The present invention relates to the field of nerve tissue engineering.
More particularly, the invention relates a novel in-vitro method for inducing cells to
produce a tissue engineered construct for nerve regeneration and repair in a synergistic
manner.
More particularly, the invention relates to a living cells-based nerve conduit/matrix, which
has tissue like properties and is capable of being used for nerve regeneration and repair.
More particularly, the invention relates to a cells-based nerve conduit/matrix for the
treatment of peripheral nerve injury (PNI), spinal cord injury and any other type of nerve
injuries.
BACKGROUND AND PRIOR ART OF THE INVENTION
The field of tissue engineering combines bioengineering methods with the principles of life
sciences to understand the structural and functional relationships in normal and
pathological mammalian tissues. The goal of tissue engineering is the development and
ultimate application of biological substitutes to restore, maintain, or improve tissue
functions. Thus, through tissue engineering, it is possible to design and manufacture a
bioengineered tissue construct in vitro. Bioengineered tissues construct include cells that
are associated with a human tissues and natural matrix/scaffold. The new bioengineered
tissue must be functional when grafted onto a host, and be permanently incorporated
within the host's body or progressively bio-remodeled by cells from the recipient host
patient. Fabrication of a tissue equivalent without a support member or scaffold leads to
scientific challenges in creating the new bioengineered tissue.
The peripheral nervous system (PNS) extends outside the central nervous system (CNS)
and provides the functions of, amongst other things, bringing sensory information to the
CNS and receiving motor commands from the CNS, coordinating body movements and
controlling the involuntary muscles. Unlike the central nervous system, the PNS is not
protected by bone and is therefore vulnerable to injuries.
Damage to nerves of the PNS can cause significant motor or sensory impairment. In
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particular, patients with acute peripheral nerve injury usually have nerve conduction
defects that can manifest as motor impairment or sensory dysfunction. Depending on the
severity of the injury and the nerve affected, a severed nerve may cause paralysis, partial
loss of mobility of the affected limb and/or a loss of sensation. Nerve and muscle atrophy
will follow if no sufficient recovery occurs or no timely treatment is provided. Similarly,
crush damage to peripheral nerves can result in reduced motor or sensory performance.
Injuries to peripheral nerves can be caused by trauma, Surgery, cancer and by congenital
anomalies. Injuries to peripheral nerves can be also caused by radiation therapy,
chemotherapy, metabolic/endocrine complications, inflammatory and autoimmune
diseases, vitamin deficiencies, infectious diseases, toxic causes, accidental exposure to
organic metals and heavy metals, drugs, amputations and disease or condition relating to
a loss of motor or sensory nerve function. Nerve injury or lesion may include nerve
transection, crush, compression, stretch, laceration (sharps or bone fragments), ischemia
and blast. In addition, nerve injury or lesion may result from damage or disruption of the
neuronal axons.
Peripheral nerve injury is a major source of morbidity and an area with significant medical
need. Indeed, only 50% of patients achieve good to normal restoration of function
following Surgical repair, regardless of the strategy. Moreover, failure of nerve
regeneration may necessitate amputation of an otherwise salvaged limb. This stems from
the inadequacy of current PNI repair strategies, where even the ―gold- standard
treatment—the nerve autograft is largely ineffective for major nerve trauma, defined as
loss of a large segment of nerve (i.e. >5 cm) or injury occurring closer to the spinal cord
(e.g., shoulder, thigh) resulting in extremely long distances for axon regeneration to distal
tar gets (e.g., hand, foot). Despite significant efforts, PNI repair has not progressed past
nerve guidance tubes (NGTs) for the Dec. 10, 2015
Surgical intervention is required if there is to be any prospect of repairing severed
peripheral nerves. One surgical technique for attempting growth of a peripheral nerve
involves providing a scaffold, usually in the form of a conduit, at the site of the nerve
damage, to facilitate and encourage the extension of regenerating axons. Specifically, the
scaffold is selected to provide an environment that will encourage nerve growth so that
nerve function can be returned. To date, success rates for peripheral nerve growth have
4
been low and it is presently not possible to achieve the extent of peripheral nerve growth
that would be required in order to repair many of the injuries experienced by peripheral
nerves.
When fabricating the next generation of nerve conduits, several aspects need to be
considered. These include the biocompatibility of the materials, and the customized
mechanical and degradation properties. Single-channel collagen conduits that possess a
mechanical strength and are easy to process have previously been used in nerve
regeneration applications.
A number of nerve conduits are FDA approved for relatively short nerve defects, such as
Integra Neurosciences Type I collagen tube, NeuraGen TM, Collagen Matrix Inc.’s
neuroflex and Synovis Surgical Innovations Gem Neurptube TM. These are single-channel
tubes which are used only for small defects of several millimeters and do not address
larger peripheral nerve injuries. In addition, axons regenerating across these single lumen
tubes assume a dispersed direction, resulting in inappropriate target re-enervation and
the co-contraction of opposing muscles or synkinesis.
In spite of the evolution of the surgical microscope and a prodigious effort into refining
techniques for accurate nerve approximation, the clinical results of surgical nerve repair
are still disappointing. Scar tissue resulting from the surgical manipulations required for
direct proximal-to-distal nerve suture frequently interferes with the growth of proximal
stump axons into the distal nerve stump. If a substantial number of axons are prevented
from crossing the anastomotic site, neuroma (painful nerve cell tumor) formation often
results. As a result, prospects for achieving significant reinnervation are reduced. The end
result is a lack of full return of motor and/or sensory function.
Additionally, the regenerative potential of the damaged proximal nerve is frequently
unpredictable and poorly understood. Severe nerve injuries have required microsurgical
grafting to span a defect. This technique involves surgically grafting a piece of a nerve,
from another part of the body, this approach too has certain limitations. The area from
which the nerve was removed is left without sensation. Moreover, the amount of nerve
tissue that can reasonably be removed for such grafts is also limited. However, suture
techniques and/or grafting have not always been sufficient for repair of a severe defect.
Still further, suture under tension, gap reduction by stretching, mobilization, flexion of a
joint, or rerouting may compromise sensitive intraneuronal vascularity, and autografts
5
induce a second surgical site with requisite risks and com plications.
Moreover, in many instances, there was either no nerve growth or only growth of
connective tissue. Thus, the functional results of surgical repair of peripheral nerve
injuries have been disappointing in spite of improved surgical techniques. Strategies have
been devised for allegedly enhancing the regeneration of peripheral nerves (those outside
the spinal cord and brain). Thus, protection of the site of a neurorrhaphy from infiltration
with fibrous tissue and prevention of neuromatous formation by the use. of wrappers,
cuffs, or tubes of various materials have been practiced since 1980. At that time, it was
attempted to interpose a drain of decalcified bone between the severed ends of a sciatic
nerve. Fibrous union without return of function, however, generally resulted. In addition
to decalcified bone and vessels, fascialata, fat, muscle, parchment, Cargile membrane,
gelatin, agar, rubber, fibrin film, and various metals have been used with varying degrees
of success. Many materials failed because they incited a foreign body reaction, produced
constricting scar tissue, were technically difficult to apply, or required secondary operation
for their removal. Various enhancements in both end tubulation and nerve wrapping have
continued in order to facilitate nerve repair.
Both biodegradable and non-resorbable materials have been used to act as a channel to
promote growth and regeneration in severed nerves which have been sutured together or
in connection with nerve grafts.
Although improved results in nerve regeneration have been obtained through the use of
tubes filled with nerve regenerating promoters, there is still much room for further
improvement. Particularly, the manufacture of tubes filled with such promoting agents is a
relatively expensive and tedious process. Moreover, it would still be desirable to provide a
means by which an even greater number of myelinated axons are regenerated, a faster
rate of nerve growth is achieved, and longer nerve gaps are spanned. A need still exists
to fulfill such a need and still reduce or eliminate problems that have been encountered
with prior art nerve repair attempts such as revascularization, excessive fibrosis,
reorientation of nerve fibres, and the final poor return of function of the end organs.
Bridging of Small gaps or come close to matching the performance of autografts. As a
result, the field is in desperate need of a transformative technology for repair of
peripheral nerve injury. The key failing of all current strategies to functionally repair
major nerve trauma is the inability to coax a Sufficient number of axons to grow a
6
Substantial distance to reinnervate distal targets (e.g., hand) and restore function. To
overcome this failing, repair strategies must address two major challenges: (1) encourage
rapid regeneration of proximal axons and (2) maintain the pro- regenerative capacity of
the distal nerve segment for regenerating axons.
Degeneration of the axon segments distal to a nerve injury site is an inevitable
consequence of transection of or injury to the nerve; however, the supporting Schwann
cells in the distal nerve segment survive and switch to a pro-regenerative phenotype to
support axon growth. This pro-regenerative phenotype includes a change in cellular
alignment to form parallel columns, providing tracts that serve as guides for regenerating
axons. Unfortunately, the natural pro-regenerative environment degrades after several
months without the presence of axons, thus depriving regenerating axons of their ―road
map' to an end target. This occurs when the time it takes to regenerate axons to infiltrate
the distal segment is greater than the time the Schwann cells can maintain their proregenerative phenotype. Often, following long or proximal PNI, the pro-regenerative
environment fails and there is incomplete functional recovery. For example, a patient with
a PNI of the upper arm may regain elbow, but not hand function, due to the distance
between the nerve injury and the end targets in the hand, which are often not reached by
proximal axons before the distal environment is no longer pro-regenerative. In another
example, a PNI is not treatable due to the large size of the nerve lesion or injury,
irrespective of the lesion or injury location.
Various techniques for prolonging the pro-regenerative capacity of the distal nerve
segment following nerve injury have been explored. These include providing neurotrophic
factors (e.g., GDNF, BDNF, and TGF-beta) to the distal nerve segment; administering
electrical stimulation to the nerve sheath in an attempt to stimulate acceleration of axon
regeneration; and transferring a foreign sensory nerve or an adjacent healthy nerve to
the denervated nerve sheath. However, such techniques are often limited by a lack of
efficacy, particularly with regard to long-term efficacy.
In addition, some of these techniques have the clear disadvantage of transecting a
healthy nearby nerve for the purpose of transferring it to the adjacent denervated nerve
stump.
Thus, there is a need in the art for more effective means of maintaining the proregenerative capacity of denervated distal nerve segments so that the effectiveness of
7
current or future means of PNI repair can be increased. There is a particular need in the
art for maintaining pro-regenerative capacity and alignment of Schwann cells in the
denervated distal nerve segment in long-term.
At present, support cells used in the tissue engineered nerves include Schwann cells and
various stem cells, which are allogeneic cells, and may cause immunogenicity, which is
not suitable for clinical applications. On the other hand, the in vivo fate and biological
effects of support cells after they are implanted into the body are not fully clear, and they
may be inactivated in the environment of the body, thus failing to achieve expected
biological effects. All the above issues limit the development of tissue engineered nerve
grafts.
Nerve damage in patients will often not regenerate naturally, and can lead to permanent
loss of sensitivity and function. For this reason, surgical and therapeutic interventions to
promote repair can be desirable.
Reference is made to GB2386841 titled Multi-channel bioresorbable nerve
regeneration conduit and process for preparing the same, by applicant IND TECH RES
INST. This invention describes a multi-channel bioresorbable nerve regeneration conduit
having a hollow round tube of porous bioresorbable polymer and a multi -channel filler
made from a porous bioresorbable polymer film located inside the tube.
Reference is made to US patent no. 5019087, titled nerve regeneration conduit, by
applicant American Biomaterials Corp. The invention discloses a method of spinning a
collagen and laminin mixture onto a mandrel to make a single channel nerve conduit.
Reference is made to U.S. Pat. No. 3,786,817, titled METHOD AND APPARATUS FOR
AIDING SEVERED NERVES TO JOIN, by applicant PALMA J. The invention describes the
use of a non-resorbable tube to aid in the alignment and joining of severed nerves. Here,
the ends of a severed nerve are inserted into the ends of a tube until the nerve ends are
close to each other or touch each other at the center of the tube. A fluid such as nitrogen
is passed though the tube to aid in regeneration.
Reference is made to In U.S. Pat. No. 4,534,349, titled ABSORBABLE SUTURELESS
NERVE REPAIR DEVICE, by applicant 3M Co. The invention is an absorbable hollow tubular
device. Which allegedly enables the sutureless repair of lacerated, severed, or grafted
nerves wherein the device is comprised of a body-absorbable polymer.
Reference is made to CN1589913, entitled A tissue engineering peripheral nerve used
8
for repairing peripheral nerve defect and its preparation method, by applicant School of
Stomatology, Fourth Military Medical University of Chinese People's Liberation Army. The
invention describes a tissue engineered nerve used for repairing peripheral nerve defect.
The tissue engineered nerve consists of a nerve conduit made of biodegradable materials
added with glial cells or stem cells having ability to differentiate into glial cells, which are
used as seed cells, and modified with microspheres for controlled release of neurotrophic
factors and with ECM molecules.
Reference is made to WO2004/087231, titled self-aligning tissue growth, by applicant
Ucl Biomedica Plc. The invention describes a self-aligning tissue growth guide. The guide
comprises a core of a biopolymer matrix which is fixed to an outer sheath at two points.
The core is seeded with cells, which generate a mechanical contractile force leading to
self-alignment of the cells within the core. This produces a cellular guidance substrate for
regenerating tissue in vivo. The tension in the core can also lead the fibres of the matrix
to align. The combination of cellular alignment and substrate alignment serves to guide
cellular regrowth in a subject. As described in WO2004/087231 , the biopolymer matrix is
preferably a collagen matrix. Cells used to seed the matrix align and contract but do not
proliferate to form organised tissue. The list of cells given in the publication as bei ng of
use includes Schwann cells. An embodiment of the guide may also include cells from the
tissue of interest seeded within the matrix, and which will grow and be guided by the
contractile cells.
Another reference is made to 9123/DELNP/2014, titled Scaffold, by applicant THE
UNIVERSITY OF SHEFFIELD. The invention provides a method for producing an
electrospun scaffold, comprising electrospinning a polymer or co-polymer onto a template
comprising a conductive collector having a three dimensional pattern thereon, wherein
said electrospun polymer or co- polymer preferentially deposits onto said three
dimensional pattern.
Another reference is made to 368/DELNP/2012, titled BIODEGDRADABLE SCAFFOLD
FOR SOFT TISSUE REGENERATION AND USE THEREOF, by applicant COLOPLAST A/S. The
present invention relates to new reinforced biodegradable scaffolds for soft tissue
regeneration, as well as methods for support and for augmentation and regeneration of
living tissue, wherein a reinforced biodegradable scaffold is used for the treatment of
indications, where increased strength and stability is required besides the need for
9
regeneration of living tissue within a patient. The present invention further relates to the
use of scaffolds together with cells or tissue explants for soft tissue regeneration, such as
in the treatment of a medical prolapse, such as rectal or pelvic organ prolapse, or hernia.
Another reference is made to 201821042296, titled PROCESS FOR PREPARATION OF
TUBULAR GRAFTS OR TUBULAR SCAFFOLDS FOR TISSUE ENGINEERING, by applicant Dr.
D. Y. Patil Vidyapeeth. The present invention relates to a process for preparing tubular
grafts or tubular scaffolds for tissue engineering, which is amenable to automation, using
which the manufacturing of tubular scaffolds or grafts derived from them can be scaled
up. More particularly, the present invention further relates to a process for preparation of
tubular grafts or tubular scaffolds, which are of uniform diameter, seamless in
construction, and are free from tears and other defects arising due to the manufacturing
process. The present invention also relates to a process for preparing tubular grafts or
tubular scaffolds for tissue engineering, using a sacrificial mould and sacrificial mandrel
approach, wherein a sacrificial mould is used for preparing a sacrificial mandrel. The
present invention further relates to a process for preparing tubular grafts or tubular
scaffolds for tissue engineering, wherein use of noncell friendly chemicals such as organic
solvents is avoided.
Another reference is made to US patent no. 10,507,187, titled Regenerative tissue
grafts and methods of making same, by applicant Jackson; Wesley M. (Albany, CA), Nesti;
Leon J. (Silver Spring, MD), Tuan; Rocky S. (Bethesda, MD. The present invention
discloses a graft containing a scaffold that includes a matrix in which are positioned
mesenchymal progenitor cells (MPCs) has the capacity to substantially improve wound
healing, including wounds resulting from injury to nerve, bone and vascular tissue. MPCs
can be harvested from debrided muscle tissue following orthopaedic trauma. The
traumatized muscle-derived progenitor cells are a readily available autologous cell source
that can be utilized to effect or improve wound healing in a variety of therapeutic settings
and vehicles.
However none of the inventions in prior art comprise novel and unique technique
of culturing human mesenchymal stem cells, mesenchymal stem cells
differentiated Schwann cells and nerve cells into a proliferating, sub- confluent
layer on a biocompatible conduit/matrix prepared from plurality of composite
polymers by using glutaraldehyde as a cross linker for direct implantation or
10
delivery. In the present invention, the cells are transferred while in a
proliferative state and the final product obtained is transported in semi-solid
medium wherein the semi-solid medium is agar medium 1% to 3% and cell
culture medium with essential growth factors. The agar medium contains HEPES
2-3gm/l and sodium bicarbonate 2-3.5gm/l. suitable for grafting and provides a
better, efficient, easy to use, cost effective ready to use biodegradable and
biocompatible artificial nerve conduit/matrix for nerve repair and regeneration
with sensory and motor function in a synergistic manner. In the present
invention, the grafts can be prepared within 12 days.
The present invention provides a novel and unique technique of nerve conduit/matrix
preparation and culturing of human mesenchymal stem cells, schwann cells and neuronal
cells and their proliferation on a biocompatible conduit/matrix suitable for nerve
implantation/grafting. The invention provides a ready to use biodegradable and
biocompatible tissue construct with autologous/allogeneic human stem cells based
product. The present invention also provides a reconstructive procedure to meet the
specific requirements necessary to achieve satisfactory healing of nerve injury and restore
functional integrity in the least time and with the least complications and morbidity. The
nerve conduit/matrix as provided by the present invention has tissue like properties and is
capable of being used for nerve regeneration and repair in a synergistic manner.
Objects of the invention:
The primary objective of the present invention is to provide artificial bioengineered nerve
conduit/matrix for peripheral nerve injury.
Another objective of the present invention is to grow cells directly on the polymeric
conduit/matrix (scaffold) for direct implantation or delivery.
Another objective of the present invention is to prepare grafts within 12 days.
Another objective of the present invention is to provide a ready to use biodegradable and
biocompatible nerve conduit/matrix with autologous/allogeneic human stem cells- based
product.
Another objective of the present invention is to provide a method of preparation of such
device.
A further objective of the present invention is to develop artificial bioengineered nerve
11
conduit/matrix for peripheral nerve injury, spinal cord injury and any other type of nerve
injury repair and regeneration in a synergistic manner.
A further objective of the present invention is to develop artificial bioengineered nerve
conduit/matrix for restoration of motor and sensory function of damaged or injured nerve.
Summary of the invention:
The present invention provides a novel and unique technique of nerve conduit/matrix
preparation and culturing of human mesenchymal stem cells, schwann cells and neuronal
cells and their proliferation on a biocompatible conduit/matrix prepared from plurality of
composite polymers by using glutaraldehyde as a crosslinker for direct implantation or
delivery. In the present invention, the cells are transferred while in a proliferative state
and the final product obtained is transported in semi-solid medium wherein the semi-solid
medium is agar medium 1% to 3% and cell culture medium with essential growth factors.
The agar medium contains HEPES 2-3gm/l and sodium bicarbonate 2-3.5gm/l suitable for
grafting. The present invention provides a ready to use biodegradable and biocompatible
tissue construct with autologous/allogeneic human stem cells-based product. The present
invention also provides a reconstructive procedure to meet the specific requirements
necessary to achieve satisfactory healing of nerve injury and restore functional integrity in
the least time and with the least complications and morbidity. The nerve conduit/matrix
as provided by the present invention has tissue like properties and is capable of being
used for nerve regeneration and repair in a synergistic manner. In the present invention,
the grafts can be prepared within 12 days.
Brief description of the drawings:
Figure 1: Figure 1 shows the Design and fabrication of biopolymer-based nerve guidance
Nerve conduit/Matrix Form.
Figure 2: Figure 2 shows Degradation behavior of Nerve conduit/Matrix in Trypsin and
collagenase.
Figure 3: Figure 3 shows Swelling ratio of Nerve conduit/Matrix in 1X PBS.
Figure 4: Figure 4 shows the FTIR spectra of glutaraldehyde cross-linked nerve
conduit/matrix
Figure 5: Figure 5 shows Surface morphology of Nerve conduit/Matrix by SEM analysis
(A) Cross-sectional view (100 X) and (B) Surface view (200 X)
12
Figure 6: Figure 6 shows MTT assay showing the biocompatibility of Nerve conduit/Matrix
with L929 cells. (PC-plain control; SC-static control; DC-dynamic control).
Figure 7: Figure 7 shows Fluorescence images showing viability of MSCs on Nerve
conduit/Matrix on day 7 in culture
Figure 8: Figure 8 shows Biocompatibility of Nerve conduit/Matrix for MSCs. (A) MSCs
pre-stained with PKH26 growing uniformly across the scaffold; (B) Calcein-AM stained
MSCs on nerve conduit/matrix scaffold; (C) Nuclear DAPI stained; (D) Merge PKH and
DAPI; (E) Merge PKH, DAPI and Calcein-AM (10 X magnification).
Figure 9: Figure 9 shows Scanning electron micrographs of MSCs seeded on the surface
of Nerve conduit/Matrix.
Figure 10: Figure 10 shows Fluorescence images showing Calcein-AM staining on
different cross-sectional sections of Nerve conduit/Matrix.
Figure 11: Figure 11 shows Calcein-AM staining on revived Nerve conduit/Matrix (stored
at -80°C after lyophilization) cryopreserved in cryomedia A, cryomedia B, cryomedia C
and cryomedia D on day 4
Figure 12: Figure 12 shows Bright field images showing morphological changes in MSCs
during their differentiation into Schwann cells at different time intervals
Figure 13: Figure 13 shows Immunostaining for the Schwann cell markers CD56
(red),p75NGFR (green), CD 104 (red), S100 (green) after 14 days of differentiation. Cell
nuclei were stained with DAPI (blue). Scale bars, 100 µm.
Figure 14: Figure 14 shows In vivo degradation of nerve conduit and matrix form
implanted subcutaneously on the dorsal site of rats at 4, 8, 12, 16, 20 and 27 weeks after
implantation.
Figure 15: Figure 15 shows Dissection (A, B) and surgical reconstruction (C) of
experimental sciatic nerve injury
Figure 16: Figure 16 shows Histological examination of regenerated sciatic nerves after 8
weeks of implantation. (A) H&E staining shown the overview of nerve morphology in each
group; (B) Myelination of regenerated nerves revealed by toluidine blue staining (10X
magnification)
Statement of the invention:
Accordingly, the present invention provides novel and unique technique of culturing
13
human mesenchymal stem cells, mesenchymal stem cells differentiated schwann cells and
nerve cells into a proliferating, sub- confluent layer on a lyophilized biocompatible
conduit/matrix prepared from plurality of composite polymers by using glutaraldehyde as
a cross-linker without any integrated harmful chemicals for direct implantation or delivery
of the said human mesenchymal stem cells, wherein in the said invention, the said cells
are transferred while in a proliferative state and the final product obtained is transported
in semi-solid medium. The said semi-solid medium is agar medium 1% to 3% and cell
culture medium with essential growth factors including HEPES 2-3gm/l and sodium
bicarbonate 2-3.5gm/l. suitable for grafting and provides a better, efficient, easy to use,
cost effective ready to use biodegradable and biocompatible artificial nerve conduit/matrix
for nerve repair and regeneration with sensory and motor function in a synergistic manner
wherein the grafts can be prepared within 12 days.
Detailed description of the invention:
It should be noted that the particular description and embodiments set forth in the
specification below are merely exemplary of the wide variety and arrangement of
instructions which can be employed with the present invention. The present invention
may be embodied in other specific forms without departing from the spirit or essential
characteristics thereof. All the features disclosed in this specification may be replaced by
similar other or alternative features performing similar or same or equivalent purposes.
Thus, unless expressly stated otherwise, they all are within the scope of present
invention. Various modifications or substitutions are also possible without departing from
the scope or spirit of the present invention. Therefore it is to be understood that this
specification has been described by way of the most preferred embodiments and for the
purposes of illustration and not limitation.
The present invention provides a novel and unique technique of nerve conduit/matrix
preparation and culturing of human mesenchymal stem cells, schwann cells and neuronal
cells and their proliferation on a biocompatible conduit/matrix suitable for nerve
implantation/grafting.
The present invention provides a ready to use biodegradable and biocompatible tissue
construct with autologous/allogeneic human stem cells based product.
14
The present invention also provides a reconstructive procedure to meet the specific
requirements necessary to achieve satisfactory healing of nerve injury and restore
functional integrity in the least time and with the least complications and morbidity. The
nerve conduit/matrix as provided by the present invention has tissue like properties and is
capable of being used for nerve regeneration and repair.
The present invention is directed to bioengineered tissue constructs of cultured cells and
endogenously produced extracellular matrix components without the requirement of
exogenous matrix components or network support or scaffold members. The invention
can thus advantageously be made entirely from human cells, and human matrix
components produced by those cells, for example, when the bioengineered tissue
construct is designed for use in humans.
The present invention is also directed to methods for producing tissue constructs by
stimulation of cells in culture, such as Mesenchymal stem cells, and Mesenchymal stem
cells differentiated into schwann cells and nerve cells to produce extracellular matrix
components without the addition of either exogenous matrix components, network
support, or scaffold members to helpful in nerve repair and regeneration.
In the present invention, further, this tissue construct can be made by seedings of
Mesenchymal stem cells and Mesenchymal stem cells differentiated schwann cells and
nerve cells to produce a cultured tissue construct that mimics the cell composition and
tissue structures for signal transduction, nerve repair and regeneration as native tissues.
The tissue constructs of the invention are useful for clinical purposes such as nerve
grafting to a patient with tissue or organ defect, such as peripheral nerve injury or any
other type of nerve injury, or for in vitro tissue testing or animal grafting such as for
safety testing or validation of pharmaceutical, cosmetic, and chemical products.
The present invention uses proliferative/preconfluent Mesenchymal stem cells (MSCs),
schwann cells, nerve cells, mesenchymal stem cells differentiated schwann cells and
mesenchymal stem cells differentiated neuronal cells whereby cells are transferred from
culture to the ready to use living nerve conduit/matrix.
In an embodiment, the cells are grown directly on the polymeric conduit/matrix (scaffold)
for direct implantation or delivery. The cells with scaffold can therefore be transferred as
15
such to the patient thus avoiding the potential damage occurring in the conventional
enzymatic separation from the culture vessel.
In an embodiment, the cells are transferred while in a proliferative state. In some
embodiments, the use of preconfluent cells aids in the adherence of such cells to the
application site as they express an integrin profile different from fully differentiated,
terminal cells.
In an embodiment, the interactive component of the invention is provided by the use of
actively proliferating Mesenchymal stem cells, schwann cells, nerve cells (unipolar or
bipolar or multipolar). During nerve repair and regeneration of damaged nerve, a number
of cytokines, growth factors etc. are released at the application site, that will helpful in
signal transduction and nerve regeneration.
In an embodiment, the cells at the application site express molecules that have both an
autocrine as well as a paracrine effect.
In an embodiment, the uses of an artificial nerve conduit graft substitute are useful for
both repair and regeneration of damaged nerve in a synergistic manner. Repair indicates
the process that a tissue undergoes to completely regenerate/reform. Allogeneic
Mesenchymal stem cells, Schwann cells and nerve cells used in this nerve conduit/matrix
will helpful in repair and regeneration of the damaged nerve.
In another embodiment, the process involves the optimization of scaffolds onto which
cells are seeded to form a uniform tissue with scaffolds that provide physical and chemical
cues to guide the process. Scaffolds may be selected from a group comprising of natural
biopolymers such as chitosan, gelatin, collagen and hyaluronic acid.
In another embodiment, the Scaffolds take forms ranging from sponge like sheets to gels
to highly complex structures with intricate pores and channels made with new materials
processing technologies. The spatial and compositional properties of the scaffold, the
porosity of the scaffold and interconnectivity of the pores are all required to enable cell
penetration into the structure as well as the transport of nutrients and waste products.
Differential porosity will helpful in the cells attachment and signal transduction.
In an embodiment, the sequential timed patterned physico-chemical treatment of the four
or more polymers is carried on to get lyophilized 3D scaffold of polyelectrolyte complex
(PEC) and also at the same time using a specifically designed aspect ratio of a system for
agitation/homogenization. The sequential timed patterned physico-chemical treatment of
16
polymers can be as dissolution of gelatin at temperature 35 – 75 °C, preferably at 60° C
using 5% of gelatin, wherein the process comprises:
a) Stirring of the gelatin solution at 2000-3200rpm at temp 15-30° C for 15- 25min.
b) Adding of 1% chitosan solution (in 0.5-2.5% glacial acetic acid solution) dropwise in
gelatin solution at temp 15-30°C and stir the solution with homogenizer for 20-30min.
c) Adding of hyaluronic solution (in milli Q water) preferably 0.1-1% dropwise in
mixture and stir for 10 minutes.
d) Adding of collagen solution type 1 or type 4 (in glacial acetic acid solution) preferably
0.1-1% dropwise in mixture and stir for 10 minutes.
e) Adding of glutaldehyde solution preferably 25-50% dropwise at final concentration
of 0.1-0.5%.
In an embodiment, once the above method of physico-chemical treatment of polymers is
complete then the process of freeze drying of the composite solution is carried on. The
composite was freeze at -80° C for 12 hrs and then lyophilize for 72 hrs at 0 ° C and 500
motor vacuum.
In an embodiment, after freeze drying conduit/matrix was neutralized with ammonia
fumes (25% ammonia solution fumes) for 12 hrs in closed chamber inside the fume hood.
In an embodiment, further mesenchymal stem cells, schwann cells and nerve cells are
seeded onto biocompatible scaffold at cell density of 0.5 x 105 to 0.8 x 105 cell/cm2. The
cells are monolayer and 80% to 100% confluent at the final stage of product formulation.
The cells used for seeding is passage 2 to passage 5. The mesenchymal stem cell,
schwann cells and nerve cells used for seeding is human mesenchymal stem cells,
schwann cells is differentiated from human mesenchymal stem cells and nerve cells is
differentiated from human mesenchymal stem cells and only pure population. The
mesenchymal stem cells, schwann cells and nerve cells have secrete several growth
factors and cytokines (extracellular matrix) helpful in nerve repair and regeneration.
In an embodiment, the final product obtained will transport in semi-solid medium. The
semi-solid medium is agar medium 1% to 3% and cell culture medium with essential
growth factors. The agar medium contains HEPES 2-3gm/l and sodium bicarbonate 2-
3.5gm/l. The semi-solid medium contains agar medium and cell culture medium in the
17
ratio of 5:5, 6:4, 7:3 and 8:2 or any one of them respectively. The semi-solid medium
maintains the cell viability of matrix between 60% to 90% at the temperature 4oC to 37oC
for 28 days.
Examples:
The following examples are for the purposes of illustration only and therefore
should not be construed to limit the scope of the invention:
Example 1: Design and fabrication of biopolymer-based nerve guidance
conduit/Matrix
Preparation of the nerve conduit/matrix:
In an embodiment, take 50 ml of 5% gelatin solution and homogenize it for 15 min at
2000 rpm. Add 25 ml of 1% chitosan solution dropwise into the gelatin solution. Continue
stir this mixture for 30 min to form a homogenous blend. Dropwise add 500 µl of 0.1%
HA solution with stirring for 10 min. To this blend, add 1 ml of 0.1% collagen solution.
After 10 min of continuous stirring, add 200 µl of 50% glutaraldehyde solution for
crosslinking. Once the mixture is homogenized cast sample in trays/conduits and freeze it
down at -80°C for 12 hr followed by lyophilization (cycle 72 hrs, drying at 0°C, vacuum
500 mtorr) to form porous scaffolds.
Mesenchymal stem cells, schwann cells and nerve cells inoculation and their
culture:
In an embodiment, 0.5 x 105 cells were seeded in the pre acclimatized scaffold (scaffold
soaked in cell culture medium) and culture the cells at the day 12-15. After/between the
day 12 - 15 scaffolds were completely filled with mesenchymal stem cells, schwann cells
and nerve cells and rich of growth factors and nutrients as shown in Figure 1.
Example2: In vitro degradation behavior of Nerve Conduit/matrix
In an embodiment, the in vitro degradation of Nerve conduit/Matrix was studied by
incubating them in an enzymatic solution and then monitoring their weight -losses at
different time points.
In an embodiment, Scaffold samples were incubated in 1X PBS containing trypsin (0.25
mg/ml) & collagenase (0.1 mg/ml) at 37°C in a shaker incubator at 60 rpm for various
18
periods of up to 21 days.
In an embodiment, at predetermined time intervals, the scaffolds were removed from the
incubation medium, washed with deionized water, then subsequently oven dry for final
weight measurement. Weight-loss was then determined as a difference between dry mass
of sample before and after the incubation, normalized to dry mass of sample before the
incubation.
In an embodiment, Scaffolds incubated in trypsin had the highest weight reduction after
two weeks of incubation compared to incubation in collagenase. In addition, in vitro
degradation of scaffolds in PBS, saline and media at 37°C was also performed to check
long term mechanical stability and degradation of scaffold material.
In an embodiment, Scaffolds were not degraded in PBS, saline and media up to four
months indicating controlled degradation behavior of nerve conduit/matrix as depicted in
Figure 2.
Example 3: Swelling test of fabricated nerve conduit/matrix
In an embodiment, to determine the percentage of water absorption, swelling studies
were performed by immersion of scaffolds in 1X PBS. The dry weight of the scaffold was
determined before immersion (Wd). Scaffolds were placed in PBS buffer solution and after
a predetermined time points, the scaffolds were taken out and surface adsorbed water
was removed by filter paper and their wet weight were recorded (Ww) as disclosed in
Figure 3. The ratio of swelling was determined using equation:
Example 4: Fourier transform infrared (FTIR) analysis
In an embodiment, the chemical structure of the fabricated Nerve conduit/Matrix was
analyzed by Fourier transform infrared spectroscopy (FTIR). The infrared spectra of the
scaffold was measured over a wavelength range of 4000–400 cm−1 as disclosed in
Figure 4.
Example 5: Morphological characterization of nerve conduit/matrix using SEM
In an embodiment, scanning electron microscopy (SEM) was performed to study the
19
surface and cross- sectional morphology of nerve conduit/matrix. The SEM image (Figure
5) showed that the nerve conduit/matrix possessed a well -defined and well integrated
porous structure that is essential for cell attachment, proliferation and survival. The
images at higher magnification indicate the presence of open and interconnected pores of
different sizes on the surface of scaffolds. Thus, nerve conduit/matrix possess sufficient
porous morphology beneficial for vascularization and nutrient exchange between the
lumen and the outer environment.
Example 6: Evaluation of nerve conduit/matrix cytotoxicity by MTT assay
In an embodiment, in vitro cytotoxicity assays were performed to test the biocompatibility
of nerve conduit/matrix. This assay is based on the measurement of viability of cells via
metabolic activity. Yellow water-soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromid) is metabolically reduced in viable cells to a blue-violet
insoluble formazan. The number of viable cells correlates to the colour intensity
determined by photometric measurements after dissolving the formazan in DMSO. The
cytotoxicity of the scaffolds was evaluated according to ISO guidelines 10993- 5-2009,
using 24 hr extraction period at dynamic condition.
To calculate the reduction of viability compared to the blank, following equation was used:
% Viability = Absorbance Extract Concentration (570 nm)
Absorbance Blank (570 nm)
Clearly, MTT data (Figure 6) revealed that scaffold nerve conduit/matrix did not induce
any cytotoxic response at any of the tested extract concentrations. Overall, nerve
conduit/matrix displayed significant biocompatibility and cell viability to L929 cells.
Example 7: Evaluation of cell attachment and proliferation of MSCs on nerve
conduit/matrix
In an embodiment, calcein-AM staining was performed to check the nerve conduit/matrix
support for cell adhesion and proliferation of MSCs. Sterile scaffold was equilibrated it in
culture medium overnight before seeding. MSCs were seeded at a density of 5 x 104 cells
per scaffold followed by incubation at 37°C in a CO2 incubator. After 5 days of culture,
media was removed from scaffold and washed with serum free media to completely
20
remove the serum esterase activity. 2 µM Calcein-AM staining solution was prepared by
adding 1 µl of 2 mM Calcein-AM to 1 ml of serum free media. Sufficient volume of CalceinAM staining solution was added to cover the scaffold surface. Cells were incubated for 30
min at 37°C. Labeled cells were imaged using fluorescence microscopy to check cell
adhesion & proliferation as disclosed in Figure 7.
Example 8: PKH26 staining of MSCs on nerve conduit/matrix
In an embodiment, PKH26 staining was performed to track the labelled MSCs on nerve
conduit/matrix after 48 hrs of culture. MSCs pre-stained with PKH26 grown uniformly
across the scaffold and exhibits adequate fluorescent signal after 48 hrs in culture. Calcein
staining further confirmed the biocompatibility of scaffold nerve conduit/matrix for MSCs
as disclosed in Figure 8.
Example 9: Morphological observation of cell adhesion through SEM
In an embodiment, MSCs were cultured at the concentration of 0.5 × 105 cells on nerve
conduit/matrix scaffold. After 5 days of cell culture, the morphology of cells seeded on the
composite scaffolds was observed using SEM. As shown in Figure 9, the MSCs were well
attached and spread out, showing more cell outgrowths. In addition, cell invasion
indicates the porous nature of conduit, which provides enough space and 3D environment
for cell growth and migration.
Example 10: Sectioning of nerve conduit/matrix scaffold to check MSCs
proliferation and migration
In an embodiment, Nerve conduit/matrix were into different sections to check MSCs cells
adherence and viability on the lumen and outside surface of conduit wall using Calcein-AM
stain. Conduit images were captured (Figure 10) in both vertical and horizontal sections
in order to ensure the cells migration.
Example 11: Cryopreservation and freeze-drying of cell seeded Nerve
conduit/matrix
In an embodiment, to check cell survival efficiency after freeze-drying (Figure 11),
trehalose was used at a single concentration of 0.5 M in combination with three different
concentration of Bovine Serum Albumin (BSA) i.e. 12.5%, 10%, 9.5%. MSCs seeded
21
scaffolds were cryopreserved using freezing media consisted of alpha MEM with 10% FBS
along with A) alpha MEM + 20% FBS + 0.5 M Trehalose dehydrate + 12.5% BSA; B)
alpha MEM + 20% FBS + 0.5 M Trehalose dehydrate + 10% BSA; C) alpha MEM + 20%
FBS + 0.5 M Trehalose dehydrate + 9.5% BSA; D) alpha MEM + 20% FBS + 0.5M
Trehalose dehydrate alone. Petri dishes containing scaffolds were placed in a freezing
solution containing isopropyl alcohol that provided a 1°C/min cooling rate when stored at
- 80°C. Freeze-drying was done by transferring frozen samples from -80°C to the
temperature-controlled shelves of a lyophilizer. Lyophilization cycle: Lyophilization cycle:
Drying 0°, Vacuum 500 mtorr, 48 hrs. cycle. Cryopreserved scaffolds under lyophilized
condition in cryomedia media A, B, C, D were revived successfully as shown by calcein
staining performed after 4th day post revival.
Example 12: In vitro differentiation of MSCs into Schwann-like cells
In an embodiment, for differentiation, several reagents and trophic factors were applied to
induce MSCs into cells with a phenotype similar to that of Schwann cells (Figure 12) by
sequential treatment: first with β-mercaptoethanol (BME), followed by all-trans-retinoic
acid (ATRA) treatment, and then culturing the cells in the presence of forskolin (FSK),
bFGF, PDGF, and HRG. Phase-contrast microscopy revealed that the differentiated MSCs
were morphologically different from the original undifferentiated MSCs. Cells cultured in
the differentiation media changed from a fibroblast-like morphology to an elongated
spindle shape (Figure 12), similar to that of Schwann cells.
In an embodiment, in order to confirm the successful SC differentiation,
immunocytochemistry of S- 100, p75, CD104 and CD56, all known as markers of
Schwann cells was performed. After induction, most of the differentiated SCs were
positive for S100, CD56, CD104 and p75 (Figure 13), in contrast to undifferentiated
MSCs.
Example 13: In vivo degradation study of Nerve conduit/matrix
In an embodiment, for in vivo degradation study, the nerve conduit/matrix in both conduit
and sheet form were implanted subcutaneously on the back of SD rats (Figure 14). At
predefined time points (4, 8, 12, 16, 20 and 27 weeks) after implantation, the test
scaffolds were photographed and harvested together with surrounding tissue for
22
histological evaluation. The nerve conduit/matrix was not degraded throughout the
implantation period, and remained stable upto 27 weeks post-implantation.
Example 14: In vivo implantation to check the efficacy of Nerve conduit/matrix
pre-seeded with differentiated Schwann cells
In an embodiment, the in vivo study was conducted to assess the efficacy of nerve
conduit/matrix pre-seeded with mesenchymal stem cells (MSCs) and MSCs differentiated
Schwann cells to repair peripheral nerve defect in rat sciatic nerve transection model. The
rat sciatic nerve transection model has been commonly used for the evaluation of tissue
engineered NGCs in promoting peripheral nerve regeneration in vivo. Before in vivo
implantation, in vitro biocompatibility of nerve conduit/matrix with MSCs was
investigated. Our initial pilot study to optimize the in vivo experiments was conducted on
15 Sprague Dawley rats. The right sciatic nerve of each animal was transected and a 10
mm segment of the nerve removed thus creating a gap that was bridged with (1) nerve
conduit/matrix seeded with either differentiated SCs (nerve conduit/matrix +DSC) or (2)
both MSCs and differentiated SCs (nerve conduit/matrix +B2+DSC). The left sciatic nerve
remained intact and used later as a control. The efficacy of nerve conduit/matrix was
investigated based on the results of walking track analysis, electrophysiology, and
histological assessment (Figure 15).
Example 15: Histological analysis of implanted nerve conduit/matrix
In an embodiment, for the histological evaluation of nerve regeneration, harvested nerve
tissue was sectioned and stained with hematoxylin-eosin and toluidine blue. Morphological
observations were carried out at 8th weeks post-operatively, detected that regenerated
myelinated fibers were smaller and showed a thinner myelin sheath in comparison to
normal nerves. The nerve conduit/matrix +DSC group showed distribution of nerve fibers
with myelin and fibrous connective tissue (Figure 16). This group showed irregular
shaped perineurium and epineurium, which was largely occupied by fibrous connective
tissue with center showing distorted axons with wavy plasma and myelin sheath. In
addition, numerous blood vessels were observed around the regenerated nerves.
Regeneration of fibrous myelinated nerve is considered important factor in terms of nerve
23
regeneration of damaged nerve. The number of regenerated nerve fibers distal to the
NGCs seeded with differentiated SC was fewer in comparison to midpoint of conduit. The
nerve conduit/matrix +B2+DSC group displayed growing nerves with less myelinated
fibers and thinner myelin sheaths in comparison to nerve conduit/matrix +DSC group. In
this group, however, far fewer nerve fibres but a large quantity of connective tissues were
visible between the nerve stumps. Besides, infiltration of inflammatory cells was found in
the proximal and middle portions of the graft, surrounded by fibrous connective tissue
fibers and nerve cell bodies.
In an embodiment, the prepared nerve conduit/matrix is potential substitute in nerve
regeneration and repair and it helps in faster restoration of motor and sensory function in
nerve injury (peripheral nerve injury, spinal cord injury and any other type of nerve
injury). Various experiments were conducted to check the said efficacy of bioengineered
nerve conduit/matrix. Cell viability, fluorescence microscopy and scanning electron
microscopy results confirm the cell growth and distribution of the cells uniformly in
matrix. In vivo studies confirmed the potential nerve regeneration and repair property of
conduit.
In an embodiment, the obtained results of the potential and properties of the product of
this invention were found considerably efficacious. It clearly indicates the technical
advancement as compared to prior art.
Thus, the present invention provides novel and unique technique of culturing human
mesenchymal stem cells, mesenchymal stem cells differentiated schwann cells and nerve
cells into a proliferating, sub- confluent layer on a lyophilized biocompatible
conduit/matrix prepared from plurality of composite polymers by using glutaraldehyde as
a cross-linker without any integrated harmful chemicals for direct implantation or delivery
of the said human mesenchymal stem cells, wherein in the said invention, the said cells
are transferred while in a proliferative state and the final product obtained is transported
in semi-solid medium. The said semi-solid medium is agar medium 1% to 3% and cell
culture medium with essential growth factors including HEPES 2-3gm/l and sodium
bicarbonate 2-3.5gm/l. suitable for grafting and provides a better, efficient, easy to use,
cost effective ready to use biodegradable and biocompatible artificial nerve conduit/matrix
for nerve repair and regeneration with sensory and motor function in a synergistic manner
wherein the grafts can be prepared within 12 days.
24
So accordingly, the present invention provides an improved biodegradable, biocompatible,
high porosity three-dimensional artificial nerve conduit/matrix based scaffold
polyelectrolyte complex (PEC) with autologous/allogeneic human stem cells for nerve
repair and regeneration, and a method of preparing thereof, said scaffold comprising of
plurality of composite polymers and using glutaraldehyde as cross-linker, wherein said
scaffold is non-adherent, has differential porosity, is able to grow cells directly on the
polymeric conduit/matrix (scaffold) for direct implantation or delivery.
In an embodiment, said grafts are prepared within 12 days.
In another embodiment, said scaffolds take forms ranging from sponge like sheets to gels
to highly complex structures with intricate pores and channels made with new materials
processing technologies such that the spatial and compositional properties of the scaffold,
the porosity of the scaffold and interconnectivity of the pores enables cell penetration into
the structure as well as the transport of nutrients and waste products with differential
porosity helping in the cells attachment and signal transduction.
In another embodiment, said method is comprising a unique technique of culturing human
mesenchymal stem cells, mesenchymal stem cells, differentiated schwann cells and nerve
cells into a proliferating, sub- confluent layer on a lyophilized biocompatible
conduit/matrix, without any integrated harmful chemicals for direct implantation or
delivery of the said human mesenchymal stem cells, wherein the cells are transferred
while in a proliferative state and the final product obtained is transported in semi-solid
medium.
In another embodiment, said nerve conduit/matrix provides repair and regeneration in a
synergistic manner.
In another embodiment, said plurality of polymers are preferably selected from but not
limited to gelatin, chitosan, collagen, hyaluronic acid, polyvinyl alcohol (PVA), poly
caprolactone (PCL), poly pyrrole, poly urethane (PU), poly allyl amine, poly ethylene
glycol 200 (PEG 200), gum acacia, guar gum and partially denatured collagen.
In another embodiment, said polymers are in the range of gelatin 1%-10% w/v, chitosan
0.5%-2.5% w/v, hyaluronic acid 0.1%-2% w/v, collagen 0.1%-10% w/v and
glutaraldehyde solution 5%-50% v/v with gelatin of 50-300 bloom strength, DAC
(dialdehyde cellulose) chitosan ranging from 75%-95.
25
In another embodiment, said semi-solid medium is agar medium in the range of 1% to
3% and cell culture medium with essential growth factors including HEPES 2-3gm/l and
sodium bicarbonate 2-3.5gm/l suitable for grafting which results in a better, efficient,
easy to use, cost effective, ready to use biodegradable and biocompatible artificial nerve
conduit/matrix for nerve repair and regeneration with sensory and motor function, in a
synergistic manner.
In another embodiment, said method of preparing the scaffold comprises physicochemical treatment; and lyophilization of freeze-dried scaffold.
In another embodiment, said method comprises sequential timed patterned physicochemical treatment of the four or more polymers to get 3D scaffold of polyelectrolyte
complex (PEC) and also at the same time using a specifically designed aspect ratio of a
system for agitation/homogenization.
In another embodiment, said method comprises stabilizing the scaffold by cross linking
with glutaraldehyde solution and freeze drying.
In another embodiment, said obtained freeze-dried 3D scaffold is stabilized and
neutralized by ammonia fumes (5%-25%) for 12-24hrs in closed chamber to make the
stable and functional scaffold for cell seeding.
In another embodiment, said obtained scaffold is freeze-dried to make the stable scaffold
for seeding of autologous or allogeneic mesenchymal stem cells (MSCs derived from bone
marrow or umbilical cord) Schwann cells and neuronal cells (differentiated form
mesenchymal stem cells).
In another embodiment, said mesenchymal stem cell, Schwann cells and neuronal cells
are seeded onto biocompatible scaffold at cell density of 0.5 x 105
to 0.8 x 105
cell/cm2
.
In another embodiment, said cells are monolayer and 80% to 100% confluent at the final
stage of product formulation.
In another embodiment, said cells seeded on scaffold are cultured-in with serum and
without serum medium.
In another embodiment, said mesenchymal stem cells are autologous or allogeneic or
both.
In another embodiment, said mesenchymal stem cells, schwann cells and neuronal cells
secrete several growth factors and cytokines (extracellular matrix) helpful in nerve
regeneration and repair.
26
In another embodiment, the final product is transported in semi-solid medium and/or
liquid medium or in frozen condition, such that the semi-solid medium/liquid medium
provides nutrients and support to matrix and maintains the cell viability of matrix between
70% to 95% at the temperature 4oC to 37oC for 15 days.
In another embodiment, said conduit/scaffold is in sheet form and/or hollow cylindrical
conduit form.
Advantages of the invention:
o The scaffold of the present invention helps in the nerve regeneration and
repair.
o The present invention comprises of improved healing.
o Can be manufactured in any size and shape as per the requirement.
o Easy to handle.
o Environment friendly as it is degradable easily.
o It ensures rapid healing in peripheral nerve injury.
o It ensures faster recovery and repair of motor and sensory function.
o The grafts can be made within 12 days.
o It is economical and offers an alternative treatment to the standard nerve
injury treatment methods.
o There is a dramatically reduced risk of transmission of infectious disease due
to rigorous process controls.
o It helps in restoration of motor and sensory function of damaged tissue.

We Claim:

1. An improved biodegradable, biocompatible, high porosity three-dimensional artificial
nerve conduit/matrix based scaffold polyelectrolyte complex (PEC) with
autologous/allogeneic human stem cells for nerve repair and regeneration, and a method
of preparing thereof, said scaffold comprising of plurality of composite polymers and using
glutaraldehyde as cross-linker, wherein said scaffold is non-adherent, has differential
porosity, is able to grow cells directly on the polymeric conduit/matrix (scaffold) for direct
implantation or delivery.
2. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said grafts are prepared within 12 days.
3. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said scaffolds take forms ranging from sponge like sheets to highly
complex structures with intricate pores and channels made with new materials processing
technologies such that the spatial and compositional properties of the scaffold, the
porosity of the scaffold and interconnectivity of the pores enables cell penetration into the
structure as well as the transport of nutrients and waste products with differential porosity
helping in the cells attachment and signal transduction.
4. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said method is comprising a unique technique of culturing human
mesenchymal stem cells, mesenchymal stem cells, differentiated schwann cells and
neuronal cells into a proliferating, sub- confluent layer on a lyophilized biocompatible
conduit/matrix, without any integrated harmful chemicals for direct implantation or
delivery of the said human mesenchymal stem cells, wherein the cells are transferred
while in a proliferative state and the final product obtained is transported in semi-solid
medium.
5. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said nerve conduit/matrix provides repair and regeneration in a
28
synergistic manner.
6. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said plurality of polymers are preferably selected from but not limited to
gelatin, chitosan, collagen, hyaluronic acid, polyvinyl alcohol (PVA), poly caprolactone
(PCL), poly pyrrole, poly urethane (PU), poly allyl amine, poly ethylene glycol 200 (PEG
200), gum acacia, guar gum and partially denatured collagen.
7. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said polymers are in the range of gelatin 1%-10% w/v, chitosan 0.5%-
2.5% w/v, hyaluronic acid 0.1%-2% w/v, collagen 0.1%-10% w/v and glutaraldehyde
solution 5%-50% v/v with gelatin of 50-300 bloom strength, DAC (dialdehyde cellulose)
chitosan ranging from 75%-95.
8. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said semi-solid medium is agar in the range of 1% to 3% and cell
culture medium with essential growth factors including HEPES 2-3gm/l and sodium
bicarbonate 2-3.5gm/l suitable for transportation of developed nerve conduit for grafting..
9. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said method of preparing the scaffold comprises physico-chemical
treatment; and lyophilization of scaffold.
10. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said method comprises sequential timed patterned physico-chemical
treatment of the four or more polymers to get 3D scaffold of polyelectrolyte complex
(PEC) and also at the same time using a specifically designed aspect ratio of a system for
agitation/homogenization.
11. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said method comprises stabilizing the scaffold by cross linking with
glutaraldehyde solution and freeze drying.
29
12. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said obtained freeze-dried 3D scaffold is stabilized and neutralized by
ammonia fumes (5%-25%) for 12-24hrs in closed chamber to make the stable and
functional scaffold for cell seeding.
13. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said obtained scaffold is freeze-dried to make the stable scaffold for
seeding of autologous or allogeneic mesenchymal stem cells (MSCs derived from bone
marrow or umbilical cord) Schwann cells and neuronal cells (differentiated form
mesenchymal stem cells).
14. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said mesenchymal stem cell, Schwann cells and neuronal cells are
seeded onto biocompatible scaffold at cell density of 0.5 x 105
to 0.8 x 105
cell/cm2
.
15. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said cells are monolayer and 80% to 100% confluent at the final stage of
product formulation.
16. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said cells seeded on scaffold are cultured-in with serum and without
serum medium.
17. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said mesenchymal stem cells are autologous or allogeneic or both.
18. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said mesenchymal stem cells, schwann cells and neuronal cells secrete
several growth factors and cytokines (extracellular matrix) helpful in nerve regeneration
and repair.
19. The improved biodegradable, biocompatible, high porosity three-dimensional
30
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein the final product is transported in semi-solid medium and/or liquid
medium or in frozen condition, such that the semi-solid medium/liquid medium provides
nutrients and support to matrix and maintains the cell viability of matrix between 70% to
95% at the temperature 4oC to 37oC for 15 days.
20. The improved biodegradable, biocompatible, high porosity three-dimensional
artificial nerve conduit/matrix-based scaffold polyelectrolyte complex (PEC) as claimed in
claim 1, wherein said conduit/scaffold is in sheet form and/or hollow cylindrical conduit form.