Dental implant surface treatments

Introduction to implant surface treatment

One of the most important factors responsible for implant success is its primary stability. It has been shown in studies that still almost 10% failures are encountered. Implant failures are most commonly associated with poor bone quality ¹. Various properties of implant such as composition, surface topography, surface roughness and surface energy affect the stability of implant-tissue interface ^2-4. Albrektsson et al. (1981) ⁵ recognized that the implant surface, including topography, chemistry, surface charge, and wettability, is one of the important factors influencing osseointegration. It has been suggested that surface topography may affect the amount of bone formed at the implant-bone interface ⁶. Enhancing bone growth towards the implant surface has been regarded essential in cases with poor bone quality.

     Many in vivo studies have demonstrated that increased surface topography results in increased bone-to-implant contact early after implant placement ^7-11. Also, it has been shown that after loading there are more implant failures in implants with relatively smooth surfaces ^12-16, in comparison with rough-surfaced implants ^17-19. Implant surface modifications are also required because immediate loading implants are subjected to biomechanically challenging situations. It has also been suggested that texturing the implant surface may not directly contribute to the initial implant stability, but it may reduce the risk of stability loss and consequently facilitating wound healing (secondary osseointegration) ^{20, 21}. Our goals for implant surface modifications are,

• To achieve the initial stability that would improve the tolerance towards micro-movements by the implant and minimize the waiting period required for loading the implant.
• To provide a surface where the blood clot is stable and retained, favoring the healing process.
• To incorporate design factors, that would diminish the effect of shear forces on the interface (such as surface roughness related and thread features), so that marginal bone is preserved.
• To design features that may stimulate bone formation, and/ or facilitate bone healing (secondary osseointegration).

     The bioinert materials may behave differently when placed in the bone. Following terms should be known to us before we discuss the surface modifications of dental implants,

Osteogenesis:

It refers to the formation and development of bony tissue.

Osteoinduction:

Osteoinduction is the process by which osteogenesis is induced. In this case, primitive, undifferentiated and pluripotent cells are stimulated to develop into the bone-forming cell lineage, leading to the formation of new bone.

Osteoconduction:

An osteoconductive surface is the one that permits bone growth on its surface. In this case growth of bony tissue into the structure of an implant or a graft takes place.

Components of surface topography

The topography of a surface can be divided into three different categories: form, waviness, and roughness depending on the wavelength or peak-to-peak spacing of the surface morphology ²². Conventionally, the waviness and form are removed by filtering and numerical surface parameters are often calculated based on the roughness alone and are thereafter called surface roughness parameters ²². The surface roughness parameters either describe surface characteristics in two dimensions, 2D (marked by R), or in three dimensions, 3D (marked by S).
     Depending on what surface characteristics they are describing, 3D surface roughness parameters are divided into different groups which are ^23-25,

1. Amplitude.
2. Spatial.
3. Hybrid.
4. Volume and area.
5. Functional parameters.

     Amplitude refers to the amplitude property of the topographical features which is considered to be the most important property of the surface ²⁴. Spatial parameters describe the texture, randomness, and periodicity of the surface. Hybrid parameters are combinations of spatial and amplitude parameters where a change in either of the two properties affects the value of the hybrid parameter. The volume and area parameters are defined from the bearing curve of the surface and describe the bearing and void property of the topography. Functional parameters are more specific parameters which describe …………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…….

     In general terms implant surface roughness is divided, depending on the dimension of the measured surface features into macro, micro, and nano-roughness.

     Macro-roughness is defined as being in the range of millimeters to tens of microns. This scale directly relates to implant geometry, with a threaded screw and macroporous surface treatments. Macro roughness can improve implant fixation and long-term mechanical stability of the implant. It is because of enhanced mechanical interlocking between the macro-rough features of the implant surface and the surrounding bone ^{28, 29}.

     Micro-roughness is defined as being in the range of 1-10 μm. This is the ideal range, which maximizes the interlocking between implant surfaces and surrounding bone. Studies supported by some clinical evidence suggest that the micron-level surface topography results in the greater accrual of bone at the implant surface ^{29, 30}.

     Nanoscale topographies have been introduced recently involving materials that have a nano-sized topography or are composed of nano-sized materials with a size range between 1 and 100 nm. Studies have shown that nanometer roughness plays an important role in the adsorption of proteins, adhesion of osteoblastic cells and thus the rate of osseointegration ³¹.

Role of surface energy and surface charge

Surface energy is an important factor involved in the regulation of osteogenesis. The surface energy of a biomaterial is determined by the material’s surface-charge density and the net polarity of the charge. It has been said that depending on the surface energy, the surface state can either be hydrophilic or hydrophobic ³². Surface wettability is largely dependent on surface energy and influences the degree of contact with the physiological environment ^{33, 34}. In general, an increase in the surface energy causes an increase in the wettability of the surface thereby improving it’s biological activity. The energy state of the implant depends on the type of biomaterial, handling during manufacturing, mode of cleaning, sterilization, and needless to say, the handling of the implant during surgical procedure ^{35, 36}. Surface charge plays an important role during the initial biological reaction on the implant surface. In general, when the surface is positively charged, the surface turns hydrophilic and some of the plasma proteins essential for the initial osteogenic interactions adsorb to hydrophilic surfaces ^37-39.

     However, contradicting results have been found in other studies in which it was found that on a negatively charged biomaterial surface, cells proliferate more actively; meanwhile, multiple layers of cells and enlarged colonies of osteoblast-like cells were also observed. In contrast, cell adhesion and proliferation on positively charged biomaterial were found to be …………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…….

     At the nanoscale, a more textured surface topography increases the surface energy which in turn increases the wettability of the surface to blood, adhesion of cells to the surface, and facilitates binding of fibrin, matrix proteins, growth and differentiation factors. Therefore, nano-topo-graphy by modulating cell behavior can influence the process of cell migration, proliferation, and differentiation. These surfaces, thus enhance the process of osseointegration by hastening the wound healing following implant placement ⁴⁵.

Implant surface modification techniques

On an implant surface, irregularities can be produced by two methods: ablative/subtractive procedures or additive procedures.

Various implant surface treatments

Ablative procedures	Additive procedures
Surface blasting Acid etching Anodic oxidation Shot/laser peening	Plasma spraying Electrophoretic deposition Sputter deposition Sol-gel coating Pulsed laser deposition Biomimetic precipitation

Ablative procedures

These are surface subtractive procedures where the implant is subjected to various physical and chemical agents which make the implant surface rough by removing the implant biomaterial from its surface. These are as follows,

Surface blasting

One of the most frequently used treatments for creating a rougher surface on a titanium dental implant is surface-blasting. Within surface blasting procedure, there are 2 main groups: those created by grit blasting, such as aluminum oxide or titanium oxide and those using calcium phosphate as a resorbable blast media (RBM). The latter method creates a textured surface by blasting a traditional machined titanium implant with calcium phosphate ceramic, which is then passivated without acid etching to remove residual media.

     Grit blasting consists of bombarding a surface with small, hard, and biologically inert ceramic particles, like alumina (Al₂O₃) or silicon carbide (SiC), titanium oxide and calcium phosphate ^{46, 47}. Compressed air is used to project the particles through a nozzle at a very high velocity; depending on the size of the particles. There are some basic requirements of the blasting material which include: it should be chemically stable, should not affect the osseointegration property of the implant and should be biocompatible.

     The roughness values produced by the grit blasting technique depend mostly on the size of the particles used. A titanium surface grit-blasted with 600 μm alumina particles has a mean surface roughness of 4 μm and improves osteoblast cell response ⁴⁸. However, it has been shown in studies that presence of particle remnants on the implant surface, even after a thorough cleaning, may influence detrimentally the physicochemical properties of the surface ^{49, 50}. The problem with alumina particles is that they …………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…….

      Other media used for grit blasting include calcium phosphates such as HA, β-tricalcium phosphate and their mixtures. These materials have a particle size in the range of 75-85 μm ⁵¹ and most importantly, they are restorable, biocompatible and osteoconductive which enhances the implant osseointegration properties.

Acid etching

One of the most important advantages of acid etching is that it produces a highly clean and detailed surface, free of surface impurities and entrapped surface material. Studies have shown an enhanced bone apposition response around acid-etched implants during healing ⁵². This procedure has been shown to greatly enhance implant osseointegration especially in the early stages of peri-implant bone healing ⁵³. We also have a dual etching procedure where implant surface is subjected to the application of hydrochloric and sulfuric acid. Small peaks and valleys are formed in a uniform pattern with a peak-to-peak distance of 1-3 μm and a peak-to-valley distance of 5-8 μm ¹⁹. Osseotite (3i/implant innovations, west palm beach, FL) is commercially available dual etched implant.

Anodic oxidation

Anodic oxidation is an electrochemical process that increases the TiO2 surface layer and roughness. Here, micro- or nanoporous surfaces is produced by potentiostatic or galvanostatic anodization of titanium in strong acids (H₂SO₄. H₃PO₄, HNO₃, HF) at high current density (200 A/m²) or potential (100 V). The result of the anodization is to thicken the oxide layer to more than 1,000 nm on titanium. When the implant is immersed in any of the above electrolytes, it becomes an anode in an electrochemical cell. When a potential is applied to the sample, ionic transport of charge transfers through the electrochemical cell, and an electrolytic reaction takes place at the anode, resulting in the growth of an oxide film. The surface roughness of the TiUnite implant …………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…….

Shot/laser peening

In this process, the implant surface is bombarded with small spherical media called shot. Each piece of shot striking the material acts as a tiny hammer, imparting to the surface small indentations or dimples. In order to create a dimple on the surface, the surface fibers of the material must be yielded in tension. Below the surface, the fibers try to restore the surface to its original shape, thereby producing below the dimple a hemisphere of cold-worked material highly stressed in compression ⁶⁴. In this procedure, a honeycomb-like uniform surface with small pores is achieved.

     Recently, laser peening method has been introduced which is a non-contact, no-media, and contamination-free peening method ⁶⁵. Laser peening drives a high amplitude shock wave into a material surface using a high energy pulsed laser. The effect on the material being processed is achieved through the mechanical “cold working” effect produced by the shock wave, not a thermal effect from heating of the surface by the laser beam.

     Before the treatment, the surface is covered by a protective ablative layer (paint or tape) and a thin layer of water. After this, a high-intensity (5-15 GW/cm2) nanosecond pulses (10-30 ns) of laser light beam (3-5 mm width) striking the ablative layer generate a short-lived plasma which causes a shock wave to travel into the implant surface. This shock wave induces the compressive residual stresses which, when released produce a dimple leading to the formation of rough surface on repeated application of laser beam. It also causes improvements in fatigue life and retards the occurrence of stress corrosion cracking ^66-68.

Various commercially available implants and their surface treatment

Commercially available implant	Surface modification
Nobel Active: (TiUnite) Nobel Biocare, Zurich. Switzerland	Anodic oxidation. Phosphate enriched titanium oxide
Ankylos Plus. XiVE. Frialit: Dentsply, Friadent. GmbH	Sandblasted, large grit blasted, acid-etched.
Leader Italia: TiXos Line.	Laser Sintered Titanium
Leader italia: Traditional implant line	Biological Organic Acid Treatment
Tioblast: AstraTech Dental. Molndal. Sweden	Grit blasted with titanium oxide
Zimmer Screw Vent	Microtextured hydroxyapatite surface
Laserlok Surface-Biohorizon. Birmingham, Alabama	Laser peening
Adin implant system	Titanium plasma sprayed
Adin implant system. OsseoFix™	Calcium phosphate biocompatible resorbable blast media (RBM)
Straumann Bone Level: Institut. Straumann AG, Switzerland	SL Active: Sandblasted. Large grit blasted followed by acid etching
NanoTite: Biomet 3i, Palm Beach Gardens. Florida. USA	Calcium phosphate by discrete crystal deposition.
GSIII: Osstem. South Korea	Resorbable blast media (RBM): Calcium phosphate hydroxyapatite
3i/implant innovations (Osseotite), west palm beach, FL	Dual etching

Additive procedures

In these procedures, the implant surface is coated with various biomaterials which improve the biological and biomechanical properties of implants. The implant surface is coated with calcium phosphates mainly hydroxyapatite (HA). When the implant is placed in the bone, the release of calcium and phosphate in the surrounding tissue increases the saturation of the ions in the tissue fluid causing precipitation of biological apatite onto the surface of the implant.

Plasma spraying

This technique is based on thermal spray technology, which uses a device to melt and coat a material at a high velocity onto the substrate ⁷⁰. The plasma spray method has been widely accepted as the apatite coating method because it gives tight adhesion between the apatite coating and titanium. Plasma-sprayed HA coatings on commercial dental implants consist of mixtures of calcium phosphate phases, predominantly a crystalline calcium phosphate phase, HA, and an amorphous …………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…….

     The major advantages of this technique are its simplicity, rapid deposition rate, low substrate temperature, low cost and variable coating porosity. However, studies have shown the loss of HA coating from the surface of implants with due course of time after implant placement ⁷¹. There are two points to be remembered about HA coatings: First, HA would tend to fuse better to the bone than to the implant itself, and with high occlusal loads (excessive biting forces in some individuals/sites in the mouth), the HA would break off of the implant. Secondly, HA coating is very susceptible to penetration by dental plaque, consisting of oral bacteria and their endotoxins. If the roughened HA surface of a dental implant becomes exposed, which can be due to exposure of cervical area of implant while placement or peri-implantitis associated bone loss, there is a tendency of further bone loss because the implant surface cannot be adequately cleaned or decontaminated, leading to more inflammation and eventually greater bone loss.

     Various implant surface modifications have been studied for their bone-implant contact. One study reported that the highest extent of bone-implant interface was observed in sand-blasted and acid etched surfaces (large grit; HCl/H₂SO₄) with mean values of 50-60%, and HA-coated implants with 60-70%. It was minimum for electropolished, as well as the sand-blasted and acid pickled (medium grit, HF/HNO₃) implant surfaces with mean values ranging between 20 and 25%. For sand-blasted implants, with a large grit, and titanium plasma-sprayed implants the mean bone contact was 30-40% ⁵⁴.

     So, it was suggested that sand-blasting and chemical etching with HCl/ H₂SO₄ as well as HA coating, seemed to be the most promising alternatives to titanium implants with smooth or titanium plasma-sprayed surfaces ⁵⁴. The advantage of the plasma spray method is, it is widely accepted as an apatite coating method because it gives tight adhesion between the apatite coating and titanium. The disadvantage of this method is the requirement of extremely high temperatures (10,000-12,000 °C) during the coating process. Unfortunately, it results in potentially serious problems including

1. An alteration in structure.
2. Formation of apatite with extremely high crystallinity.
3. Long term dissolution and the accompanying de-bonding of the coating layer.

To overcome these drawbacks, methods like sol-gel coating have been developed.

Titanium plasma spray Coating

Coating of pure titanium coatings on Ti-6Al-4V alloy have been used in order to improve the biocompatibility of functional titanium-based alloys. These coatings are believed to favor the osseointegration of the bone because of the inherent roughness of such coatings, although they do not induce osteogenesis per se.

Radiofrequency (RF) magnetron sputtering

Radiofrequency (RF) magnetron sputtering is a recent technique used in the modification of implant surface. The application of magnetron sputtering offers a flexible approach to surface improvement of metal implants. This technique facilitates to the production of dense and well-adhered films with controlled elemental composition ⁷².

     In this technique, a magnetic field is created by permanent magnets, electromagnets or a combination of both. This configuration acts to trap electrons. Thus, in a magnetron sputtering discharge, atoms are sputtered from the cathode target by magnetically confined plasma ⁷³. The target material is sputtered by the bombardment of high energy ions accelerated over the cathode sheath potential. Secondary electrons are emitted and accelerated away from the target surface as a result of the ion bombardment. These electrons play an important role in maintaining the plasma. A magnetic field confines the ionizing energetic electrons near the cathode allowing operation at high plasma densities and low pressures.

Electrophoretic deposition (EPD)

EPD is a special colloidal processing technique that uses the electrophoresis mechanism for the movement of charged particles suspended in a solution under an electric field, to deposit them in an ordered manner on a substrate to develop thin and thick films, coatings and free-standing bodies ^74-76. EPD is usually carried out in a two-electrode cell. The motion of charged particles …………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…….

     The disadvantage of this treatment is the requirement of post-deposition heat treatment to densify the coating. In the case of HA coating, the feedstocks require the temperature of at least 1200⁰C to be densified. Another problem is, temperatures above 1050°C affect the oxide layer and mechanical properties of a stainless steel or titanium alloy as well as decompose HA affecting the interfacial strength between the metal and coating.

Sol-gel coating

The sol-gel process is based on the hydrolysis and condensation of bioactive material on the implant surface. In comparison to plasma spray method, the sol-gel technique offers certain advantages. Because of the high chemical homogeneity, fine grain structure and low crystallization temperature of the resultant coating is achieved. As compared to plasma coating it is both an economical and technically simple procedure to perform. Bioactive glass (BG) composites also have been used to coat implant surface by this method. This method has also been used to deposit TiO2 layer on the implant surface. The sol-gel-derived HA and TiO2 films, with thicknesses of about 800 and 200 nm, adhered tightly to each other and to the CpTi (grade 2) substrate.

Pulsed laser deposition (PLD)

PLD has been used to deposit HA ceramic over the titanium substrate with an interlayer of titania. PLD is an alternative method to coat metal substrates (such as implant surface) with HA in order to improve both the chemical homogeneity and the mechanical properties of calcium phosphate coatings ⁷⁹. PLD has successfully produced HA coatings with various compositions and crystallinity ⁸⁰. Moreover, PLD can synthesize thin HA coatings, adequate for high fatigue resistance.

Biomimetic precipitation

The term ‘biomimetic’ refers to biomaterial having the ability to induce differentiation of the appropriate cells (i.e., endothelial and osteoblastic cells) for the formation of new bone; it should be easily synthesized or produced, rather than having to be extracted from allograft materials (to eliminate all risks of disease transmission); it should be easily and quickly resorbed as the osteogenic response occurs; it should have no immune-provoking properties; it should be easily transported and stored; and it should be reasonably cost-effective ⁸¹. The incorporation of recombinant bone morphogenetic proteins (BMPs), which are produced by genetic engineering onto the implant surface, is the future of implant surface modifications. These methods are presently under research. Because these methods are done at molecular levels, the evolution of mechanisms to make these techniques biologically efficient and economically acceptable is a major task in this direction.

Future trends

Chemical modification of the titanium oxide layer on the implant surface for more favorable biological response is under extensive research. Various methods have been employed for this purpose which includes treatment in simulated body fluid ⁸², covalent attachment of biological molecules ⁸³, changes in the surface ion content ⁸⁴, glow discharge ⁸⁵, or alkali treatment ⁸⁶. It has been demonstrated that these modifications alter the cellular response during healing after implant placement.

Chemical modification by fluorides

Fluoride has specific attraction for calcium and phosphates and the known beneficial effects of clinical importance that can be observed when this ion is in contact with calcified tissues ^{87, 88}. It has been shown that the trabecular bone density is increased and calcification of bone is induced when fluoride is present during bone remodeling ^{89, 90}. Using radioactively labeled phosphorous 32P ⁹¹, it has been shown that uptake of phosphate is significantly higher at the fluoride-modified implant surfaces than at the non-modified titanium dioxide surfaces, demonstrated by an elevated level of radioactivity at these surfaces.

     Fluorides also seem to induce bone cell growth factors which are required during osteogenesis ⁹². Fluoride is suggested to have an …………Contents available in the book …………Contents available in the book…………Contents available in the book…………Contents available in the book…………Contents available in the book…….

     The low concentration of hydrogen fluoride has been used on titanium dioxide without changing the surface microtexture of the implant significantly. Small amounts of fluoride will then be incorporated into the crystal structure of the titanium dioxide. Further research is going on in this direction to better understand the role of fluoride and its use in implant dentistry.

Use of bisphosphonates

All bisphosphonates have a common P-C-P structure, which is similar to the P-O-P structure of native pyrophosphate. These agents inhibit osteoclastic bone resorption of the bone. Bisphosphonates attach to HA binding sites on bony surfaces, especially surfaces undergoing active resorption. When osteoclasts begin to resorb bone that is impregnated with the bisphosphonate, the bisphosphonate released during resorption impairs the ability of the osteoclasts to form the ruffled border, to adhere to the bony surface, and to produce the protons necessary for continued bone resorption. A detailed description of the mechanism of action of these drugs is available in, “Host response modulation therapeutic agents in periodontics”.

     Research work has shown a significant increase in bone-implant contact when the implant surface was coated with pamidronate or zoledronate. The clinical application of these agents incorporated on implant surface is still awaited.

Use of growth factors

Many studies have been done to investigate the effect of different growth factors on peri-implant osseointegration ^94-99. Most important of them are the BMP’s which belong to TGF-β superfamily. These proteins control both, the cell growth and differentiation, according to cell type and state of differentiation ¹⁰⁰, and also directly affect the expression of receptors involved in surface recognition and cellular attachment to titanium implants ⁹⁷. Unfortunately, most of the research done on the incorporation of BMP’s on implant surface has not reached the clinical practice so far . The reason for this problem is that growth factors are by nature free-floating, short-lived signals that initiate or modulate small, incremental changes in cells as a part of cascade reactions. Another factor that should be kept in mind is that the effect of these signals varies dramatically with cell type, stage, and location, with the condition of the surrounding tissues, and with the presence of other growth factors.

Tissue engineering

Tissue engineering is the use of a combination of cells, engineering materials, and suitable biochemical factors to improve or replace biological functions 101. It is an interdisciplinary approach where the principles of engineering and life sciences are combined toward the development of biological substitutes that restore, maintain, or improve tissue function. It involves the interaction of cells with a material surface where the behavior of the cells is studied. For example osteoblasts and chondrocytes are sensitive to subtle differences in surface roughness and surface chemistry.

     Clinically a tissue-engineered scaffold material is used to carry the bioactive molecules. This scaffold acts as artificial extracellular matrices (ECM) and as a spacer, keeping a certain open space ¹⁰². When placed in body, scaffold material has to be dissolved completely into the living body after auto-cells are regenerated with artificial extracellular matrices ¹⁰². Various materials have been investigated to be used as scaffold which include blended-polymer scaffolds, collagen-based scaffolds, and composite scaffolds of polyhydroxybutyrate-poly-hydroxyvalerate with bioactive wollastonite (CaSiO₃) ¹⁰³. A three-dimensional printing (3DP) technology can be used to fabricate porous scaffolds which can be supplemented with bone morphogenetic proteins (BMPs).

     Electrospinning is another method by which scaffolds are made from the synthetic and natural polymers ¹⁰⁴. Research work is going on in this field to make these complicated procedures easy and clinically applicable. In future, we have to do more research in this field to find out the clinical applications and drawbacks of these techniques.

Conclusion

The long-term stability and functionality of dental implants is our ultimate goal. Surface modifications of dental implant play an important role in this endeavor. Adding bioactive surface layers to implants could improve implant biocompatibility, osseointegration, and durability, all important qualities required for long-term performance. Present research is focused on identifying the bio-molecule carriers, which can transport the bio-molecules like bone morphogenetic proteins in active form during implant placement and promote initial healing and bone formation. It should be noted that the implant first comes in contact with fluid (blood) during placement, which is a major factor for initial cell recruitment onto the implant surface. Recruiting growth factor producing cells to the bone-implant interface through focal attachment and molecular recognition may help in faster bone formation.

References

References available in the hard-copy of the website.

Suggested reading

1. Qu Z, Rausch‐Fan X, Wieland M, Matejka M, Schedle A. The initial attachment and subsequent behavior regulation of osteoblasts by dental implant surface modification. Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials. 2007 Sep 1;82(3):658-68.
2. Avila G, Misch K, Galindo-Moreno P, Wang HL. Implant surface treatment using biomimetic agents. Implant dentistry. 2009 Feb 1;18(1):17-26.
3. Junior JF, Verri FR, de Faria Almeida DA, de Souza Batista VE, Lemos CA, Pellizzer EP. Finite element analysis on influence of implant surface treatments, connection and bone types. Materials Science and Engineering: C. 2016 Jun 1;63:292-300.
4. Kilpadi DV, Lemons JE. Surface energy characterization of unalloyed titanium implants. Journal of biomedical materials research. 1994 Dec;28(12):1419-25.
5. Yeo IS. Reality of dental implant surface modification: a short literature review. The open biomedical engineering journal. 2014;8:114.