Introduction to root surface biomodification

To achieve new attachment on the diseased root surfaces affected by periodontal disease, it is necessary to eliminate calculus, bacterial plaque and other cytotoxic substances on or within the root surface. Periodontitis-affected root surfaces are hypermineralized and contaminated with cytotoxic and other biologically active substances 1. The non-surgical periodontal therapy, which involves scaling and root planing, is directed at the removal of these deposits on the root surface as well as the removal of diseased exposed cementum. It has been shown that periodontal instrumentation leaves behind a smear layer on the root surface which remains interposed between the gingival flap and the root surface 2. This layer has been found to be composed of microorganisms, cementum fragments, plaques, calculus and cementum matrix components and ranges in thickness from 2-15 µm 2. This layer impedes ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ………

Periobasics: A Textbook of Periodontics and Implantology

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Classification of root surface bio-modification agents

Various chemicals have been used on the root surface to remove the smear layer, thus promoting healing and further enhancing clinical outcomes. Along with this, application of growth factors to root surface to enhance regeneration has also been the focus of research. The root surface bio-modification agents are broadly classified into following categories,

1. Root surface conditioners,
     Citric acid.
     Tetracycline HCI.
     EDTA.
     Fibronectin.
     Laminin.
     Doxycycline.
     Minocycline.
     Polyacrylic acid.
     Phosphoric acid.
     Formalin.
     Chlorhexidine.
     Hydrogen peroxide.
     Cetyl pyridinium chloride and sodium-n-lauroyl sarcosine.
     Cohen’s factor.
     Bile salts and plasma fractions.
2. Dentin bonding conditioners.
3. Enamel matrix proteins.
4. Platelet-rich plasma.
5. Recombinant human growth factors.
6. Lasers.

Root surface conditioners

These agents are applied on the root surface to remove the smear layer, open and widen the dentin tubules, and expose the dentin collagen matrix. Following is the detailed description of root conditioners,

Citric acid

As already discussed in “History of periodontal regenerative therapy”, citric acid was initially suggested as root bio-modification agent by Register (1973) 4, and since then it has been studied extensively. Citric acid has been recommended for removing smear layer and exposing collagen in order to retard gingival epithelium down-growth 5-7. The application of citric acid on exposed root surfaces may prevent the apical migration of dentogingival epithelium which may be attributed to the early fibrin linkage of the root surface, thereby enhancing new attachment formation 8. Furthermore, citric acid demineralization of underlying dentin may enhance new connective tissue attachment by either accelerating cementogenesis or by its bactericidal properties 5, 9. In brief , following mechanisms have been suggested regarding the effects of citric acid application on the root surface,

  • It causes the removal of smear layer.
  • It causes partial demineralization of the root surface which enhances new attachment/reattachment and re-generation.
  • It has got the antibacterial effect.
  • It causes root surface detoxification.
  • It causes exposure of root collagen and opening of dentinal tubules
  • It also causes initial clot stabilization.

     Citric acid has been shown to produce more clot stabilization on the dentin surface than tetracycline hydrochloride, ethylenediaminetetraacetic acid (EDTA), sodium citrate or a saline solution 10.

Technique of application:

Different techniques have been used to apply citric acid to the root surface, including using cotton pellets 11, 12, immersing the specimen in the solution itself 13 and using a camel hair brush as an applicator 3. The rubbing technique was suggested by Register (1975) 5. Miller (1982, 1985) 14, 15 used the brushing technique for root bio-modification during root coverage procedures. It has been demonstrated that application of citric acid (pH= 1) for ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……….



Drawbacks of citric acid:

Some drawbacks associated with the citric acid application include the formation of the extremely acidic environment in the surrounding tissues, which may result in unfavorable wound healing responses 1. Its low pH has also been shown to induce cytotoxic effects when in direct contact with periodontal cells 18. The factors influencing the effects of citric acid on root surface include the concentration of the acid, pH of the acid, duration of application and mode of application.

Tetracycline hydrochloride

Tetracyclines are a group of bacteriostatic antimicrobials effective against a wide range of microorganisms. The unique property of drugs of this group is their ability to modulate the host response. This anti-microbial group of drugs has been shown to have matrix metalloproteinase inhibitory and anti-inflammatory properties. Along with this, tetracycline hydrochloride inhibits microbial attachment and has root surface conditioning properties 23. It has been demonstrated that tetracycline conditioning of the root surfaces not only removes the surface smear layer, but also inhibits collagenase activity and bone resorption 16. In a comparative study, the effect of tetracycline root conditioning and flap surgery was compared with flap surgery alone. The histological analysis revealed 0.27 mm of average increase in connective tissue attachment and also cementogenesis was seen in tetracycline-treated sites 24.

Thus, in brief, the properties of tetracycline hydrochloride, which make it a suitable root surface bio-modification agent are,

  • It enhances attachment and growth of gingival fibroblasts, thus facilitating regeneration.
  • It has anti-collagenase activity.
  • It has anti-inflammatory properties.
  • It has high substantivity.
  • It inhibits parathyroid hormone-induced bone resorption.

Know More…

Can pure tetracycline hydrochloride powder be obtained from commercially available tetracycline capsules?

No, for root surface conditioning a pure formulation of tetracycline hydrochloride is required. The commercially available tetracycline capsules have a significant amount of filler and other substances which may contaminate the root surface. Various pharmaceutical companies may provide a pure formulation of tetracycline hydrochloride.

     It also has an indirect relation to regeneration. Application of low pH tetracycline increases fibronectin and other extracellular matrix glycoprotein binding to the root surface, thereby enhancing fibroblast attachment and growth on the root surface. At the same time, it suppresses the proliferation and growth of epithelial cells. Tetracycline hydrochloride has a sustained release from the root surface for at least 48 hours and up to 14 days, which provides its anti-bacterial properties during healing period 25. Wikesjo et al. (1986) 26 demonstrated that 10 or 100 mg/ml solutions of tetracycline hydrochloride were sufficiently concentrated to remove the smear layer and expose a regular pattern of ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ………

Ethylenediaminetetraacetic acid (EDTA)

Use of acidic agents to demineralize the root surface had a drawback of adversely affecting the surrounding tissues. So, a chemical agent that could remove the smear layer and demineralize the tooth surface at neutral pH was required. EDTA is a chelating agent which is widely used during endodontic treatment. It exerts its demineralizing effect through chelating divalent cations at neutral pH. Studies have shown that application of 18% EDTA on the root surface improves fibroblast attachment and migration on the root surface and also facilitates the development of an oriented fiber attachment system between the demineralized surfaces 29, 30. On the contrary, results of some other studies which used 24% of EDTA (pH 7.0-7.2) and applied it to root surfaces for 2-3 minutes showed no difference in probing depth, clinical attachment level and probing bone levels between EDTA treatment and control root surfaces 31, 32. It was also demonstrated in one study that the use of EDTA gel as a ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ………

Fibronectin

Fibronectin is a high molecular weight extracellular matrix glycoprotein with a molecular weight of approximately 440 KDa. It is a dimer consisting of two nearly identical monomer strands linked by a pair of disulphide bonds. This glycoprotein exists in two main forms: 1) as an insoluble glycoprotein dimer that serves as a linker in the ECM (extracellular matrix), and; 2) as a soluble disulphide linked dimer found in the plasma (plasma FN) 34. The plasma form is synthesized by hepatocytes, and the extracellular matrix form is made by fibroblasts, chondrocytes, endothelial cells, macrophages, as well as certain epithelial cells.

     It is involved in many cellular processes, including tissue repair, embryogenesis, blood clotting, and cell migration/ adhesion. It has a chemoattractant effect on fibroblasts and mesenchymal cells and it also promotes cell adhesion to both collagen and scaled root surfaces. One important function of fibronectin is that it acts as a non-specific opsonin. It binds to actin and DNA, thus promoting cellular and tissue debris removal by macrophages. It has been shown that the application of fibronectin to partially demineralized root surfaces enhances new attachment and cell proliferation from periodontal ligament (PDL) and supra crestal area 35. Optimum concentration for fibronectin application has been shown to be 0.38 mg/ml of saline.

     One of the initial evidence for effects of fibronectin as root surface bio-modification agent was provided by Terranova and Martin (1982) 36, who demonstrated that after application of exogenous fibronectin, the fibroblast attachment to the root surface was improved significantly.

     It has been shown that citric acid conditioning and subsequent fibronectin application give better results as compared to exogenous fibronectin application only. One study ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ………



Laminin

The most abundant components of basement membranes are the laminins and collagen Type IV. While collagen has some adhesion-promoting activity, laminin has been demonstrated to have potent actions on cells: stimulating cell adhesion, growth, differentiation, and migration 38-40. It has been shown that laminin promotes gingival epithelial chemotaxis and in addition, movement of gingival fibroblasts. The affinity of laminin and fibronectin is not same towards the mineralized surface. A mineralized surface attracts laminin, which favors epithelial proliferation, whereas a demineralized surface attracts fibronectin and favors fibroblast proliferation 36. As laminin facilitates the down growth of epithelium, it is undesirable. The role of laminin needs to be investigated to authenticate its efficacy as root surface biomodification agent.

Doxycycline

Doxycycline belongs to the tetracycline group of drugs. It is an effective anti-microbial agent against periodontal pathogens. Along with this, it has anti-enzymatic properties. Topical application of doxycycline has shown a long-lasting substantivity on periodontally diseased root surfaces. It has been demonstrated that the anti-bacterial effect of doxycycline persists on the conditioned root surface upto to 14 days 41. A 100mg/ml solution of doxycycline can be obtained by mixing doxycycline HCl powder (100 mg) in sterile water (1 ml). It has a pH of approximately 2.2.

Minocycline

It is a semi-synthetic tetracycline having good bacteriostatic potential. Minocycline has a low pH in concentrated solution. It acts as a calcium chelator and its application results in enamel and root surface demineralization and removal of endotoxins invading untreated periodontally diseased roots 42. It possesses anti-collagenase activity and promotes fibroblast attachment to the root surface. Various studies done to evaluate the effects of minocycline on root surface when used as root surface bio-modification agent have demonstrated its efficacy comparable to other members of this group 42, 43.

Polyacrylic acid

Being a weak acid, polyacrylic acid has been used as root surface conditioning agent. Its acid etching effect removes smear layer from the root surface, making it more suitable for healing. One study compared periodontal healing after application of polyacrylic acid for 20 seconds and citric acid application for 3 minutes on the root surface. Results demonstrated a greater connective tissue adhesion to root surface in case of polyacrylic acid-treated teeth as compared to citric acid-treated teeth 44. Because a little clinical data is available regarding the effect of polyacrylic acid on the root surface and it’s biological effects, more clinical research is required to authenticate its clinical use.

     Many other agents such as phosphoric acid, formalin, chlorhexidine, hydrogen peroxide, cetyl pyridinium chloride and ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ………




Dentin bonding conditioners

The dentin bonding conditioners have also been used for root surface biomodification. In one scanning electron microscope study, the surface morphology of roots treated with dentin bonding conditioner was compared to that of routinely used root surface demineralization agents, citric acid and tetracycline hydrochloride. The results of this study indicated morphological similarities between surfaces obtained with the dentin conditioning agent and other acidic materials that are used routinely in periodontal regenerative therapy 45.

     There is relatively insufficient clinical research done on dentin bonding conditioners as root surface bio-modification agents and their regenerative potential. More comparative studies are required to investigate the behavior of fibroblasts towards root surface treated with dentin bonding conditioners as compared to traditional root surface conditioners.

Enamel matrix proteins

It is well established that organic matrix plays a key role in mineralization. The enamel matrix proteins are involved in early tooth development and play a vital role during formation of cementum, PDL, and alveolar bone. Application of enamel matrix proteins on root surface creates a biological environment similar to that during tooth development, favoring periodontal regeneration.

Biological basis:

During tooth formation, Hertwig’s epithelial root sheath starts growing in the apical direction from the cervical area to form a mould for the root. Subsequently, as the dentin formation starts, the cells of the Hertwig’s epithelial root sheath lying opposite to dentin enter into secretory phase. They start depositing enamel matrix proteins on the developing root surface. As the Hertwig’s epithelial root sheath disintegrates, cells from the surrounding connective tissue come in contact with the surface of root on which enamel matrix proteins are present. These cells then differentiate to express a cementoblast phenotype and start forming collagen and acellular cementum. In this way, cementum, PDL fibers, and alveolar bone are formed.

     It is important to note here that the mineralization of dentin is primarily on a ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ………

Evidence for regeneration with enamel matrix derivatives (EMDs):

The initial research to evaluate the potential of EMDs in periodontal regeneration was done in animal models. The first animal experiment was done by Hammarström (1997) 47 on monkeys to evaluate the potential of EMDs in initiating regeneration or reformation of acellular cementum. After a healing period of 8 weeks, the test sites had a thin layer of hard tissue with acellular extrinsic fibres and inserting collagen fibres which indicated regeneration in these areas.

     After it was shown, that EMDs were able to induce acellular cementum formation, these were applied in experimentally made dehiscence defects in monkeys. The results demonstrated evidence of periodontal regeneration 48. In this study, several drug vehicles were evaluated to determine which one of them most effectively allowed the EMD to precipitate on the treated root surface. It was seen that polyglycolic acid (PGA) was more effective than hydroxy-ethylcellulose (HEC) or dextran in carrying and allowing precipitation of EMDs.

     PGA is commonly used in food and pharmaceuticals as a thickening agent. One of the most useful properties of PGA is that its solution has a neutral pH which allows dissolving ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ………

Mechanism of action of EMD’s:

It has been demonstrated by various investigations (ellipsometry , total internal reflection fluorescence, and biospecific interaction analysis) that EMD’s adsorbs both to hydroxyapatite and collagen on denuded root surface. As a result, insoluble spherical complexes are formed which remain at the treated sites for up to 2 weeks which provides sufficient period for the proliferation of PDL cells or undifferentiated cells 49. In vitro (cell culture) exposure of fibroblasts to EMD’s was done in one study to find out fibroblast response to these proteins. It was found that EMD’s enhanced proliferation of PDL cells, but not epithelial cells. Total protein production by PDL cells was increased and mineralized nodule formation of PDL cells was also increased 50. Other potential mechanisms which potentiate periodontal regeneration upon EMD application include increase attachment of PDL fibroblasts to diseased root surfaces 51, increased production of growth factor 52, limiting epithelial down growth 53, and increased matrix formation by affecting fibroblast mRNA levels for the synthesis of matrix proteoglycans and hyaluronic acid 54.

Emdogain ®:

It is a commercially available formulation of EMDs. The main biologically active components of Emdogain® are freeze-dried enamel proteins, the amelogenin fraction. PGA acts as a vehicle to carry these biologically active proteins. The enamel matrix proteins are obtained and purified from tooth buds of porcine origin. The two components of the material i.e. enamel matrix proteins and PGA vehicle are mixed immediately prior to application to make a syringeable gel. The solubility of amelogenins is dependent on pH and temperature. At physiological pH, they are insoluble but they become ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ………



Clinical procedure:

The patient is thoroughly evaluated by clinical and radiographic examination. Pre-surgical preparation of the patient included scaling and root planing to remove plaque and calculus. In cases of tooth hypermobility due to occlusal taruma, the occlusal adjustment should be done. If occlusal adjustment is ineffective or is not feasible, splinting should be considered. The clinical procedure for Emdogain® application involves the following steps,

  • The flap is raised to get complete access to the root surface and underlying bone defect. A more conservative approach should be used to elevate the flap in order to optimize wound stability.
  • The granulation tissue and tissue tags are removed to completely expose the underlying bone. The deposits from the root surface are removed using hand and ultrasonic instruments.
  • The bleeding is controlled and the defect is isolated.
  • The root surface is then conditioned to remove the smear layer. A 24% EDTA formulation at neutral pH (PrefGel™, BIORA AB, Malmö, Sweden) has been shown to effectively remove the smear layer as well as expose the collagenous matrix of dentine and cementum by selective removal of mineral 1. The root EDTA gel is applied to the root surface for 15 seconds.
  • The area is then rinsed with saline and Emdogain® gel is applied to the root surface starting at the apical end and then covering the involved root surface completely. Any contamination with saliva or blood should be avoided.
  • The mucoperiosteal flap is replaced and sutured in such a way that primary closure and optimal wound stability are achieved.

Post-operative instructions:

The patient is given a systemic antibiotic cover for 2-3 weeks post-operatively. The patient is instructed, not to chew from the operated area or brush the area for the first 4-6 weeks. To achieve best results, the patient is recalled on a weekly or biweekly basis for professional tooth cleaning and plaque control.

Platelet-rich plasma (PRP)

Platelets are an important component of blood coagulation cascade. Major components of platelet structure are secretory granules (primary, secondary and tertiary granules), which contain ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……….

      The concept behind PRP application for periodontal regeneration is to obtain high-density platelet concentrate from patient’s own blood and then applying this concentrate in the area of periodontal wound healing where regeneration is desired. Platelet-derived growth factor (PDGF) is a major mitogen for fibroblasts, smooth muscle cells, and other cells 57. Platelets synthesize a mixture of the three possible PDGF isoforms (70% AB, 20% BB, 10% AA) 58. It has been shown that PDGF-AB is a potent stimulator of DNA synthesis in fibroblasts 59. A detailed description of PRP is available in “Application of platelet concentrates in periodontal regeneration”.

Recombinant human growth factors

Application of growth factors for periodontal regeneration is a major focus of research presently. Various growth factors which are believed to contribute to periodontal regeneration include the platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), transforming growth factor (TGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP). In general, these growth factors promote proliferation of fibroblasts from the PDL and favor bone formation. Research has provided evidence for improved cellular response following growth factor application. Gamal et al. (1998) 60 investigated human PDL fibroblast response to platelet-derived growth factor-BB (PDGF-BB) and insulin-like growth factor (IGF-1) application on tetracycline HCI (TTC) conditioned root surfaces. They concluded that there was a significant increase in fibroblasts adherence in the PDGF-BB and combination PDGF-BB/IGF-1 treatment groups as compared to the controls as well as the TTC control. The combination of PDGF-BB/IGF-1 did not significantly improve the adhesion of cells as compared to PDGF-BB alone 60.

     One study investigated the effect of human PDGF-BB on the attachment of ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ………

     The effect of PDGF-BB combined with EDTA gel on adhesion and proliferation of cells on the root surface was evaluated in another study. 8 specimens were derived from 40 periodontitis-affected teeth and were divided into 5 groups: Control group (untreated), SRP (scaling and root planing) group, EDTA (24%) group, PDGF (25 ng/ml) group and combined EDTA+PDGF group. The results of the study demonstrated significantly high cell adherence on PDGF and combined group as compared to control and SRP group 62.

      A detailed description of growth factors and their application in periodontal regeneration is available in “Application of growth factors in periodontal regeneration”.




Lasers

Lasers have been studied for their effect on the root surface 63-69 as well as for their effects on the behaviour of PDL cells 70-72. Although, laser therapy has been widely studied in periodontics for pocket debridement, wound healing and in various surgical approaches but there is insufficient data available regarding its efficacy in root surface bio-modification when compared with other traditionally used root surface conditioners. The detailed description of lasers and their application in various periodontal treatments has been given in “Lasers in periodontics”.

     A scanning electron microscopic study evaluating the effect of the Nd:YAG laser radiation on the removal of root surface smear layer after root planing in comparison with citric acid treatment demonstrated that the Nd:YAG laser effectively removed the smear layer, opened dentinal tubules, and exposed collagen fibers on the root surface without widening the diameter of tubules after root planing 64. Another in vitro study evaluated the effects of Nd:YAG laser (80 mJ at 10 pulses/second for 1 minute) on fibroblast attachment on the endotoxin-treated root surface. Results of the study concluded that ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ……. Contents available in the book ………   

      One study was aimed at evaluating the effect of Er:YAG laser irradiation at 100 mJ, 15 pps on root surface using scanning electron microscope, to determine the laser’s ability to remove lipopolysaccharides using infrared spectrometry. The results of the study showed that Er:YAG laser could remove 83% of lipopolysaccharide from the root surface, suggesting Er:YAG laser as a useful root conditioner 68.

     Laser application on root surface has a significant effect on fibroblast attachment. In an in vitro study, the effect of Nd:YAG laser (at energy 75 mJ at 20 pulse/sec using a 320 μm contact fiber for 1 minute) was evaluated on fibroblast attachment to the non-diseased root surfaces. The study concluded that laser exposure denatures the surface protein which inhibits fibroblast attachment 74.

Conclusion

The rationale for root surface biomodification is to remove the smear layer on the root surface, uncover and widen the dentin tubules, and unmask the dentin collagen matrix. Many agents have been used for this purpose, which has been described in previous sections. Root surface biomodification is usually combined with other procedures such as guided tissue regeneration and bone grafting to achieve best results. Presently, our focus of research is on the application of growth factors on root surface to achieve an environment, which is most conducive for regeneration. There are many difficulties in growth factor application which include purification and extraction of these growth factors, identification of appropriate carrier for them, and achieving their appropriate local concentration in the area of application for a considerably long duration of time during the healing period. Most importantly, our major goal is to make them easily available and cost effective.

References

References are available in the hard-copy of the website.

Periobasics: A Textbook of Periodontics and Implantology

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