Root surface bio-modification


To achieve new attachment on 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 removal of these deposits on the root surface as well as 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 1. This layer has 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  new  connective  tissue  attachment  to  the  root  following  periodontal  reconstructive procedures. Root surface bio-modification involves application of a weak acid on the root surface which remove this smear layer from the root surfaces, exposes the collagen fibers, produces a zone of demineralization and opens and enlarges the dentinal tubules. The exposed dentinal collagens are supposed to act as chemoattractant for periodontal fibroblasts 3.  

Classification of root surface bio-modification agents:

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

  1. Root conditioners
    • Citric acid
    • Tetracycline HCI
    • EDTA
    • Fibronectin
    • Laminin
    • Doxycycline
    • Minocycline
    • Polyacrylic acid
    • Phosphoric acid
    • Formaline
    • Chlorhexidine
    • Hydrogen peroxide
    • Cetyl pyridium chloride and sodium-n-lauroyl sarcosine
    • Cohnns factor
    • Bile salts and plasma fractions
  1. Dentin bonding conditioners
  2. Enamel matrix proteins
  3. Platelet-rich plasma
  4. Recombinant human growth factors
  5. 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 4, and since then………………..


In brief following mechanisms have been suggested regarding effects of citric acid application on root surface,

  • It causes removal of smear layer.
  • It causes partial demineralization of the root surface which enhances new attachment/reattachment and regeneration.
  • It has got 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 dentin surface than tetracycline hydrochloride, ethylenediaminetetraaceticacid (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 pallets 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 5. Miller used brushing technique for root bio-modification during root coverage procedures 14, 15. It has been demonstrated that application of citric acid (pH= 1) for 3 minutes using a rubbing pressure on the surface results in a complete removal of smear layer and exposure of smoother root dentin surface with relatively wide dentinal tubular openings (flared or funnel shaped) even up to 8.88 µm 16, 17.

Drawbacks of citric acid:

Some drawbacks associated with citric acid application include formation of extremely acidic environment in the surrounding tissues, which may result in un-favourable 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 concentration of the acid pH of the acid, duration of application and mode of application.

Know more……….

Effect of citric acid and tetracycline hydrochloride on fibroblast behaviour:
Citric acid, tetracycline hydrochloride and tetracycline derivatives such as minocycline hydrochloride and doxycycline hydrochloride are frequently used for root surface bio-modification. These are mild acids which remove the smear layer from the root surface and expose the organic components such as type I collagen, proteoglycans, fibronectin and growth factors 19. Studies have demonstrated that they can also influence fibroblast behaviour thereby improving their attachment and migration on root surface. The proposed mechanisms by which these influence fibroblast behaviour are 5, 20-22,

  • Induced cementogenesis
  • Collagen splicing
  • Fibronectin fibrin-collagen binding thereby inhibiting epithelial apical migration
  • Enhanced fibroblast chemotaxis, migration and attachment


Tetracycline hydrochloride:

The tetracyclines are a group of bacteriostatic antimicrobials effective against a wide range of organisms. 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 and by its local antimicrobial effects 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,


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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 sustained release from 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. 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 open dentinal tubules. A 100 mg/ml tetracycline hydrochloride solution with pH 1.6 can be prepared by mixing 100 mg of tetracycline hydrochloride powder in 1 ml sterile water. The application time suggested is 2-3 minutes 27 since long etching time of 3 minutes and above has been shown to impair periodontal healing 28.


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.


Ethylene-diamine-tetra-acetic-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. Ethylene-diamine-tetra-acetic-acid (EDTA) is a chelating agent which is used widely during endodontic treatment. EDTA exerts its demineralizing effect through chelating divalent cations at neutral pH. Studies have shown that……………..


It was also demonstrated by one study that use of EDTA gel as a root surface conditioning agent negatively affected the outcome of root coverage 33. Research done on clot stabilization demonstrated that clot adherence to root surface bio-engineered with EDTA obtained worse results when compared to citric acid and tetracycline hydrochloride 10.  


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 ECM 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 partial demineralized root surfaces enhances new attachment and cell proliferation from periodontal ligament and supra crestal area 35. Optimum concentration for fibronectin application has been shown to be 0.38 mg/ml saline.

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

It has been shown that citric acid conditioning and subsequent fibronectin application gives better results as compared to exogenous fibronectin application only. One study investigated the effect of citric acid and fibronectin application on post operative attachment gains after GTR procedure. The results of the study demonstrated significant gains in clinical attachment and probing depth reduction 37. However, more clinical data is required to authenticate these results.


The most abundant components of basement membranes are the laminins and type IV collagens. 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 are not same towards mineralized surface. A mineralized surface attract laminin which favours epithelial proliferation whereas a demineralized surface attract fibronectin and favour fibroblast proliferation 36. As effect of laminin is down growth of epithelium, it is undesirable. The role of laminin needs to be investigated to authenticate its efficacy as root surface bio-modification agent.


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 14 days 41. A 100 mg/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.


It is a semi synthetic tetracycline having good bacteriostatic potential. Minocycline has a low pH in concentrated solution, acts as a calcium chelator and its application results in enamel and root surface demineralization and removal of endotoxin invading untreated periodontally diseased roots 42. It possesses anti-collagenase activity and promotes fibroblast attachment to 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 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 effect of polyacrylic acid on root surface and its biological effects, more clinical research is required to authenticate its clinical use.

Many other agents such as phosphoric acid, formaline, chlorhexidine, hydrogen peroxide, cetyl pyridium chloride and sodium-n-lauroyl sarcosine, Cohnns factor and bile salts and plasma fractions have also been investigated as root surface bio-modification agents, but there is scanty evidence available for their use in comparison to routinely used agents such as citric acid, tetracycline hydrochloride or EDTA.

Dentin bonding conditioners:

The dentin bonding conditioners have also been used as root surface conditioners. In one scanning electron microscope (SEM) 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 behaviour 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 play a key role in mineralization. The enamel matrix proteins are involved in early tooth development and play a vital role during formation of cementum, periodontal ligament and alveolar bone. Application of enamel matrix proteins on root surface creates a biological environment similar to that during tooth development favouring periodontal regeneration.

Biological basis:

During tooth formation, the Hertwig’s epithelial root sheath starts growing in 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 of the developing root surface. As the Hertwig’s epithelial root sheath disintegrates,………………………


Evidence for regeneration with enamel matrix derivatives (EMD):

The initial research to evaluate the potential of enamel matrix derivatives in periodontal regeneration was done in animal models.  The first animal experiment was done on monkeys to evaluate the potential of EMD in initiating regeneration or reformation of acellular cementum 47. After 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 enamel matrix derivatives (EMD) 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 propylene glycol alginate (PGA) was more effective than hydroxyethyl cellulose (HEC) or dextran in carrying and allowing precipitation of EMD.

Propylene glycol alginate (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 EMD even at room temperature. The thixotropic property of PGA solution allows formation of a viscous formulation. Upon application of shear forces, the viscosity of the material decreases which allows its easy application and complete coating of the root surface.

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 sits upto 2 weeks which provides sufficient period for proliferation of periodontal ligament 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 periodontal ligament 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 synthesis of matrix proteoglycans and hyaluronic acid 54.


It is the commercial available formulation of EMD. The main biologically active components of Emdogain® are freeze-dried enamel proteins, the amelogenin fraction. Propylene glycol alginate (PGA) acts as a…………………………



Know more…………

Why there is no foreign body reaction against enamel matrix proteins?

Being high molecular weight proteins derived from different species, body should generate an immune response against enamel matrix proteins. But, there is no foreign body reaction against them. This is because of the reason that these are one of the extremely conserved proteins during evolution for at least 350 million years 55.

So, these proteins are not considered as foreign by our body and there is no foreign body reaction against these proteins. So far, no preclinical local irritation or clinical sensitization trial has found any adverse reaction against these proteins 56.


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, 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 following steps,

  • The flap is elevated to get complete access to the root surface and underlying bone defect. A more conservative approach should be used to elevate the flap as compared 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 (PrefGelâ„¢, 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 on 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 complete involved root surface. 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 is achieved.

Post-operative instructions:

The patient is given a systemic antibiotic cover for 2-3 weeks post-operatively.  Patient is instructed not to chew in the operated area or brush in this 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:

Platelets are important component of blood coagulation cascade. Major components of patelet structure are secretory granules (primary, secondary and tertiary granules), which contain growth factors, coagulation proteins, adhesion molecules, cell-activating molecules, cytokines, integrins, inflammatory molecules, and some other molecules, which are synthesized in megakaryocytes and packaged into the granules through vesicle trafficking processes.

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 platelet rich plasma 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 periodontal ligament and favour bone formation. Research has provided evidence for improved cellular response following growth factor application. Gamal et al. investigated human periodontal ligament fibroblast…………………


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The effect of PDGF-BB combined with EDTA gel on adhesion and proliferation of cells on 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 “Growth factors in periodontal regeneration”.


Lasers have been studied for their effect of root surface 63-69 as well as for their effects on the behaviour on periodontal ligament 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 “Application of lasers in periodontal therapy”.

A scanning electron microscopic study evaluating the effect of the Nd:YAG laser radiation on removal of the 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 endotoxin treated root surface. Results of the study concluded that the treated root surfaces demonstrated areas of charring, crater formation, cementum meltdown and tracking. The organic matrix showed the signs of burning leaving behind re-solidified lava like substance. Authors concluded that the laser treatment adversely affects the bio-compatibility of the root surface making it un-favourable for fibroblast attachment 73.

One study was aimed at evaluating the effect of Er:YAG laser irradiation at 100mJ, 15pps 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 non diseased root surfaces. The study concluded that laser exposure denatures the surface protein which inhibits fibroblast attachment 74.


The rationale for root surface bio-modification 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 have been described in previous sections. Root surface bio-modification 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 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 area of application for considerable long duration of time during the healing period. Most importantly, our major goal is to make them easily available and cost effective.  

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