Printable biomaterials for 3D brain regenerative scaffolds: An in vivo biocompatibility assessment

Abstract

Background: Brain regeneration after injury is a challenge being tackled by numerous therapeutic strategies in pre-clinical development. There is growing interest in scaffolds implanted in brain lesions. Developments in 3D printing offer the possibility of designing complex structures of varying compositions adapted to tissue anatomy.

Methods: This feasibility study assessed the cerebral biocompatibility of four bioeliminable Digital Light Processing (DLP) printed materials in the rat model: gelatin methacrylate (GelMA), poly(ethylene glycol)diacrylate (PEGDA) mixed with GelMA (PEGDA-GelMA), poly(trimethylene carbonate) trimethacrylate (PTMC-tMA) and an ABA triblock copolymer of polypropylene fumarate-b-poly γ-methyl ε-caprolactone-b-polypropylene fumarate (P(PF-MCL-PF)). Their tolerance was compared to that of polydioxanone Ethicon (PDSII), a neurosurgery suture component commonly used in clinical practice. A one-month MRI and behavioral follow-up aided in safety assessment.

Results: High-resolution T2 MRI imaging effectively captured the scaffold structures and demonstrated its non-invasive utility in monitoring degradability. PDSII served as a control of the acceptable inflammatory response to implantable foreign bodies. GelMA, PEGDA-GelMA and PTMC-tMA did not affect the permissive glial barrier, promoted cell migration, and neovascularization without additional perilesional microglial inflammation (median mean of 6.5 %, compared to 8.2 % for the PDSII control). However, the GelMA scaffold core was not colonized and allowed a limited neuronal progenitors recruitment. The rigidity of PTMC-tMA facilitated insertion, but posed histological issues. The brain hardly reacted to the P(PF-MCL-PF).

Conclusion: All these materials can serve as a basis for brain regeneration. PEGDA-GelMA emerged as a promising candidate for intracerebral implantation, combining biophysical and bioprinting advantages while maintaining an acceptable level of inflammation compared with clinically used suture, paving the way for innovative therapies.

Keywords: 3D printing; Brain repair; MRI; Scaffold; Tissue bioengineering.

Graphical abstract

Fig. 1

Study protocol overview. The time-points shown on the top line illustrate the key experimental steps. 3D printing of biomaterials (grey line): The protocol begins with the optimization of printing methods, followed by the printing and decontamination of scaffolds for in vivo implantation. Day zero of the protocol is launched with the injection of malonate for lesion induction. One week later, scaffolds are implanted in the lesion. The bottom line shows the kinetics of the behavioral tests (pink line): for training, tests started before the brain injury and were performed at different times after brain lesion: three days and one, two, three and four weeks. MRI acquisitions (logo screen): three successive MRI sessions: post-lesion = pre-implantation, post-implantation and pre-sacrifice at 1 month. Animal sacrifice: for histological analysis.

Fig. 2

Scaffold CAD model and design after printing. A [1]: 3D model for GelMA and PEGDA-GelMA, including a handle on top for handling [2]. image of the structure and pores under a white light microscope x20 [3]. image of Eosin-stained PEGDA-GelMA under a white light microscope x20. B [1]: 3D model for PTMC-MA, including a handle on top for handling. Corners are cut so that the implant can pass through the 5 mm diameter round hole drilled in the skull. The scaffold height without the handle is 6 mm [2]. Printed scaffold held by its handle with custom-made tool (left); Scaffold on a glass slice (top); Close-up on the scaffold (bottom), with visible canals [3]. Side view of the scaffold under a light microscope, with the handle positioned at the top (out of frame). The channels running through the structure are clearly observable, and the bright areas at some of the intersections correspond to points of contact between the scaffold and the glass slide, hence altering the light path [4]. Micro-CT imaging. The size of the scaffold can be adapted to the size of the lesion (model A or B).

Fig. 3

Behavioral tests. (A) Grip strength test of the dominant front paw, contralateral to the injected hemisphere, compared to the pre-injury value, expressed in percent. (B) The NSS score shows sensorimotor deficits after malonate injury (score rated out of 16). The graphs show the score curves obtained for each animal in each group at the different time points of the protocol. GelMA: n = 2; PEGDA-GelMA: n = 2; PTMC-tMA: n = 2; PDSII: n = 1; Injured: n = 1. The grey and beige bands represent data sets from Injured (n = 8) and Sham (n = 8) reference groups at 1 month, respectively.

Fig. 4

T2 MRI and histological sections of control and implanted lesioned rat brains. The left panel showed T2 MRIs at one-week and one-month post-implantation, respectively. The right panel showed hematoxilin-eosin-stained sections of rat brains at 1 month. (A–B) T2 MRIs after the implantation of GelMA, bregma 0.48 mm. (C) GelMA rat brain HE sections showing the fragmented scaffold at the lesion site, bregma 0.48 mm. (D–E) Focus on the peri-scaffold and GelMA scaffold area, respectively. Perilesional brain tissue showed a chronic inflammatory reaction and granulation tissue composed of macrophages, fibroblasts and capillaries. (F–G) T2 MRIs after the implantation of PEGDA-GelMA, bregma 0.24 mm. (H) PEGDA-GelMA rat brain HE sections showing part of the scaffold at the lesion site, bregma 0.24 mm. (I–J) Focus on the peri-scaffold and PEGDA-GelMA scaffold area, respectively. Perilesional brain tissue showed a weak chronic inflammatory response and granulation tissue comprising macrophages, fibroblasts and capillaries. (K–L) T2 MRIs after the implantation of PTMC-tMA, bregma 0.84 mm. (M) PTMC-tMA rat brain HE sections showing tissues that have migrated into the scaffold at the lesion site, bregma 1.08 mm. (N–O) Focus on the peri-scaffold and PTMC-tMA scaffold area, respectively. Perilesional brain tissue showed a weak focal chronic inflammatory reaction. (E/J/O) 40x magnification on capillaries (black arrow). (P–Q) T2 MRIs after the implantation of PDSII sutures, bregma 0.24 mm. (R) PDSII rat brain HE sections, bregma −0.00 mm. (S–T) Focus on the peri and suture threads area respectively. (U–V) T2 MRIs after the lesion, bregma 0.24 mm. The hyperintense lesion stabilized after 1 month. The ventricle is dilated due to striatum atrophy. (W) 1-month control rat HE brains section, bregma 0.96 mm. (X) The peri-injured brain tissue shows a thin, stabilized reactive meshwork in contact with the lesion, with few vessels. Normal brain tissue can be seen at a distance all around the injured area. (Y) Boxed area in X, magnification showing no colonization of the lesioned area by cells Scale bar: 5 mm (MRI); 1000 μm (left 20X histology); 100 μm (for 20X histology zoom middle and right) except for (X), 50 μm. HE: hematoxylin-eosin.

Fig. 5

I - Histology of trichrome stained brain sections and characterization of perilesional and implanted areas. (A, D, G, J, M): topography of lesions at low magnification on coronal sections stained with Masson's trichrome (left). The collagen deposit is identified by the blue coloration surrounding the scaffolds. Higher magnification shows perilesional brain tissue (orange boxed area) and peri-scaffold reactions (green boxed area). For GelMA (B–C), the peripheral staining is weak and physiologic with few loose fibers in the tissue, and collagen rings around the vessels can be seen. For PEGDA-GelMA (E–F), the staining is weak and physiologic with few loose fibers in the tissue, and collagen rings around the vessels can be seen. For PTMC-tMA (H–I), there was a presence of a fine collagenous mesh at the peripheral tissue-scaffold interface. For PDSII (K–L), normal-appearing peripheral tissue, with only a collagen ring at the suture section edge. For the injured rat (N–O), slight reorganization around the lesion, at the forming glial scar level, with few collagen fibers at the lesion edge and collagen around the vessels. II - Histology of Glial fibrillary acidic protein (GFAP) stained brain sections and characterization of the glial barrier after lesion and implantation. (A, D, G, J, M): topography of lesions at low magnification on coronal sections stained with Glial fibrillary protein (GFAP) (left). The GFAP deposit is identified by the brown coloration of astrocytes(/glial cells) surrounding the scaffolds. Higher magnification shows perilesional brain tissue (orange boxed area) and peri-scaffold reactions (grey boxed area). For GelMA (B), heterogeneous glial scarring can be seen at the scaffold/host tissue interface. (C) No astrocytic population appears to be present within the implant. For PEGDA-GelMA (E), a discrete glial barrier is visible at the implant/host tissue interface. (F) No astrocytic population appears to be present within the implant. For PTMC-tMA (H), a glial barrier molded to the edges of the scaffold can be seen at the scaffold/host tissue interface. (I) Some astrocytes appear to be present inside the scaffold. For PDSII (K–L), a strong astrocytic reaction visible at the lesion edge with a thin barrier around the sutures, reconstructing tissue poor in astrocytes. For the injured rat (N–O), thin glial barrier at the lesion/host tissue interface, astrocytes aligned parallel to the lesion. Scale bar: 1000 μm (left histology (slide scan x20), whole slice); 100 μm (for histology zoom middle and right) except for (O), 50 μm.

Fig. 6

Characterization of lesion edges: glial barrier and microglia. (A) Representative images of glial barrier observed on the GFAP-stained sections. (B) Quantification of brains glial barrier thickness. GelMA: n = 2; PEGDA-GelMA: n = 2; PTMC-tMA: n = 2; PDSII: n = 1; Injured: n = 1. Measurements made in 4 independent fields of one representative slice for each rat separated on the x axis. (C) Representative images of the microglial dispersion observed at the lesion edge on the different immunofluorescence sections labelled with Iba1. (D) Estimated number of Iba1+ cells (%) around scaffold or lesion. Measurements made in 4 independent fields for each rat on two different level slices. The points on the graphs represent the data measured per field for each rat separated on the x axis, with their respective medians and interquartile. Scale bar (slide scan x20): (A) 50 μm; (C) 100 μm. GFAP: glial fibrillary acidic protein.

Fig. 7

Characterization of colonizing tissue in the scaffold: Neural, glial and vascular cells. Representative scaffold images for each marker. Measurements taken in 3–6 independent fields per rat on two to three slices. One graph per estimated cell type (A, B, C). The points on the graphs represent the data measured per field for each rat, with their respective medians and interquartile. Scale bar: 100 μm, 20X magnification. S: Scaffold; C: Cortex; V: Ventricle; DCX: Doublecortin; GFAP: glial fibrillary acidic protein.