WO2016193991A1

WO2016193991A1 - A scaffold for corneal regeneration

Info

Publication number: WO2016193991A1
Application number: PCT/IN2016/000131
Authority: WO
Inventors: Soma Guhathakurta
Original assignee: Soma Guhathakurta
Priority date: 2015-06-05
Filing date: 2016-05-24
Publication date: 2016-12-08

Abstract

The invention attempts to disclose few examples, few embodiments along with few illustrations for the purpose of understanding the concept of developing a novel scaffold derived from a specific breed of fish scales as a novel material possessing excellent mechanical strength a requirement for corneal regenerative application. However all variations and modifications as will be obvious to skilled persons in the art is well within the scope and spirit of the invention.

Description

TITLE : "A SCAFFOLD FOR CORNEAL REGENERATION"

FIELD OF INVENTION

The field of invention is fish scale derived scaffold for corneal regeneration.

OBJECTS OF INVENTION

The main object of the invention is to achieve a fish scale derived novel scaffold for corneal regeneration.

The second object of invention is to achieve a novel process for achieving a scaffold derived from a fish scale. This fish scale scaffold is not only restricted to cornea with a particular kind of scale, but also another specific fish scale can develop into cartilage. Iri general fish scale can be used in wound dressing as well, scalp repair and intervertebral disc repair.

The third object of invention is to use fish scales, which was not commercially exploited previously.

The fourth object of invention is to use Hilsha (Tenualosa ilisha) fish scales for generating scaffold for corneal regeneration.

The fifth object of invention is to use Rohu (Labio rohita) and vetki (Lates calcarifer) fish, scale for cartilage preparation.

DESCRIPTION OF INVENTION

The invention addresses selection of a particular fish scale which exhibits thinness, transparency and possesses desired shape and form, which is most suitable for generating scaffold for coined regeneration.

In prior art Telapia (Oreochromis spp) fish and Rohu with small size scale had been addressed without much result and suffered many disadvantages.

The invention proves beyond doubt that specific fish scales (Hilsha) can be harvested from the commercial seller of fish and used in the invention. The method : 10 such scales purchased from Chennai commercial market weighing 5gm altogether was transferred to 50 ml of (1-4) Normal Sodium Hydroxide solution after washing it with microbe free water for three rinses. The material was kept for a period falling in the range 4-50hours in the solution mentioned above.

Thereafter it was washed well with PBS (BUFFERED SALINE) for three rinses. Now the simply washed scales are ready for decalcification. Though the prior art repeatedly teach the sequence of decellularisation followed by decalcification, the invention reverses the sequence.

The process of decalcification: This procedure is done first because the hard scale softens out so that decellularisation is easier and porosity can be developed in the scaffold for cell deposition and growth.

The process of decalcification involves 2-10% nitric acid of 50ml used for 15-60 hours on a rocker for ten scales to get rid of the calcium salts embedded in the scaffold at room temperature (20-25degree centigrade).

Thereafter it is rinsed well with buffered saline sterile for three rinses.

The next step immersing the rinsed scales in 50ml of 2-20% EDTA for 3-6 days with renewal of solution at intervals preferably every 48 hours, at room temperature (20- 25degree centigrade).

Thereafter it is rinsed well with buffered saline sterile for three rinses.

Subsequently 50 ml of Nitric acid (1-10%) 50 ml solution was poured into the 10 scales and kept for 10-48 hours.

Once again it is rinsed well with buffered saline three times.

Steps of decellularisation follows the decalcification :

(1-6%) Sodium deoxycholate solution was prepared with a pH of (6.6-9.0) 50 ml for 10 scales and was put on a rocker for 48 hours, at room temperature, washed in buffered saline and renewal of the same procedure for 24 hours.

Washed in buffered saline thrice and taken for next step. Formalin(2-20%) solution of 50 ml were taken to treat the scales further for crosslinking under UV- medium wave for (1/2-8hours) and then they continued to be in the same formalin solution in staged manner at lower percentage to start with and increasing the percentage subsequently which is 10% formalin solution, not only for crosslinking but also for fixation and sterilization. The pH of this solution was maintained 7-9. The temperature kept 4-8 degree Centigrade.

Washed well three times with buffered saline.

Glutamic acid (0.5-5%) immersion of the scales is done for 2hours as detoxification stage for formalin as well as this is also a stage for sterilization.

Washed well thrice with buffered solution.

Taken into preservation in 70% alcohol (60-90%) with Ethanol preservation to control calcification preferably 70% Ethanol preservation and most preferably heparin along with Neomycin sulphate to ensure that calcification cannot happen but it remains sterile and bioactive.

The fish scale derived scaffold can be fabricated that satisfies the needs of current corneal prostheses. The decalcified and acellularized fish scale exhibit a favourable cell proliferation and biosynthetic activity on cultivation. Hence the invention teaches that Tenualosa ilisha fish scales - derived method can be a new and novel scaffold in corneal tissue engineering field.

This particular Tenualosa ilisha fish scale is a good substitute for artificial cornea, which exhibits long-term transparency, as scaffold provided as per invention also exhibits transparency and, long term transparency. The fish scale even after decalcification and decellularization continues to be hydrophilic and permeable to oxygen, the most desired features demanded in an artificial cornea scaffold.

As such the invention proposes successfully inducing regeneration of damaged corneas by implantation of decalcified and decellularized fish scale as scaffolds which replicate the role of a typical natural extracellular matrix (ECM) of scaffolding of a cornea. The invention teaches the method of decalcified and decellularized corneal scaffold preparation, method of examining the surface morphology of such a corneal scaffold, evaluation of histo pathological evaluation, implantation techniques, monitoring and diagnosis of corneal recovery.

The Optical property of processed cornea is tabled below

Few illustrations are given below :

Fig. 1- Digital image of Corneal graft

Fig. 2 A-D- The transparency of artificial corneal scaffolds differed with processing method employed. Methodologies have been standardized to achieve better transparency of artificial corneal button as seen in figure C (client to check).

Fig. 3- Before processing, the fish scale had active calcium deposits as demonstrated by Von kossa positive stain

Fig. 4- The fish scale was completely decalcified by our processing methodology and was Von kossa stain negative

Fig. 5 A-D- Biocompatibility was demonstrated by epithelial cells proliferation on corneal scaffolds. The cells (Hela) extensively grew on the scaffold over a period of time in culture. Fig. 6 A-B- Biocompatibility : Human Bone marrow mesenchymal cells were grown on the scaffold. The shape of cells which grew on the scaffold had no significant differences with the cells before they climbed on the scaffold. Arrow indicate mesenchymal stem cells growing on the scaffold. (200X magnification)

The invention attempts to disclose few examples, few embodiments alongwith few illustrations for the purpose of understanding the concept of developing a novel scaffold derived from a specific breed of fish scales as a novel material possessing excellent mechanical strength a requirement for corneal regenerative application. However all variations and modifications as will be obvious to skilled persons in the art is well within the scope and spirit of the invention.

Claims

WE CLAIM

1. A process for preparing fish scales for use as scaffold for corneal regeneration comprising :

a. decalcificating the harvested scales, and

b. thereafter decellularating the decalcified scales

characterized in that the fish scales referred herein is Hilsha fish scales and decellularating step follows decalcification step.

2. The process as claimed in claim 1 wherein the decalcifying step further involves the following steps :

a. harvesting scales from Hilsha fish,

b. rinsing the harvested scales with microbe free water three times,

c. immersing the washed scales at room temperature in normal nutric acid for a predetermined duration to eliminate calcium salts,

d. rinsing the soaked scales in PBS buffered saline sterile three times, e. immersing the rinsed scales in EDTA at room temperature for predetermined duration with renewal of solution at specific intervals,

f. rinsing buffered saline steriles three times,

g. immersing the rinsed scale in nutric acid for a predetermined duration, and h. rinsing buffered saline steriles three times.

3. The process as claimed in claim 1 wherein the decellularising step further involves the following steps :

a. preparing sodium deoxycholate solution with PH in the range 6.6 - 9.0, b. immersing the decalcified scales in the prepared sodium deoxycholate solution for a predetermined duration,

c. washing the scales in buffered saline,

d. immersing the scales in buffered saline,

e. washing the scales in buffered saline three times,

f. cross linking the scales in formalin solution under UV medium wave plurality of times, and

g. washing with buffered saline three times. The process as claimed in claim 1 wherein the process may include detoxification step which comprises of

immersing the decalcified and decellularised scales in glutanic acid for predetermined duration, and

- washing with buffered solution three times.

The process as claimed in claim 1 & 4 wherein the process may include preservation step which comprises of

- preserving the decalcified, decellurised and detoxified scales in alcohol selectively with ethanol, heparin and neomycin sulphate or in combination therewith

The process as claimed in claim 1 - 5, wherein the process may include fabrication step of decalcified, decellularised, detoxified and preserved scale into collogen scaffolds.

A scaffold derived from fish scales as prepared in claims 1 - 6 for corneal regenerative applications.