < 0.05*). Significant differences were observed between the NGCs+FGF2 and NGCs groups at weeks 3, 4, and 7 (p < 0.01**), respectively. Additionally, there were significant differences at weeks 8 and 12 when comparing the NGCs+FGF2 and NGCs groups (p < 0.05*), as shown in Figure 31. The findings indicate that the Autograft and NGCs+FGF2 groups with controlled release showed an equivalent reduction in hyperalgesia intensity, possibly controlling neuropathic pain over 12 weeks.Muscle and Body Mass

[0149] During the assessment of muscle mass, significant differences were observed between the normal contralateral tibial and gastrocnemius muscles when compared to the Autograft, NGCs, and NGCs+FGF2 groups, respectively (p < 0.001**), at week 12. In the comparison between Autograft and the NGCs and NGCs+FGF2 groups, there were significant differences at week 12 in the cranial tibial muscle (p < 0.01**) and gastrocnemius muscle (p < 0.001**), respectively. On the other hand, no significant differences were observed between the NGCs and NGCs + FGF2 groups in both the cranial tibial and gastrocnemius muscles (p > 0.05), as illustrated in Figure 32. The findings correlate with increased neuromuscular reinnervation in the autograft-treated group.

[0150] Regarding body mass, there were no significant differences during 12 weeks when comparing the Autograft, NGCs, and NGCs + FGF2 groups (p > 0.05), as illustrated in Figure 33.The findings show that the surgical procedures did not present systemic side effects and the experimental animals maintained a linear growth curve. Sciatic nerve immunostaining

[0151] During the evaluation of Schwann cell reactivity by means of S100 protein immunostaining, significant differences were observed in the comparison of the contralateral nerve with respect to the Autograft, NGCs and NGCs-FGF2 groups. Indicating an increase in immunostaining in all experimental groups. Among the experimental groups, a significant increase in S100 immunostaining was observed in NGCs+FGF2 when compared to Autograft (P < 0.05*), as shown in Figures 34 and 35. However, there were no significant differences between the NGCs and NGCs+FGF2 groups (p> 0.05). The findings indicate an increase in Schwann cell reactivity in the NGCs group with controlled FGF2 release.

[0152] During the evaluation of cytoskeletal organization using neurofilament protein, significant differences were observed when comparing the contralateral nerve with respect to the Autograft, NGCs, and NGCs-FGF2 groups. This indicates increased immunostaining in all experimental groups. Among the experimental groups, there were no significant differences between the Autograft, NGCs, and NGCs+FGF2 groups (p> 0.05), as shown in Figures 34 and 35. The findings indicate increased cytoskeletal organization after injury and repair in all experimental groups, being more evident in the Autograft and NGCs+FGF2 groups.

[0153] During the evaluation of macrophage reactivity after nerve repair using IBA-1 protein immunostaining, significant differences were observed when comparing the contralateral nerve with respect to the Autograft, NGCs, and NGCs-FGF2 groups. This indicates increased immunostaining in all experimental groups.Among the experimental groups, a significant increase in IBA-1 immunostaining was observed in the NGCs and NGCs+FGF2 groups when compared to Autograft (P < 0.05*), as shown in Figures 36 and 37. However, there were no significant differences between the NGCs and NGCs+FGF2 groups (p> 0.05). The findings indicate an increase in macrophage reactivity in the groups subjected to NGC implantation in the absence or presence of FGF2.

[0154] During the evaluation of P75 neurotrophin receptor immunostaining. NTRSignificant differences were observed when comparing the contralateral nerve with respect to the Autograft and NGCs-FGF2 groups (p < 0.01**). This indicates an increase in immunostaining in the groups submitted to autograft and treated with NGCs with controlled release of FGF2. However, a significant increase in immunostaining was observed in the NGCs+FGF2 group when compared to the Autograft (p < 0.01**) and NGCs (p < 0.001*) groups, as shown in Figures 36 and 37. The findings indicate greater expression of neurotrophin receptors after treatment of the injured nerve using NGCs with controlled release of FGF2.

[0155] In the qualitative evaluation of myelinated axon formation, the regenerated nerve within the tubular structure was evaluated for myelin sheath staining using fluoromyelin, showing complete reconnection of the nerve stumps in the Autograft, NGCs, and NGCs+FGF2 groups.However, the Autograft and NGCs+FGF2 groups showed greater red fluorescence, indicating a greater quantity and organization of myelinated axons, when compared to the NGCs group. Furthermore, in the NGCs+FGF2 group, an increase in DAPI-stained nuclei was noted, indicating greater cell proliferation, as shown in Figure 38. Morphological analysis

[0156] During the ultrastructural evaluation using transmission electron microscopy, better organization of nerve fibers was observed in the Autograft and NGCs+FGF2 groups when compared to NGCs, as shown in Figure 39, items (A)-(D). Although the axons in the NGCs+FGF2 group were of smaller caliber, an increase in number was observed when compared to Autograft and NGCs, as shown in item (D) of Figure 39.The diameter of the motor axons (white circle) surrounded by the myelin sheath (black circle) shows a larger diameter and myelin sheath thickness similar to the contralateral side in the Autograft and NGCs+FGF2 groups, when compared to the NGCs group, as shown in Figure 39, items (A)-(D). The collagen fibers within the endoneurium, consisting of thin cylindrical sheets, are distributed around the motor axons, as shown in Figure 39, item (E). A greater quantity of collagen fibers was noted in the groups treated with NGCs when compared to the Autograft group, as shown in Figure 39, items (E)-(H).

Claims

1 / 4 CLAIMS 1. Process for obtaining a biocompatible neuroprotective conduit with controlled release of bioactive molecules for nerve regeneration, characterized in that it comprises the steps of: (i) 3D printing of a sterile NGC and guide tube from the biomaterial PCL; (ii) sterilization of the NGC; (iii) preparation of sterile GelMA functionalized with the growth factor FGF-2; (iv) insertion of the guide tube into the NGC; (v) application of the GelMA functionalized with the growth factor FGF-2; (vi) photocrosslinking with a UV light head; and (vii) removal of the guide tube.

2. Process according to claim 1, characterized in that step (i) further comprises: a. characterization of bandages of the polycaprolactone (PCL) biomaterial; b. definition of the parameters for printing the NGC; and c.Continuous spiral deposition of PCL biomaterial with the following dimensions: inner diameter (ID) of 1845 ± 37.65 μm, outer diameter (ED) of 2307 ± 44.47 μm with a wall thickness of 461.3 ± 9.60 μm.

3. Process, according to claim 2, characterized in that the optimized parameters for printing the NGC consisted of a metal nozzle of 0.3 to 0.4 mm, 200 to 210 kPa pressure; 180 to 200. o C cylinder head temperature and 10 o Temperature range (C) of the print bed. 2 / 4 4. Process according to claim 1, characterized in that step (ii) further comprises: a. immersion of the NGC in 70% ethanol for 15 seconds; and b. washing with sterile DPBS, drying at a temperature of 20 to 22 0C and irradiation of the NGC with UV light in a wavelength range of 200 to 280 nm for 30 min.

5. Process according to claim 1, characterized in that step (iii) further comprises: a. diluting under stirring 10% (wt / v) porcine skin-derived gelatin in DPBS (pH = 7.4) at 50°C; b. adding 6% (wt / v) methacrylic anhydride at a rate of 0.5 mL / min under stirring conditions at 2500 rpm, allowing it to react for 60 to 180 min at 50°C; c. diluting the solution in two volumes of ultrapure or deionized water at 40°C; d. Dialyze the GelMA in dialysis membranes (12 kDa) for one week at 40°C to remove reaction byproducts and adjust the pH to 7.4 using a 1 M NaHCO3 solution; e. Filter the solution through 0.2 μm polystyrene vacuum filters; f. Freeze the precursor solution by flash freezing in liquid nitrogen (-196 0 C) and transfer to the freezer (-80 0C); g. freeze-dry the frozen precursor solution for a period of 5 to 7 days and store at -20ºC protected from light and moisture; 3 / 4 h. Weigh and dilute the lyophilized GelMA in DPBS at a concentration of 10% (w / v) and keep in an incubator at 37°C. o C for 30 minutes; I add the previously diluted 0.5% (w / v) Irgacure 2959 photoinitiator (70 o C / 30 minutes) and transfer the GeLMA+IC2959 precursor solution to the refrigerator for 12 hours (2 to 4 oC); j. incubate the GelMA+IC2959 precursor solution for 15 to 30 minutes to reverse gelation; and k. add 2 μg / mL of recombinant human protein FGF2-STAB previously reconstituted in DPBS.

6. Process according to claim 1, characterized in that step (v) comprises applying 50 μl of previously prepared and photocrosslinked 10% GelMA hydrogel with a UV light head for 240 s at a wavelength of 365 nm, allowing crosslinking and encapsulation of FGF-2 to the NGC.

7. Biocompatible neuroprotective conduit with controlled release of bioactive molecules for nerve regeneration, characterized in that it comprises a hybrid nerve guide conduit (NGC) consisting of an outer PCL wall coated with GelMA hydrogel and FGF-2. 8.Use of the neuroprotective conduit, as defined in claim 7, characterized by being for applications such as traumatic injuries due to complete laceration of the nerves of the upper and lower extremities and face, generating short-gap defects (<2.5cm) or long-gap defects (>2.5 cm); nerve injuries due to external crushing from trauma; nerve injuries subjected to decompression (carpal tunnel syndrome or cubital tunnel syndrome); adjuvants. 4 / 4 in nerve injuries undergoing end-to-end neurorrhaphy; adjuvants in nerve injuries undergoing autograft; compressive lesions of the nerve roots of the cauda equina; chronic paralysis resulting from spinal cord injury (SCI).

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