WO2019021320A2

WO2019021320A2 - A neural conduit to reconstitute and regenerate the degenerated or damaged nervous system

Info

Publication number: WO2019021320A2
Application number: PCT/IN2018/050496
Authority: WO
Inventors: Sandeep Kumar Vishwakarma; Aleem Ahmed Khan; Avinash Bardia; Syed Ameer Basha Paspala; Md. Aejaz Habeeb
Original assignee: Dr Habeebullah Life Sciences Limited
Priority date: 2017-07-27
Filing date: 2018-07-27
Publication date: 2019-01-31
Also published as: WO2019021320A3

Abstract

A neural conduit to reconstitute and regenerate the degenerated or damaged nervous system comprising of acellularized meningeal scaffold repopulated with functional neuronal cells. The said neural conduit can be used as a biological implant in neurological conditions for filling the tissue gaps and in spinal cord injuries. A method for producing the neural conduit, comprising of repopulating the acellularized meningeal scaffold with regenerative cells to produce a large number of functional neuronal cells to augment the damaged/dysfunctional neural circuits

Description

A neural conduit to reconstitute and regenerate the degenerated or damaged nervous system

This invention relates to implantable neural conduit developed using acellularization and neuronal cells repopulation technology. In particular, this invention relates to acellular neural conduit, their application for repair of damaged nerves/nerve junctions and the methods of preparing the same.

Human nervous system consists of brain, spinal cord, sensory organs and nerves which communicate information throughout the other body parts. This communication system involves two major type of cells named neurons and glia. Neurons are very specialized type of cells in the body which are also called as nerve cells. These nerve cells are responsible for transmitting the information in form of electrochemical signals.

The damage in nervous system results in impairment in electrochemical signaling that leads to several devastating neurological conditions. The earlier attempts to reconnect the damaged nervous system didn’t show significant improvement due to the absence of natural microenvironment and growth factors. Examples of methods used in such earlier attempts are described in US 7,255,871 and PCT/US01/03122.

Further, several synthetic biomaterials are disclosed in the prior art which use single tissue engineering or mechanical conduits. But all these biomaterials provide either electrical or only chemical stimuli from extrinsic sources and lack natural three dimensional extracellular brain matrix and growth factors. In addition the diversity in regenerative mechanisms resulted in unfavorable biological conditions to restore the damaged nerves system.

Some acellularized extra-cellular matrix-based biomaterials have been reported earlier. However, all acellularized scaffolds can’t be used in tissue regeneration. In case of neurological conditions, this particular perception is more complicated. Hence, bioscaffolds having capacity of similar regenerative mechanisms with host tissues and ability to provide more suitable microenvironment is of utmost importance during the development of such biomedical implants. Accordingly, understanding the components and complex natural biochemical stimuli existing in such scaffolds are the key factors to enhance their potential therapeutic applicability.

Therefore, the approaches or the methods disclosed in the prior art suffer with one or more draw drawbacks. Hence, there is need for a neural conduit which could mimic the robust nervous system regenerative mechanisms.

Accordingly this invention provides a neural conduit and a method for the development of the neural conduit using human brain meninges to reconstitute and regenerate the degenerated or damaged nervous system. The neural conduit described herein can be defined as a biological system which provides natural orchestral platform and suitable biological niche for connecting the neuronal circuits through intact terminal axons for proper electrochemical signaling.

The human brain meninges used in the neural conduit as described herein is a protective covering of the CNS which consists of three layers named dura matter, arachnoid matter and pia matter. These layers have a very complex anatomy and are completely different from the other membranes in the body.

The neural conduit as described herein comprises of an acellularized meningeal scaffold and regenerative cells. The acellularized meningeal scaffold comprises of cell deficient extra cellular matrix (ECM) and vascular bed that acts as a natural platform to choreograph the long-distance axonal guidance and organized neuronal cell growth and survival. Both the internal and external surfaces of the acellularized meningeal scaffold are three-dimensional and highly intact after acellularization. The acellularized meningeal scaffold represents a repository of several important brain cell stimulatory molecules which act as natural chaperons to guide, and support the long-term cell survival and function. The acellularized meningeal scaffold has significant mechanical strength and retains biological activity to promote regeneration of damaged/dysfunctional tissues. The acellularized meningeal scaffold further comprises of an array of ECM proteins and cytokines which helps to promote tissue regrowth/regeneration at the lesion-site.

The regenerative cells as described herein are human neuronal cells which are highly proliferative and possess unique ability to produce large number of functional neuronal cells for regenerative applications.

The method for producing the neural conduit as described herein comprises of repopulating the acellularized meningeal scaffold with regenerative cells to produce a large number of functional neuronal cells to augment the damaged/dysfunctional neural circuits. The neurogenic differentiation of stem cells on the acellularized meningeal scaffold provide unique natural platform for cellular engraftment, proliferation and generation of intact axonal neurons, and is useful to regenerate the constant vitality and architecture at the lesion site.

This invention also provides a method for producing acellularized meningeal scaffold. The method for developing the acellularized meningeal scaffold as described herein is based on the retrograde change of acellularization reagents using five different cell-damaging factors such as (a) mechanical cell damage with increasing the incubation time which favors the destruction of cells and its components; (b) cells lysis due to isotonic stress; (c) shear stress which allows detachment of cells and debris; (d) enzymatic detachment and (e) different gradients of detergent solutions which remove debris. The acellularization method described herein removes the cellular components from these tissues while preserving the intact ECM and vasculature bed. The acellularized meningeal scaffold generated using acellularization technology can be utilized for stem cells repopulation to generate functional neural conduit for regenerative applications. Mammals from which brain membranes can be obtained includes but not limited to rodents, rabbits, goats, pigs, dogs, cattle and humans. Brain tissues used herein are discarded human materials. The acellularization method described herein efficiently maintains the anatomical structures and active biological molecules responsible for cells attachment and survival on meningeal scaffold. This invention also provides a simplified method for increased production of natural meningeal scaffold as described in example 1.

In addition, the materials, methods and examples described in this invention are illustrative only and are not intended to be limited. All the research publications, patents and other relevant references mentioned are incorporated herein and in case of conflict, the specifications of the present invention including definitions will control.

The detailed embodiments of the present invention are set forth in accompanying the drawings and described below. Other information, objectives and advantages of this invention would be apparent from the drawing and described in detailed and claimed for their applicability.

Fig.1

shows the detailed schematic representation for the development of natural meningeal scaffold using acellularization technology which could be used to cultivate neurological cells to generate functional neural conduits for biomedical applications in spinal cord injury and other neurological conditions. This representation was drawn to provide realistic overview for the applicability of unique properties of meningeal scaffolds which provides native 3D-ECM, essential growth factors for the neural cells proliferation, organization, differentiation and directed axonal guidance which is required to develop extended neuronal conduits for nerve tissue regeneration.

Fig.2

demonstrates structural differences between native and acellularized meninges (A) microscopic observation showing the changes in the phenotype during acellularization process and (B) immune-histochemical staining with Hematoxylene and Eosin of micro-sections showing elimination of nuclear contents and preservation of intact ECM and 3D-architecture following acellularizationat different time points. (Scale bar: 40µm; Resolution: 10X)

Fig.3

shows (A) quantification of residual DNA content in native and acellularized meninges which reveals continuous decrease with increasing the time of acellularization and was almost absent in 240min acellularized meningeal scaffold (DM/240) (B) the tensile strength of acellularized meninges reduced up to 50% after complete acellularization (after 240min, DM/240). (C) preservation of glycosaminoglycan’s (GAGs) in acellularized meningeal scaffolds showed continuous decrease with increase in time of acellularization process and reduced up to 50% after 240min (DM/240). (D) Alcian blue staining of native and acellularized meninges showed positive staining. The staining intensity was reduced with increasing the acellularization process but the similar pattern of staining was observed throughout the material. (Scale bar: 40µm; Resolution: 10X).

Fig.4

shows (A) scanning electron microscopy (SEM) images for ultra-structure analysis of acellularized meningeal scaffolds showing intact architecture of ECM and 3D-organization to provide suitable microenvironment for neuronal cells survival and expansion (B) fibrous micro-structures of completely acellularized brain tissues. The 3D network of connective tissue fibresare arranged in specific manner in native form through the network of ECM proteins. All the three different meningeal layers are depicted by star marks in different colors.

Fig.5

shows the quantitative assessment of retained cytokines (C) vascular endothelial growth factor (VEGF) and (D) basic fibroblast growth factors (bFGF) in acellularized meningeal scaffolds showing significant higher amount of retention even after 240min of acellularization which was almost similar to native form.

Fig.6

demonstrates the schematic representation of the structural and functional cues directing the formation of follower new long stretched axonal tracts with respect to the existing pioneer axons on acellularized meningeal scaffolds which could be used to effectively bridge the lesion-sites or injuries.

Fig.7

shows significant higher percentage of neuronal cell (A) survival and (B) expansion on complete acellularized meningeal scaffolds (240min) as compared to the control condition (on fibronectin coated cover slips)

Fig.8

shows (A) immunofluorescence staining of differentiated neurons on acellularized meningeal scaffolds with positive expression of neuronal specific marker β-tubulin III (red) and green fluorescent protein (GFP, green) (Scale bar: 40µm; Resolution: 10X)(B) high resolution fluorescence images of neurons differentiated on acellularized meningeal scaffolds showing structural cues directing the long axonal outgrowth along the pioneer axonal tract and formation of new synapses due to spatial configuration of neurons and scaffold in support of growth factors and cell adhesion molecules. (Scale bar: 40µm, Resolution: 20X and Scale bar: 100µm, Resolution: 40X& 60X).

Fig.9

shows microscopic investigation of the engineered biological neural conduits (A) highly specialized arrangement of neural cells with high adherence was observed at day 7 under the influence of mechanical and structural cues (B) the formation of more follower neurons were observed at day 14 and (C-D) the differentiated neurons showed formation of new synapses and well-connected structural organization with long-stretched axonal tracts and well connectivity at day 21 (Scale bar: 100µm; Resolution: 10X).

The reference symbols in drawings and figures are represented as: FM: fresh/native membrane, DM/30: acellularized membrane after 30min, DM/60: acellularized membrane after 60min (one hour), DM/120: acellularized membrane after 120min (two hours), DM/240: acellularized membrane after 240min (four hours).

The invention described herein provides neural conduit developed by using acellularized meningeal scaffold repopulated with human neuronal cells to augment the damaged/dysfunctional neural circuits. This particular conduit is useful to regenerate the constant vitality and architecture at the lesion site. The acellularized meningeal scaffold of this invention has both chemotactic and mechanical cues.

The neural conduit as described herein provide preformed neuronal constructs with differentiated human neuronal cells and extended axonal tracts. This promising strategy for neuro-structured meningeal conduit may be simultaneously capable of physical restoration of damaged axonal connections, addressing the neuronal cell replacement and neuronal circuit modulation which may challenge the current quo of hardware-based neural remodulation.

The invention described herein is premised on the grace of bioengineered neural networks, whereby neurons have intrinsic ability to sense and respond to local activity. The generation of intact long-axonal tract in follower neuron in correspond to the pioneer neuron suggests that these bioengineered tissue constructs could be transplanted directly to rebuilt the damaged axonal tracts and activate the host neuronal network to fasten the regenerative processes.

The utility of this invention could offer the resistant neurological construct to the underlying pathophysiology of neurodegenerative conditions. Another important aspect of this invention is to bridge the long-gaps formed between neurons due to the developmental defects or injury. In view of limited success in this perspective, the growth of neuronal axons on acellularized membranes provide preserved plasticity of the biologically active scaffolds in directed guidance for arrangement and physical wiring of long-stretched axonal tracts.

Human brain tissues have three major components, the ECM, neurological cells and embedded vasculature. The acellularization technology described herein removes the cellular components from these tissues while preserving the intact ECM and vasculature bed. The acellularized meningeal scaffold generated using acellularization technology can be utilized for stem cells repopulation to generate functional biological neural conduits for regenerative applications. Mammals from which brain membranes can be obtained includes without limitation, rodents, rabbits, goats, pigs, dogs, cattle and humans. Brain tissues used herein are discarded human materials.

Herein, the acellularization method accompanied for meningeal membranes utilizes gradients of acellularization reagents which allow homogeneous removal of cellular components much faster. The acellularized meningeal scaffold generated herein has enormous potential for clinical translation as biological and mechanical support for restoration of damaged nervous system. This strategy could overcome on currently available approaches for neuronal replacement, neuromodulation and axonal tract regeneration further to establish electrochemical signaling.

More detailed description of methods useful for developing meningeal neural conduit according to the invention described herein is provided in the following examples. While certain methods for exemplified below, the invention is not so limited and skilled artisan can extend its wide range of applicability upon consideration thereof.

Example 1

Development of acellularized meningeal scaffold

Herein an exemplary method for developing the acellularized meningeal scaffold has been described for its applicability according to the invention described herein. This example includes generation of brain membrane scaffold by utilizing a gradient of reagents while keeping the tissue in a shaking incubator at 37°C temperature. Initial acellularization was performed by incubating meninges with 1X PBS supplemented with 0.05% Tween-20. Second incubation was followed with 0.1% SDS in 1X PBS with NaCl for 5 min and the final incubation was done in 0.5% SDS until the removal of entire cellular components. The acellularized meningeal scaffold was then washed thrice with 1X PBS to remove SDS and other chemicals remained inside the membrane.

This method was opted to generate acellularized meningeal scaffold without catheterization and continuous flow of reagents inside the vessels. The acellularization of meninges was completed in less than four hours of incubation which was identified by translucent nature due to dissolution of cells. The method based on the retrograde change of reagents successfully removes the cellular components from the membrane while retaining its intact vasculature and ECM.

Example 2

Characterization of acellularized meningeal scaffold

In view of the application of the current invention, the acellularized meningeal scaffold was characterized at different levels such as complete removal of nucleic acids, retention of intact ECM and preservation of natural architecture.

To identify the residual nucleic acid content in meningeal scaffold during acellularization process, lysate of membrane ECM was prepared after 30min, 60min, 90min, 120min, 150min, 180min, 210min and 240min of acellularization process after digestion in papain solution (0.1%), 1mM EDTA, 7.0mM cystein and 1M NaCl in 1X PBS at 60°C for 48h on incubator shaker. The existence of residual nucleic acids was quantified by spectrophotometric analysis at 260 and 280nm.The absence of nucleic acids in acellularized meningeal scaffold was analyzed by the absence of DNA material through quantification. The absorbance of residual nucleic acid content in lysate showed continuous decrease in DNA content with increasing the acellularization time and was almost negligible after 240min.

To identify the anatomical structures before and after acellularization of meningeal scaffold microscopic analysis was performed at different time points which demonstrated the clearance of cellular materials with increasing the incubation time with different gradients of the solutions. Fresh membranes have intact vascular web with red blood cells and other cells on the ECM which gives it very blunt appearance under light microscope. However, during acellularization process the amount of RBCs and other types of cells gets reduced with the time due to lysis. After 4h (240min) of acellularization, the brain membranes showed clear, intact and transparent vascular network in all three dimensions. The membrane vasculature and ECM were competed translucent after 240min of acellularization.

To identify the presence of residual nucleic acid and intact ECM, acellularized meningeal scaffold was fixed in 4% formalin solution at room temperature for 15-20 min. Following to fixation, the paraffin blocks were prepared for acellularized membranes. 3-5µm thin sections were prepared and stained with H&E stain. Histological evaluation by H&E staining of acellularized brain membranes showed gradual decrease in the presence of cells and their components with increasing the time and was completely absent after 240min. Histological analysis also revealed preserved native ECM and vascular architecture within the acellularized membranes.

Immunofluorescence staining was performed to identify the retained ECM after acellularization. The antigen retrieval was performed for the micro sections of acellularized membranes and further stained with Alcian blue. The existence of native GAGs and ECM was the paramount goal during development of acellularized membranes. The histological analysis revealed the preservation of GAGs during acellularization process as demonstrated by Alcian blue staining. Further the quantitative analysis of GAGs present in ECM of DM was quantified which revealed almost 50% existence of GAGs in complete acellularized membranes (240min). The preservation of GAGs in acellularized membranes demonstrates the retention of naturally present cytokines and their activity.

SEM examination was performed to assess the ultra-structure of acellularized meningeal scaffolds. For this the acellularized meningeal scaffold was lyophilized as per the standard processing protocol. Fixation and dehydration was not performed before the SEM. The cross sections were prepared, mounted and subjected for SEM analysis using JOEL-JSM 5600 SEM at RUSKA Lab’s College of Veterinary Science, SVVU, Rajendranagar, Hyderabad, India (Bozzola and Russell, 1998). The surface and cross-section demonstrated the fibrous micro-structures of membrane tissues which appeared to be maintained in native form. The key architectural structures such as vascular tracts, and ECM were clearly recognized with their peculiar features and distribution within the acellular membranes. The 3D network of connective tissue fibres arranged in specific manner in native form through the network of ECM proteins. Overall, the SEM analysis confirms the preservation of 3D microanatomy and ultrastructure following acellularization.

To determine the mechanical integrity, tensile strength analysis of acellularized membranes was performed according to ASTM D1708 (Yoshioka et al. 2007). Prior to this analysis acellularized meningeal scaffold was completely hydrated in 0.9% saline. The tensile strength was estimated by pulling the samples at 50mm/min of failure using a mechanical test stand and reported in N/cm of sample width. The tensile strength of native membranes was higher (4.8±0.75 N/cm) than the acellular membranes. This strength was continuous decreased with the acellularization time and was almost 50% less after complete acellularization process (240min) representing tensile strength of approximately 2.8±0.51 N/cm.

Example 3

Biological active molecules of interest in acellularized meningeal scaffold

The retention of natural growth factors and cytokines in acellularized meningeal scaffold showed significantly high amount of VEGF and bFGF which was almost similar to the native membranes. The retention of VEGF and bFGF cytokines in ECM of acellular brain membranes similar to the native form is an important aspect of its biological activity as a cellular implantable biological material. The amount of residual cytokines in acellularized brain membranes was identified by ELISA using VEGF (RAB0507-1KT, Sigma Aldrich) and FGF kits (RAB0182-1KT, Sigma Aldrich).

Residual GAGs post-acellularization were identified byimmuno-histochemicalanalysis. The rehydrated acellularized ECM and native meningeal paraffin sections were stained with Alcian blue (B8438-250ML, Sigma Aldrich) to compare the distribution of residual GAGs in acellularized meningeal ECM. The histological analysis revealed the preservation of GAGs throughout the acellularization process as demonstrated by Alcian blue staining. Further the quantitative analysis of GAGs present in ECM of acellular meningeal scaffold was quantified which revealed almost 50% existence of GAGs after complete acellularization (240min). The preservation of GAGs in acellular meningeal scaffold demonstrates the retention of naturally present cytokines and their activity which is of utmost important for cellular proliferation and regeneration.

Example 4

Culture of neurological cells on acellularized meningeal scaffold

The following example describes the culture of human neuronal cells on acellularized meningeal scaffold to generate the biological meningeal neural conduit according to the invention. Cryopreserved human neural precursor cells (hNPCs) were cultured on sterilized acellularized ECM of meningeal scaffold. Briefly, 1.5 X 105hNPCs were seeded on per cm2 area of the membranes in neural differentiation medium supplemented with Retinoic acid and 2% FBS as described earlier (Vishwakarma et al. 2013). At

day

3, 7, 14 and 21 the cells on membrane ECM were fixed in 4% buffered formalin for further analysis. The composition and configuration of acellular meningeal scaffold provides orchestral natural platform for cells for conductive growth which are well identified in the art.

To identify the neuronal cell adhesion and viability on acellularized meningeal scaffold, after 24h of cell seeding MTT assay was performed as per the standard protocol described by Mosmann (1983). Cells on fibronectin coated coverslips (GG-12-fibronectin, NeuVitro, USA) were used as control to compare the difference in cell adhesion and viability percentage. In addition to this, MTT cell proliferation assay was performed at

day

3, 7, 14 and 21 to identify the percentage cell growth as compared to the control condition. The analysis of percentage cell viability on acellularized meningeal scaffold demonstrated higher cell viability (95±4%) after 24h of cells seeding as compared to the control condition (86±5%, fibronectin coated cover slips). Further the growth of neuronal cells during ex-vivo differentiation after retinoic acid mediated stimulation showed higher growth potential of differentiating neurons on acellularized meningeal scaffold as compared to the control. This method was followed to obtain more number of neurons as compared to glial cells to generate implantable and intact biological neural conduits in SCI and other neurological conditions.

Example 5

Generation of meningeal neural conduit

To investigate the development of meningeal neural conduit, neuronal differentiating ability of hNPCs on acellularized meningeal scaffold was identified at day 14 by immunofluorescence staining using β tubulin-III antibody (ab78078, Abcam, USA) and further labeled with green fluorescencent protein (GFP). The fluorescence images were captured and documented using Axiocam software (version 4.1.2) in inverted fluorescence microscopy (Carl Ziess, Germany). Staining with β-tubulin III confirmed the well organized and aligned differentiated neuronal cells on 3D-natural acellular membranes. The expression of green fluorescence protein (GFP) by neuronal cells and axonal process of neurons stained with β-tubulin III showed coalesced into discrete 3D-acellular membrane architecture spanning the cellular population directly adhered to the meningeal scaffold.

Generation of neuronal cells with long-axonal tracts on acellular membranes supposed to provide hepatotactic, chemotactic and neurotrophic cues for long-term cells survival, efficient cells proliferation and differentiation. To demonstrate this, cells on acellularized meningeal scaffold were observed under inverted microscope and carefully investigated the cellular networks on the scaffold at day 7, day 14 and day 21 post-neurogenic differentiation in conditioned medium. The light microscopic images were captured in 10X to identify the long-distance neurons growing in guiding manner.Further, high resolution microscopy analysis revealed that acellularized meningeal scaffold support the generation of long neuronal axons first termed as pioneer axon which provides the cues for following generating neurons by forming new synapses. This event is crucial to provide cues for the development of new long follower axonal tract and host axons during regeneration process and passing the electrochemical signals.

As is evident from the foregoing description, certain aspects of the invention described herein are not limited by the particular details of the examples illustrated herein, and it is therefore contemplated that other modifications and applications, or equivalents thereof, will occur to those skilled in the art. It is accordingly intended that the claims shall cover all such modifications and applications that do not depart from the sprit and scope of the invention described herein. Other aspects, objects and advantages of the invention described herein can be obtained from a study of the drawings, the disclosure and the appended claims.

Citation List follows:

Philip H and Harvey A. Fishman.Nanotube mat with an array of conduits for biological cells. 2007, US 7,255,871 B2.

HADLOCK Theresa A and SUNDBACK Cathryn A. Neural regeneration conduit. 2001, WO 01/54593A1.

Bozzola JJ and Russell LD. In: Electron microscopy principles and techniques for biologists, second ed., Jones and bartlertt publishers, Sudbury, Massachusetts. 1998, pp. 19-24, 54-55, 63-67.

Yoshioka I, Saiki Y, Sakuma K, Iguchi A, Moriya T, Ikada Y, Tabayashi K. Bioabsorbable gelatin sheets latticed with polyglycolic acid can eliminate pericardial adhesion, Ann Thorac Surg. 84 (2007) 864e70.

Vishwakarma Sk, Paspala SAB, Bardia A, Tiwari SK, Srinivas G, Avinash R, Khan AA. Isolation and characterization of neural precursor cells from different regions of human fetal brain: Assessment of in vitro proliferation and differentiation, Int J Adv Res 1 (2013) 782-793.

Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, J. Immunol. Meth. 65 (1983) 55–63..

Claims

A neural conduit to reconstitute and regenerate the degenerated or damaged nervous system comprising of acellularized meningeal scaffold repopulated with functional neuronal cells.
The neural conduit according to claim 1, wherein the acellularized meningeal scaffold comprises of cell deficient extra-cellular matrix (ECM) and vascular bed.
The neural conduit according to claim 1, wherein the acellularized meningeal scaffold comprises of an array of ECM proteins and cytokines which helps to promote tissue regrowth/regeneration at the lesion-site.
The neural conduit according to claim 1, wherein the internal and external surfaces of the acellularized meningeal scaffold are three-dimensional and highly intact after acellularization.
The neural conduit according to claim 1, wherein the acellularized meningeal scaffold is obtained from the brain membranes of mammal.
The neural conduit according to claim 5, wherein the mammal is rodent, rabbit, goat, pig, dog, cattle or human.
The neural conduit according to claim 1, wherein the regenerative cells are neural stem cells.
The neural conduit according to claim 1, wherein the neural conduit can be used as a biological implant in neurological conditions for bridging the gaps formed between neurons due to the developmental defects or injury.
The neural conduit according to claim 1, wherein the neural conduit is used as a biological implant in spinal cord injury.
The neural conduit according to claim 1, wherein method for producing acellularized meningeal scaffold comprises of retrograde change of acellularization reagents using five different cell-damaging factors such as (a) mechanical cell damage with increasing the incubation time which favors the destruction of cells and its components; (b) cells lysis due to isotonic stress; (c) shear stress which allows detachment of cells and debris; (d) enzymatic detachment and (e) different gradients of detergent solutions which remove debris.
A method for producing the neural conduit of claim 1, comprising of repopulating the acellularized meningeal scaffold with regenerative cells to produce a large number of functional neuronal cells to augment the damaged/dysfunctional neural circuits.