GB2555901B - Method of preparation of a novel stem cell-biocomplex for clinical implantation in periodontal space - Google Patents

Description

TITLE

METHOD OF PREPARATION OF A NOVEL STEM CELL-BIOCOMPLEX FOR CLINICAL IMPLANTATION IN PERIODONTAL SPACE

FIELD OF THE INVENTION

The present invention relates to the field of regenerative dentistry, particularly relates to a method of novel cell-implant clinical grade stable biocomplex preparation by seeding or culturing mesenchymal stem cells on to titanium or zirconium or collagen based dental implant materials.

BACKGROUND AND PRIOR ART DISCUSSION

One of the aims of regenerative medicine is to reconstruct tissues through the combination of biomaterials and cells to remodel the damaged sections of the functional human body. The ultimate goal of tissue regeneration is to replace the lost or damaged regions. Stem cell-based tooth engineering is deemed as a promising approach in regenerative dentistry as a model. Dental pulp stem cells (DPSCs) represent a kind of adult cell colony which has the potential of self-renewing and multi-lineage differentiation. These DPSCs seem to be the source of odontoblasts that contribute to the formation of dentin-pulp complex. Some studies have proved that DPSCs are capable of producing dental tissues in vivo including dentin, pulp, and crown-like structures. Whereas other investigations have shown that these stem cells can bring about the formation of bone-like tissues.

Guided tissue regeneration (GTR) is focused on development of hard tissues in addition to the soft tissues for periodontal attachment. At present, guided bone regeneration is predominantly applied in the oral cavity to support new hard tissue growth on an alveolar ridge to allow stable placement of dental implants. Used in conjunction with dental implants and GTR by stem cells, owing to the remarkable advantage of regeneration of only a small amount of tissue is highly beneficial to the patient in the field of periodontal disease management. For decades, successful osseointegration has provided thousands of restorations that maintain normal function in bone disorders. With the increasing need to utilize implants in spinal injuries and cardiac milieu for growing patients and enhance their function to simulate normal physiology and nerve connection, there appears to he an urgent need for bio-implants enriched with live human stem cells that have the trans-differentiation capability to form three (3) critical tissues required for tissue regeneration namely bone, neurons and connective tissue cells.

The cell-implant bio-complexes functional output is considered most optimal for long-term stability and survival of implants. Among the progenitors, mesenchymal stem ceils (MSCs) have been shown to be a very promising cell type with their clinically proven trans-differentiation capacities in to lineages. The sources of MSCs are numerous, hut the choice of clinically significant source for harvesting with good yield expressing the retained characteristic markers profile would aid the implantation procedure to overcome the issues with cell number and density of seeding. Human umbilical cord tissue is one of the richest sources of clinical grade progenitor mesenchymal cells. The flow cytometric analysis of these MSCs revealed that the cells are positive for CD29, CD44, CD71, CD73, CD90, and CD105. Furthermore, these cells expressed HLA-A, B, C (MHC-I), hut not HLA-DP, DQ, DR (MHC-Π), or co-stimulatory molecules such as CD80 and CD86. The cultured MSCs from cord tissue were shown to possess potential to differentiate into adipogenic, osteogenic and chondrogenic lineages in vitro upon induction along with meseodermal lineage specific cell types. The same cells were shown ideal seed cells for bone tissue engineering as well.

The technology of dental implants has advanced rapidly in recent years to provide a solution to the aesthetic, functionality and health issues resulting from missing teeth. But conventional dental implants are not the perfect solution for replacing missing teeth as the healing process extends for many months and rejection of the implant occurs in about five percent (5%) of patients. Despite much advancement in implant technology conventional implants do not provide a truly permanent solution for periodontal integration a missing tooth. But the answer could lie in a highly researched new dental technique - stem cell dental implants biocomplexes which could well be the future of implant dentistry accelerating the osseointegration and propiception at the site.

Guided bone regeneration (GBR) encourages new bone growth to replace areas of damage and can be used alongside guided tissue regeneration (GTR) to rebuild soft tissue in a patient’s body. The technologies and practices behind these techniques are subject to constant refinement, and clinical studies examined the possible application of these techniques to other regions of the body. GBR involved epithelial and connective tissue exclusion and space creation to allow the cells of the odontal ligament to repopulate the root surface and to allow bone cells to grow into the area of the defect. US20090208907 entitled “Dental Implant” discloses a cylinder-shaped dental implant comprising a titanium-made base provided with a cavity for a porous structure and a canal for administering medications, used for two-stage implantation into the alveolar bone. This invention makes it possible to create the conditions for successful ingrowth of bone and soft tissues into the implant obtainable by the best ingrowth of the bone tissues into the internal porous structure and the soft tissue ingrowth into a surface porous 3D structure in the area of the alveolar bone. US 20100098670 entitled “Autologous dental pulp stem cell-based bone graft substitute” discloses a method for promoting the differentiation of dental pulp stem cells into osteogenic cells in vitro by contacting the dental pulp stem cells with a biocompatible calcified substrate having a collagen mimetic deposited thereon in a predetermined amount. US20150196371 entitled “Endosseous dental implant assembly” discloses a dental implant assembly comprising a dental implant comprising an abutment portion for connecting to a tooth crown and a hollow base portion defining a cavity therein, said abutment portion integrally with said hollow base portion and a bio-supportive or biodegradable scaffold carried by said hollow base portion of the dental implant wherein the scaffold is impregnated with regenerative stem cells, growth factor or bone graft material.

Although spinal (Biomaterials 23 (2002) 3369-3376) biodegradable implants and implantable cardioverter defibrillator have been in clinical practice, impregnating stem cells in the implants for guided regeneration of the damaged tissue is a new and logical direction owing to the ability of clinical grade stem cells to regenerate the damaged tissue after implantation and the implants already in clinical practice.

SUMMARY OF THE INVENTION

The main objective of the present invention is to prepare a novel progenitor cell-implant by seeding or culturing mesenchymal stem cells on to an inorganic and synthetic implant material biologically active and dynamic with live stem cells impregnated in it which will be useful for the purposes of clinical implantation in periodontal space enhancing osseointegration and propioception. Accordingly, the present invention provides a method of preparation of a bio complex dental implant enhancing osseointegration and proprioception in repaired tissue, comprising the steps of: (a) Culturing and expanding mesenchymal stem cells as per the method described in WO 2014/135949 (corresponding Indian Application No: 932/CHE/2013, published as 32/2015), the relevant portion of which is herein incorporated by reference; (b) Trypsinizing the cultured mesenchymal stem cells that are attached to an adherent plate surface; (c) Pelleting and reconstituting cells in sterile culture expanded osteocytes extract; (d) Coating dental implant surface with reconstituted mixture at room temperature ; and (e) Air drying the bio complex obtained for 2 minutes at room temperature in the class 100 cabinet to attain stability.

Accordingly, the present invention provides a method of preparation of bio complex dental implant enhancing osseointegration and proprioception in repaired tissue, comprising the steps of: (a) Culturing and expanding mesenchymal stem cells as per the method as described in WO 2014/135949 (corresponding Indian Application No: 932/CHE/2013, published as 32/2015), the relevant portion of which is herein incorporated by reference; (b) Trypsinizing the cultured mesenchymal stem cells that are attached to an adherent plate surface; (c) Pelleting and reconstituting cells in autologous protein rich plasma; (d) Coating dental implant surface with reconstituted mixture at room temperature; and (d) Air drying the bio complex obtained for 2 minutes at room temperature in the class 100 cabinet to attain stability.

Accordingly, the present invention discloses a method of preparing such a novel stem cell implant complex derived from human umbilical cord, dental pulp, adipose and stromal tissues by seeding or culturing mesenchymal stem cells on to titanium or zirconium or collagen based dental implant materials. The method of preparation of such implant 100 comprising the steps of: harvesting any mesenchymal stem cells 101 as described in WO 2014/135949 (corresponding Indian Application No: 932/CHE/2013, published as 32/2015), the relevant portion of which is herein incorporated by reference; trypsinizing the attached mesenchymal stem cells from the adherent plate surface upto 10 million; pelleting down 10 million mesenchymal stem cells and reconstituting it in 500 micro litres of sterile culture expanded osteocytes extract or autologous plasma rich plasma 102; coating dental implant surface (3 mm) with 500 micro litres of 10 million cells at room temperature in 3 minutes by dispensing the solution in 3 layers superimposed 103; air drying the complex for 2 minutes at room temperature in the class 100 cabinet, to attain stability 104. The obtained bio-complex is applied surgically into the periodontal space 105.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the embodiments of the present invention, reference is now made to the following description taken in connection with the accompanying in which:

Figure 1 illustrates a flowchart describing the steps involved in the preparation of the active biocomplex 100.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to exemplary embodiments and methods of the present invention as illustrated in the accompanying drawings, in which like reference characters designate like or corresponding parts throughout the drawings. It should be noted, however, that the invention in its broader aspects is not limited to the specific details, representative devise and methods and illustrative examples shown and described in connection with the exemplary embodiments and methods.

The clinical research conducted in this invention particularly concentrates on the MSCs derived from cord tissues and dental pulp. This method does not involve surgery or any in-vivo operational activities to collect the above mentioned tissues rather; they are collected form the discarded tissues or donated by a consented donor. The MSCs both from cord tissues and dental pulp, in equal proportion are isolated, cultured and expanded by the novel method as described in WO 2014/135949 (corresponding Indian Application No: 932/CHE/2013, published as 32/2015), the relevant portion of which is herein incorporated by reference.

Figure 1 shows the flow chart illustrating the steps involved in the preparation of the bio complex/ dental implant surgically placed in the tooth cavity for regeneration of dental tissues. The method of preparation such bio complex 100 comprises the following steps. Initially, the mesenchymal stem cells selected from the group comprising of human umbilical cord, dental pulp, adipose and other stromal tissues are culture expanded 101 as described in WO 2014/135949 (corresponding Indian Application No: 932/CHE/2013, published as 32/2015), the relevant portion of which is herein incorporated by reference. 10 million culture harvested MSCs are reconstituted in 500 micro litre of sterile culture expanded osteocytes extract or autologous protein rich plasma 102. Further the reconstituted cells are seeded or coated on dental implant surface made of titanium, zirconium and collagen based dental implant materials 103. Finally the obtained bio complex is air dried for 2 minutes in the room temperature 104 and the same shall be used to replace in the dental socket through surgical method 105.

The above performed method for producing a biocomplex makes the inert material bioactive which in turn makes the material catalyzing osseointegration and propioception in the microenvironment of periodontal socket, which is the invention step involved in the present invention. 10 million mesenchymal stem cell concentration is seeded or coated on 3 mm dental implant surface ratio in order to make it bio active when place in the dental cavity.

Example 1

The patient's left side was chosen as the test side. The surgical procedure was carried out under local anesthesia containing 2% lignocaine hydrochloride in 1:100,000 epinephrine. The biocomplex was placed at the osteotomy site with IF of 40 N cm and measured twice from two different angles, around 90 degrees and parallel to the crestalline. The two measurements buccolingually and mesiodistally are recorded and an average of both the values were taken on record. Patient safety evaluation in terms of unusual pain, swelling, allergic reaction, inflammation, outgrowth observed for 2 weeks post implantation was recorded to be negative while biocomplex stability measurements with Ostell ISQ device was recorded as >70.

Claims (8)

WE CLAIM:

1. A method of preparation of a bio complex dental implant enhancing osseointegration and proprioception in repaired tissue (100), comprising the steps of: a. Culturing and expanding mesenchymal stem cells (101); b. Trypsinizing the cultured mesenchymal stem cells that are attached to an adherent plate surface; c. Pelleting and reconstituting cells in sterile culture expanded osteocytes extract (102); d. Coating dental implant surface with reconstituted mixture at room temperature (103); and e. Air drying the bio complex obtained for 2 minutes at room temperature to attain stability (104).

2. A method of preparation of bio complex dental implant enhancing osseointegration and proprioception in repaired tissue (100), comprising the steps of: a. Culturing and expanding mesenchymal stem cells (101); b. Trypsinizing the cultured mesenchymal stem cells that are attached to an adherent plate surface; c. Pelleting and reconstituting cells in autologous protein rich plasma (102); d. Coating dental implant surface with reconstituted mixture at room temperature (103); and e. Air drying the bio complex obtained for 2 minutes at room temperature to attain stability (104).

3. The method according to claims 1 and 2, wherein the mesenchymal stem cells for culturing are selected from a group comprising of human umbilical cord, dental pulp, adipose and stromal tissues.

4. The method according to claims 1 and 2, wherein the dental implant surface is made of a material selected from a group comprising of titanium, zirconium, and collagen based materials.

5. The method according to claims 1 and 2, wherein it includes trypsinizing, pelleting and reconstituting 100 million mesenchymal stem cells in 500 micro litres of sterile culture expanded osteocytes extract or autologous plasma rich plasma.

6. The method according to claims 1 and 2, wherein it includes coating 3mm of a dental implant surface with 10 million reconstituted cells at room temperature in 3 minutes by dispensing the solution in 3 layers superimposed.

7. A bio-complex dental implant for enhancing osseointegration and proprioception in repaired tissue comprising a bio inactive dental implant coated with superimposed layers of reconstituted culture expanded mesenchymal stem cells.

8. A bio-complex dental implant according to claim 7, wherein the cultured cells are reconstituted in sterile culture expanded osteocytes extract or autologous plasma rich plasma.