TECHNICAL FIELD

The present disclosure is in relation to scaffolds for various applications. More particularly, the disclosure relates to a scaffold for tissue engineering, wound dressing and therapeutic drug delivery.

BACKGROUND OF THE DISCLOSURE

Tissue engineering is a new and exciting technique which has the potential to create tissues and organs de novo. It is a multidisciplinary field which involves the 'application of the principles and methods of engineering and life sciences towards the fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes that restore, maintain or improve tissue function' (Shalak and Fox, 1988, Preface. In: Tissue Engineering). It involves the in-vitro seeding and attachment of human cells onto a scaffold. These cells then proliferate, migrate and differentiate into the specific tissue while secreting the extracellular matrix components required creating the tissue. It is evident, therefore, that the choice of scaffold is crucial to enable the cells to behave in the required manner to produce tissues and organs of the desired shape and size.

Chronic Wound is any non healing wound and/or ulcer that has been present at least for 3-4 week duration and has not responded to conventional therapies. Non healing wounds occur due to tissue hypoxia, i.e., a lack of healing oxygen to the area. This is common in diabetics and in people who are bed-ridden, but could happen in any one with comprised blood flow (blood carries oxygen to a wound for healing but also carries dead cells away from the wound). A compromised blood flood would be due to a narrowing of the vessels that carry blood as in cardiovascular disease. Diabetics in particular often suffer from non healing wounds in the extremities, especially the feet. The further away a wound is from the heart in some one with compromised blood flow, the more difficult the healing process.

Collagen: Proteins are natural polymers and make up almost 15% of the human body. Collagen is the major protein of the extra cellular matrix (ECM) and is the most abundant protein found in mammals, comprising 25% of the total protein and 70% to 80% of skin (dry weight). Collagen acts as a structural scaffold in tissues. The central feature of all collagen molecules is their stiff, triple -stranded helical structure. Types I, II, and III are the main types of collagen found in connective tissue and constitute 90% of all collagen in the body.

Function of Collagen in Wound Healing: Previously, collagens were thought to function only as a structural support; however, it is now evident that collagen and collagen-derived fragments control many cellular functions, including cell shape and differentiation, migration and syntheses of a number of proteins. Findings suggest that cell contact with precise extra cellular matrix deposition. Type I collagen is the most abundant structural component of the dermal matrix; migrating keratinocytes likely interact with this protein. Collagenase (via formation of gelatin) may aid in dissociating keratinocytes from collagen-rich matrix and thereby promote efficient migration over the dermal and provisional matrices. Cellular functions are regulated by the ECM. The information provided by ECM macromolecules is processed and transduced into the cells by specialized cell surface receptors. Evidence demonstrates that the receptors play a major function in contraction of wounds, migration of epithelial cells, collagen deposition, and induction of matrix degrading collagenase. Although keratinocytes will adhere to denatured collagen (gelatin), collagenase production is not turned on in response to this substrate. Keratinocytes have been known to recognize and migrate on Type I collagen substratum, resulting in enhanced Collagenase production. Collagen plays a key role in each phase of wound healing.

Collagen-based Wound Dressings: There are a number of different collagen dressings available, which employ a variety of carriers/combining agents such as gels, pastes, polymers, oxidized regenerated cellulose (ORC), and ethylenediamine tetraacetic acid (EDTA). The collagen within these products tends to be derived from bovine, porcine, equine, or avian sources, which is purified in order to render it non antigenic. The Collagen dressings are comprised of Type I (native) collagen; whereas, other collagen dressings contain denatured collagen as well, a given collagen dressing may contain ingredients, such as alginates and cellulose derivatives that can enhance absorbency, flexibility, and comfort, and help maintain a moist wound environment. Collagen dressings have variety of pore sizes and surface areas, as well. All of these attributes are meant to enhance the wound management aspects of the dressings. Many collagen dressings contain an antimicrobial agent to control pathogens within the wound. Collagen dressings typically require a second day dressing (see table 1 for a summary of currently available collagen-based wound dressings).

Table 1: Collagen-based Dressings Currently on the Market

There are no wound covers made from human collagen. Non-human collagen are immunogenic though not strongly besides may harbor other pathogens. Therefore, screening is very essential.

OBJECTIVES

The first objective of the present disclosure is to provide a process of preparing a scaffold for tissue engineering, therapeutic drug delivery and for wound covering and healing purposes.

Another objective of the present disclosure is to prepare the scaffold for tissue engineering, therapeutic drug delivery and for wound covering and healing purposes using umbilical cord as a starting material.

Yet another objective of the present disclosure is to provide slices of the paste prepared from umbilical cord for adsorbing various therapeutic ingredients to achieve release in a regulated manner.

Still another objective of the present disclosure is to provide a transdermal patch prepared from umbilical cord using the process of instant disclosure.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1: Umbilical cord patches stored in poly-ethylene pouches
Figure 2: Umbilical cord paste in a syringe

Figure 3: Scaffold sufficiently translucent for tissue engineering purpose

SUMMARY OF THE DISCLOSURE

Accordingly, the present disclosure provides a process for preparing scaffold for tissue engineering and therapeutic drug delivery, comprising steps of: obtaining collagen rich source devoid of extrinsic material; immersing the collagen rich source in alcohol, drying and again immersing in alkaline solution to neutralize endotoxins; washing, cutting and thawing the collagen rich source followed by grounding into a paste; and layering the obtained paste on a surface made of a polymer to obtain the scaffold for tissue engineering and therapeutic drug delivery.

DETAILED DESCRIPTION OF THE DISCLOSURE

Before explaining any one embodiment of the present disclosure by way of drawings, experimentation, results, and pertinent procedures, it is to be understood that the disclosure is not limited in its application to the details as explained in below embodiments set forth in the following description or illustrated in the drawings, experimentation and/or results. The disclosure is further capable of other embodiments which can be practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary—not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

The present disclosure is in relation to a process for preparing scaffold for tissue engineering and therapeutic drug delivery, comprising steps of: obtaining collagen rich source devoid of extrinsic material; immersing the collagen rich source in alcohol, drying and again immersing in alkaline solution to neutralize endotoxins; washing, cutting and thawing the collagen rich source followed by grounding into a paste; and layering the obtained paste on a surface made of a polymer to obtain the scaffold for tissue engineering and therapeutic drug delivery.

In an illustrative embodiment, collagen rich source is natural and/or synthetic, but not limiting to umbilical cord from animal or human subjects.

In an illustrative embodiment, said immersion is performed in absolute alcohol for about 1 minute and the alkaline solution is IN NaOH.

In an experimental embodiment the cutting is performed under laminar air flow followed by transferring to a liquid nitrogen tank for storage.

In an illustrative embodiment said grounding is performed in a blender for a time period of about 5 minutes at an rpm ranging from about 1000 to 5000.

In an illustrative embodiment said paste is stored in a syringe at a temperature of about 4° C or can be made into films and stored in sterile ply-ethylene pouches.

In an illustrative embodiment said scaffold can be used as a wound cover for healing wounds.

The present disclosure is related to a scaffold for tissue engineering and therapeutic drug delivery, wherein said scaffold is obtained by the process as described in the above embodiment.

In an illustrative embodiment said therapeutic compound is a peptide, protein, nucleic acid or a drug and are formulated as transdermal patches for slow and controlled release of therapeutic compound.

In an illustrative embodiment tissue engineering comprises culturing cells selected from a group consisting of umbilical cord cells, stem cells, embryonic stem cells, pluripotent cells, multipotent cells, chondrocytes, osteoblasts, osteocytes, fibroblasts, bone marrow cells, stromal cells, chondrocyte progenitors, osteoclasts, endothelial cells, macrophages, adipocytes, monocytes, plasma cells, mast cells, leukocytes, epithelial cells, myoblasts, and precursor cells derived from adipose tissue.

In an illustrative embodiment, it is provided that the crux of the method of preparing the wound cover and the tissue engineering scaffold is that growth factors present in the umbilical cord is preserved, which will help in the healing of wound, neoangiogenesis and supporting three dimensional growth of tissues 'in-vitro'. The novelty and inventive step of the instant disclosure does not lie in a single step in the process, it is in fact the combination of all the steps which help in achieving the success of the instant disclosure.

In an yet another illustrative embodiment, is that this preparation uses human collagen, which is immuno compatible and does not harbor uncharacterized pathogens, probably present in collagen of non human origin.

Umbilical cord which develops during gestation of the human embryo is the main structure which provides oxygenated blood and nutrition, while removing metabolic waste to the developing fetus; it is predominantly made up of collagen which is made by mesenchymal cells.

Traditionally "Umbilical Cord" is discarded after division from the infant at birth, the umbilical cord is composed of a vein and two arteries surrounded by a sticky, jelly-like substance called Wharton's jelly, all encased in the surrounding tissue. The cord varies in length from inches to over three feet in length and is highly flexible. The umbilical cord is fetal tissue in a primitive state, giving it the advantage that antigenicity is lower than in adult tissue.

The umbilical cord is obtained as a waste of child birth. The umbilical cord is initially screened for infectious diseases viz. HIV-1 & 2, Hepatitis B & C and Syphilis. Based on the screening results the decision will be taken for usage. Based on the need, the umbilical cord may be used fresh, or it may be preserved for future use. The cord may be freeze-dried, refrigerated, chemically stored or preserved in other known ways. It may require treatment with antibiotics, chemicals, drugs, X-rays and temperature to ensure that it is sterile when ready for use. It is antigenic and may require chemical or other known treatment to remove any antigen substances. Coiled at the time of delivery, the cord can be straightened out by mechanical or chemical techniques. Cords obtained from mammals, premature babies, early or terminated pregnancies can also be used to repair smaller vessels.

The disclosure is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope of the present invention. On the contrary, it is to be clearly understood that various other embodiments, modifications, and equivalents thereof, after reading the description herein in conjunction with the drawings and appended claims, may suggest themselves to those skilled in the art without departing from the spirit and scope of the presently disclosed and claimed invention.

Example 1; Screening and Cleaning of Umbilical Cord - a source of collagen
The umbilical cord obtained as a waste of child birth. Initially, the umbilical cord is screened for infectious diseases viz. HIV-1 & 2, Hepatitis B & C and syphilis. All the screening tests are performed as per the existing known methods. Based on the screening results, a decision is taken to use the umbilical cord for further processing. If the results are favorable, then the cord is collected fresh within 24 hrs of delivery for further processing. The process begins with thorough cleaning in sterile saline several times to remove externally sticking material or any other extrinsic material. The cleaned cord is then immersed in absolute alcohol for a time period of about l min and then dried in clean air under a laminar flow hood for a time period ranging from about 5 minutes to 10 minutes.

Example 2; Processing source of collagen

The source of collagen as obtained in example 1 is then immersed in IN NaOH for a time period of about l min to neutralize endotoxin. This step is followed by washing it several times in sterile distilled water till the wash tests neutral. Thereafter, the cord is cut into 4-6 pieces using sterile instrument under laminar flow hood and transferred to a liquid nitrogen tank and stored there till further process. The stored cord is taken out and allowed to thaw to room temperature in a laminar flow hood. It is then ground to a paste in a sterilized warning blender for a time period of about 5 minutes at an rpm ranging between 1000 - 5000. The paste is then taken into 20 ml syringes and stored at 4°c till use.

Example 3; Storage and packaging of the product

The paste as obtained in example 2 can also be made into films. The thickness of films can be variable. These films are dried sterile in a freeze drier. They are packed in sterile poly-ethylene pouches to be taken out when needed for use as a wound cover (Fig: 1). Tensile strength of the film can be increased by cross linking with formaldehyde treatment. The paste in syringe can also be directly applied, if the wound is irregular in shape (Fig: 2)

Example 4: Preparation of scaffold for tissue engineering

The preparation as obtained under example 2 can also be used for making an excellent scaffold for tissue engineering purpose. The paste can be layered on to the surface of petri dishes and multi well plates made of polystyrene and other plastics used for making disposable tissue culture ware. The thickness can be controlled so as to make the scaffold sufficiently translucent so that cells growing on the scaffold become visible, a necessity for tissue engineering application (Fig: 3). Similarly, it can be used to prepare wound coverings.

Example; 5 Adsorbing therapeutic compounds and formulating as transdermal patches for drug delivery

The preparation as obtained from examples 1 to 3 is used in this example. It is evident that the umbilical cord by virtue of being rich in extra cellular matrix components, are capable of adsorbing various therapeutic compounds such as peptide, protein, nucleic acid or a drug. Examples include but not limited to hormones, insulin, growth factor and such substances of therapeutic importance. The substances adsorbed to the preparation can be formulated as transdermal patches. Based on the preliminary studies it was found that the adsorbed therapeutic substances were released in a regulated manner and therefore, this preparation will be used in slow release transdermal patch or similar such application of therapeutic importance.

Example; 6 Comprehensive process of preparing scaffold for tissue engineering/ wound covering/ transdermal patch for therapeutic drug delivery

The umbilical cord can be obtained as a waste of child birth. Initially, the umbilical cord is screened for infectious diseases viz. HIV-1 & 2, Hepatitis B & C and syphilis. All the screening tests are performed as per the existing known methods. Based on the screening results, a decision is taken to use the umbilical cord for further processing. If the results are favorable, then the cord is collected fresh within 24 hrs of delivery for further processing. The process begins with thorough cleaning in sterile saline several times to remove externally sticking material or any other extrinsic material. The cleaned cord is then immersed in absolute alcohol for a time period of about 1min and then dried in clean air under a laminar flow hood for a time period ranging from about 5 minutes to 10 minutes.
This is then immersed in IN NaoH for a time period of about 1min to neutralize endotoxin. This step is followed by washing it several times in sterile distilled water till the wash tests neutral. Thereafter, the cord is cut into 4-6 pieces using sterile instrument under laminar flow hood and transferred to a liquid nitrogen tank and stored there till further process. The stored cord is taken out and allowed to thaw to room temperature in a laminar flow hood. It is then ground to a paste in a sterilized waring blender for a time period of about 5 minutes at an rpm ranging between 1000 - 5000.

The paste is then taken into 20 ml syringes and stored at 4°c till use. The paste as obtained can also be made into films. The thickness of films can be variable. These films are dried sterile in a freeze drier. They are packed in sterile poly-ethylene pouches to be taken out when needed for use as a wound cover (Fig: 1). Tensile strength of the film can be increased by cross linking with formaldehyde treatment. The paste in syringe can also be directly applied, if the wound is irregular in shape (Fig: 2). The preparation as obtained can also be used for making an excellent scaffold for tissue engineering purpose. The paste can be layered on to the surface of petri dishes and multi well plates made of polystyrene and other plastics used for making disposable tissue culture ware. The thickness can be controlled so as to make the scaffold sufficiently translucent so that cells growing on the scaffold become visible, a necessity for tissue engineering application (Fig: 3). Similarly, it can be used to prepare wound coverings. The preparation as obtained is capable of adsorbing various therapeutic compounds such as peptide, protein, nucleic acid or a drug. Examples include but not limited to hormones, insulin, growth factor and such substances of therapeutic importance. The substances adsorbed to the preparation can be formulated as transdermal patches. Based on the preliminary studies it was found that the adsorbed therapeutic substances were released in a regulated manner and therefore, this preparation will be used in slow release transdermal patch or similar such application of therapeutic importance.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

We claim;

1) A process for preparing scaffold for tissue engineering and therapeutic drug delivery, comprising steps of:

a) obtaining collagen rich source devoid of extrinsic material;

b) immersing the collagen rich source in alcohol, drying and again immersing in alkaline solution to neutralize endotoxins;

c) washing, cutting and thawing the collagen rich source followed by grounding into a paste; and

d) layering the obtained paste on a surface made of a polymer to obtain the scaffold for tissue engineering and therapeutic drug delivery.

2) The process as claimed in claim 1, wherein said collagen rich source is natural and/or synthetic, but not limiting to umbilical cord from animal or human subjects.

3) The process as claimed in claim 1, wherein said immersion is performed in absolute alcohol for about 1 minute and the alkaline solution is IN NaOH.

4) The process as claimed in claim 1, wherein the cutting is performed under laminar air flow followed by transferring to a liquid nitrogen tank for storage.

5) The process as claimed in claim 1, wherein said grounding is performed in a blender for a time period of about 5 minutes at an rpm ranging from about 1000 to 5000.

6) The process as claimed in claim 1, wherein said paste is stored in a syringe at a temperature of about 4° C or can be made into films and stored in sterile ply-ethylene pouches.

7) The process as claimed in claim 1, wherein said scaffold can be used as a wound cover for healing wounds.

8) A scaffold for tissue engineering and therapeutic drug delivery, wherein said scaffold is obtained by the process as claimed in claim 1.

9) The scaffold as claimed in claim 10, wherein said therapeutic compound is a peptide, protein, nucleic acid or a drug and are formulated as transdermal patches for slow and controlled release of therapeutic compound.

10) The scaffold as claimed in claim 10, wherein tissue engineering comprises culturing cells selected from a group consisting of umbilical cord cells, stem cells, embryonic stem cells, pluripotent cells, multipotent cells, chondrocytes, osteoblasts, osteocytes, fibroblasts, bone marrow cells, stromal cells, chondrocyte progenitors, osteoclasts, endothelial cells, macrophages, adipocytes, monocytes, plasma cells, mast cells, leukocytes, epithelial cells, myoblasts, and precursor cells derived from adipose tissue.