Although Regeneration is not a new word in the field of medicine/dentistry, very few clinical aspects of regenerative endodontics are practiced.
Regeneration is a continuous natural process in the human body, billions of RBCs needs to be replaced each day through the haemopoeitic stem cells. Small intestinal lining continuously shed and regenerate, as do the cells of skin, hair and bone.
Wound healing and secondary dentine formation is a natural regenerative process to a stimulus, injury or disease.

What we will be discussing here is a part of regenerative endodontics---APICAL REGENERATION AND REVASCULARIZATION.
Various ways by which regenerative endodontics can be achieved-
1-APICAL REGENERATION & REVASCULARIZATION (IN VIVO)
2-POST NATAL STEM CELL THERAPY-
To fill in a mm scale defect we need 500000 MSCs , so technically we need to isolate stem cells from source (skin, buccal mucosa, fat and bone, pulps) , cultivate ,expand them in vitro in a lab and then implant in vivo.
3-PULP IMPLANTS-
Three dimensional pulp tissue expanded and grown in a lab and then transplanted into a disinfected root canal system.
4-SCAFFOLD IMPLANT-
Quite similar to pulp implants except the three dimensional pulp body is grown on a porous polymer self resorbing scaffold and then delivering it into the disinfected root canal system.
5-CELL PRINTING-
Three dimensional cell printing techniques uses layers of cells suspended in a hydrogel scaffold to recreate the structure of tooth pulp tissues, we have the advantage of precisely placing cells so as to mimic natural pulp tissue structures.
6-GENE THERAPY-
Simply delivers mineralizing genes into the pulp tissue space to promote tissue mineralization.
APICAL REGENERATION & REVASCULARIZATION.
We need to ask a few questions? What is apical regeneration & revascularization, when to do it, why do we do it, and how do we do it?
What is apical regeneration & revascularization.
It is a self defying word-Biologically based procedure designed to replace damaged -diseased structures including dentin and root structures as well as cells of the pulp-dentin complex.
In tooth with an immature apex, with a necrotic and infected pulp this procedure simply give us the chance for continuous root end development and apical closure along with continuous strengthening of dentinal walls thus significantly improving the prognosis of the tooth and survival


WHEN TO DO IT?

Human immature tooth is developing organ. Any trauma (fracture, caries, anatomic anamolies, etc) at this stage leading to necrotic, infected pulp is a candidate to be considered for apical regeneration and revascularization.
WHY?


Immature avulsed tooth has a SHORT APEX, OPEN APEX, BLUNDERBASS CANALS, difficult to clean & shape, intact but NECROTIC PULP, WEAK WALLS.
With the current treatment modalities -
1-Calcium hydroxide (vitapex, metapex)
2-MTA


Appears to be the options available to successfully treat such cases and with proven successes, but they have some inheritent disadvantages,
1- Long treatment time, multiple appointments with calcium hydroxide
2-doesnt strengthen the weak root walls, so more prone to fracture
3- Poor crown to root ratio
4-high ph of calcium hydroxide of up to 14
5- Technique sensitive and weak apical closure
Long term use of calcium hydroxide intra canal has been shown to further weaken the tooth.
MTA has the advantage of far fewer appointments 1- 3 appointments, but we have chances of over extrusion & the apex is more over difficult to pack completely.
Rationale of endodontic treatment is to prevent and treat apical periodontitis, for apical periodontitis to be present pulpal necrosis and infection must be present, therefore vital (non infected)pulp ensures no apical periodontitis or at least prevents the spread to a certain extent.
The potential to regenerate an injured /necrotic pulp would always be the best root canal filling possible as we might be replacing it with the natural as close as possible.

With apical regeneration & re-vascularization, we can achieve complete apical closure, thus ensuring a proper crown to root ratio. Continuous strengthening of dentinal walls throughout the procedure can be observed, thus normal fracture resistance returns. Simple, inexpensive, requires no special skills & armamentarium and materials and prognosis significantly improved.
Studies have shown the formation of new fibrous pulp like tissues, by as soon as 5 weeks pulp test start to respond, a perfect natural seal is thus formed. long term fallow up of such cases needs to be done as chances of infection may be there ,but what apical regeneration has done is significantly improved the prognosis of such tooth so as to last for a life time. further research is going on as the source of the tissue is yet to be confirmed.
If no apical regeneration has been achieved after 3 months ,we can always revert back to other treatment modalities. Only disadvantage being discoloration at the CEJ due to use of minocycline has been reported. Intra canal bleach post revascularization and apical regeneration is the best option also crowns appears to be a good options.

DISCOLURATION AFTER 3 WEEKS
HOW?


STEP BY STEP PROTOCOL-
Rubber dam isolation during the treatment is a standard for all endodontic procedures.

RUBBER DAM ISOLATION PREOP FIRST APPOINTMENT
LA can be given if desired, no root canal instrumentation needed as this will further weaken the already fragile dentin walls, a 10 -15 no ISO 2% taper file or a small broach can some time be used carefully to remove the pulp. Proper careful debridement with copious irrigation protocol with 5.2% sodium hypo chlorite, 2% endodontic hexidene and normal saline thus becomes very important first step in apical regeneration and revascularization. care should be taken not to use sodium hypo and hexidene concomitantly as formation of carcinogenic compounds been noted so after every use of sodium hypo wash it off with saline and vice versa ,care should be taken also not to forcefully force the solution through the open wide apex, this might damage the apical papilla a key to revascularization. Although not necessary a vitapex dressing for seven days not touching the apex can be given. By this time most of the signs of inflammation ,infection subsides, intra oral sinus subsides.
Now packing a TRI ANTIBIOTIC PASTE, into the canals and leaving it there for further 3 weeks. Continuous follow up of the patient required by this time signs of repair becomes evident on our x-rays.

TRIPLE ANTIBIOTIC PASTE APPLIED
Peri apical area in such cases is devoid of blood supply so oral antibiotics are unable to reach the infected root canal space. According to a study by Hoshino et al this particular combination of antibiotics in the desired dosage was found to be the most effective way of disinfecting the root canal space.
After 3 weeks the most important step in revascularization can be carried out .Now, apical area of such tooth may retain some vital pulp tissues (APDCs), APICAL Papilla, also the periapical bone which can be stimulated to bleed and form a blood clot into the canal, wait for some time for clot formation, use of collaplug just 3-4 mm below the CEJ to prevent bleeding recommended. Since apical papilla is located at the tip of the root and receives blood supply from the surrounding tissues SCAP (stem cells from apical papilla) may survive even after pulp necrosis of endontontic treatment and continues to form root dentin.


BLEED INITIATED IN THE CANALS

COLLAPLUG APPLIED OVER THE CLOT
Very importantly this procedure is to be carried out under LA without a vasoconstrictor and a rubber dam. A large file, endondontic sharp explorer can be used to induce bleeding. The blood plaque remains intact below the CEJ at this level 3-4 mm thick layer of MTA has to be applied, next day recall a resin composite double seal is mandatory. Cavit should be used as a intermediary restoration whenever necessary.
What this procedure does is to induce the MSCs to promote continuous root end development and allows continuous thickening of dentinal walls .


MTA applied over the collaplug

TEMPORARY SEAL WITH CAVIT RESIN COMPOSITE SEAL
TRIPLE ANTIBIOTIC PASTE
Simply put this paste has been found to be the most effective way to disinfect the canal.
1-Ciprofloxacin-200mg
2-Metronidazole-500mg
3-Minocycline-100mg
carrier- Macrogol ,proplylene glycol,saline.
Don’t forget to remove the sugar coat with a sharp scalpel or blade. Crush each drug separately in a motor ,mix equal ratio of each drug along with the carrier on a sterile mixing pad .The paste can be carried into the canal via a lentulo or any other special devices used for delivering the mix into the canal ,desired consistency of the paste can be achieved accordingly.
MESENCHYMAL STEM CELLS-(MSCs)
NOW, what this procedure does is induces the mesenchymal stem cells from the apical area of a immature permanent tooth which contains apical papilla ,which can be stimulated to promote bleeding and thus inducing the MSCs cache to revascularize & regenerate the lost ,damaged dentin structures. Further, some pulpal tissue may also retain some vitality also the periapical bone may be stimulated too .Further studies needs to be done to confirm the source of MSCs as such we are at this stage not sure whether it is the apical papilla, pulpal remnants retaining some vitality, or the peri apical area which induces the release of MSCs. MSCs are multipotent stem cells which can develop into other body parts such as bone ,cartilage, and fat. 1-osteoblasts 2-chondroblasts 3-adipocytes 4-neural cells.
STEM CELLS
Undifferentiated cells that can differentiate and divide into all the cell types continuously are stem cells ,thus all our tissue and organ have a common origin. Stem cells can be Embryonic(fetal) or Adult(post natal).Stem cells & progenitor cells acts as a repair system for the body. Further stem cells can be divided according to there ability to form and differentiate, that is plasticity into--------------
1-TOTIPOTENT-first division of fertilized egg (each cell type can develop into a new individual)
2-PLURIPOTENT-can differentiate into all the specialized cell types
3-MULTIPOTENT-can differentiate into a number of cell types
4-OLIGOPOTENT-can differentiate into a few cell types
5-UNIPOTENT-can differentiate into only a single cell type
Stem cells undergo numerous cycles of cell division while maintaining the undifferentiated state, they have a capability to copy different cell type and self revival. MSCs are being used by researchers in the field of regenerative medicine & tissue engineering to artificially construct human tissue which has been previously damaged by injury or disease, neoplasm etc. MSCs are unique in their ability to develop cells which can contribute to replacing muscle tissues or internal organs. They can grow in vitro, in a lab under controlled condition or by using mediation to stimulate new cell growth within the human body. Some sources of MSCs are-bone marrow, fetal umbilical cord blood, pulp tissue, skin etc. MSCs differ from ESCs by the fact that they can be created without the need of an embryo but, such cells can’t develop into different types of cells in the body. Further, use of ESCs is controversial , unethical to many and banned by most countries as embryo is also considered life has to be destroyed. Now a day fertilized IVF embryo which goes as medical waste has gained approval for stem cell research.
The wisdom tooth, an excellent source of DPSCs (dental pulp stem cells) and also (SHED) human exfoliated deciduous teeth pulp has a excellent source of MSCs and are preferred from the ones taken from bone marrow as they can be easily stored ,preserved ,show greater activity and plasticity. ESCs are more versatile and plastic but their development can’t be controlled and ethical issue limits their use, adult stem cells are less proliferative , require high compatibility between donor and recipient ,they can self revive.

Some say we need ESCs to study the early phase of human development, which might be crucial in finding cure for numerous life threatening diseases incurable today ,Diabetes ,blindness ,vision impairment, deafness tooth loss through pro teeth stem cell development ,blood transplants ,baldness ,cancer developing a better drug delivery system to name a few ,ethical issues will always be involved limiting their use but now MSCs has been show to have better plasticity than previously thought and also by PLURIPOTENCY TRANSDUCTION continuous development and concepts are evolving, umbilical cord blood bank have come up its like buying a insurance policy if needed in the future for better survival. Because human third molars are discarded as medical waste , colonally expanded MSCs derived from dental pulp are valuable cell source for the generation Of Ips ( induced pluripotent stem cells) . Ips closely resembles human embryonic cells in many aspects including morphology, gene expression ,surface marker expression ,epigenetic states and ability to differentiate into three germinal layers (endoderm, mesoderm, and ectoderm) in vitro and in vivo.
Concept of tissue engineering was first conceived by Langer and Vacanti in the early 1990s. The most promising cell source for tissue engineering are stem cells. Tooth buds are also used as a source of stem cells for dental tissue regeneration, tooth buds contains both dental epithelial cells and MSCs and studies have reported the formation of bioengineered teeth with anatomically corrected tooth –crown shape and enamel, dentin and pulp tissues using dental cell reaggregated tooth bud cells. Dental stem cell banks have been established and patients have started to cryopreserve their DSCs. iPSCs (induced pluripotent cells) can be used for autologous tissue regeneration.The mechanism of how a blood clot benefits the processes of regeneration and revascularization is not entirely clear , one possibility being SCAP cells from the apical papilla may migrate into the root canal and produce dental pulp complex like tissue. Platelet derived growth factor delivered into the blood clot can also help maintain a sufficient number of MSCs into the canal to promote regeneration and revascularization. The blood clot can also act as a natural scaffold for attachment , proliferation and differentiation.
Science is like a ever flowing river ,nothing is static, what we are doing now might just be obsolete in the future, new materials ,drugs ,concepts and techniques are continuously being bombarded , endodontics is evolving ,we have to accept changes adopt them master the technique and put them into practical use incorporating them in your routine dental practice.
Regenerative endodontics has the capability to change the way we look at our treatment, we should keep our eyes and arms open adopt and accept the changes all for the benefit of our patients.
THANK YOU.