Biomembrane for guide bone regeneration

Barrier membranes for GTR and GBR applications

The strategy of utilizing a mat-like material (resorbable or non-resorbable) for the isolation of the periodontal defect, which would function as a physical barrier to avoid gingival cell invasion led to the development of GTR/GBR (Karring et al., 1993; Magnusson et al., 1988). Some of the common characteristics that both GTR/GBR membranes exhibit include:

Biocompatible – absence of eliciting inflammatory response from the host tissues

Dehydration profile to match the new tissue formation

Balanced mechanical and physical properties to allow its placement in vivo

Membrane strength to ensure their barrier function without collapsing (Polimeni, et al., 2006; Wikesjo et al., 1995)

Based on their degradation characteristics the GTR/GBR membranes are divided to either non-resorbable and a resorbable membrane.

5.2. Principles of guided bone regeneration

The fundamental theory of GBR was initially established almost five decades before, when cellulose acetate filters were tried out for the renewal of sinews or tendons and nerves (Ashley et al., 1959). Consequently, cellulose acetate (Millipore™ membrane filter) improved curing deficiencies of bone tissues of ribs, radial bone or bone of the forearm and thigh or femoral bone (Rüedi & Bassett, 1967). presently, a succession of animal research offered substantiation to illustrate that GBR can as expected assist bone renewal in minimum-sized bone tissues deficiencies (Hämmerle et al., 1997), (Marouf & El-Guindi, 2000) and (Donos et al., 2004), also curing of bone flaws surrounding teeth implants by supplementing the breadth and height of atrophic alveolar points preceding to implant placing (Froum et al., 2012), (Zellin et al., 1995) and (Buser et al., 2008).

The fundamental theory of GBR (Fig. 2) entails the positioning of perfunctory blockades to take care of blood clots and to segregate the bone deficiency from the nearby connecting tissue, thereby allowing bone – developing cells the way in to an isolated area meant for bone renewal (Buser et al. 1994). This theory uses a membrane that acts like a barricade and is beneficial to help growth of alveolar ridge deficiencies, stimulate bone renewal, give better outcome for bone-grafting and take care of deteriorating implants (Sottosanti, 1997).

Fig 2: The principle of guided bone regeneration using mechanical barriers (membranes) to seal off the bone defect from the surrounding soft connective tissue into a secluded space by which cells only from the surrounding bone can migrate.

Membranes that act as barriers, when utilized as a remedial tool, have to comply with five major pattern standards, as explained by Scantlebury (Scantlebury, 1993): biocompatibility, space-creation, cell-occlusiveness, tissue assimilation and medical manageability.

5.2.1. Biocompatibility

The membrane must have the ability of a material to perform with an appropriate host-response in a specific situation. The contact between the substance and tissue must not have a harmful effect on the neighboring tissue, the projected curative outcome, or the general wellbeing of the patient concerned.

5.2.2. Create a space for in growth

The membrane must contain a sufficient rigidity to generate and uphold an appropriate gap for the projected osseous renewal. This feature is mostly connected to the membrane width. Additionally, a membrane must offer a most advantageous gap which could be sustained for tissue’s inward growth and yet offer sufficient backing to the tissue, in big deficiencies also. The substance must in addition be suitably supple to offer the precise arrangement necessary for practical renewal, yet remain adequately rigid to endure the force applied from outside, like as mastication in jaw renewals (Heinze, 2004). In the event of the membrane caving into the fault gap, the section for renewal is condensed and a maximum medical result cannot be realized.

5.2.3. Occlusivity

The best possible blockade ought to be adequately occlusive to evade fibrous tissue development, that might stop or interrupt bone development. Occlusivity is hence directly connected to porous nature of the membrane; this feature has a main impact on the possibility for cell incursion (Salzmann et al., 1997). Certainly, a membrane’s barrier occlusivity is as significant as the feature as retaining a gap when bone deficiencies are being renewed (Lundgren et al., 1997).

The design of the spongy formations on the whole, and not the kind of substance utilized has been recommended to present the biological action of a substance (Dickey et al., 2005). The pores or holes in the membrane assist the dispersal of oxygen, fluids, nutrients and bioactive materials needed for cell development that is fundamental for bone and soft tissue renewal. Nevertheless, these holes or pores should also be resistant to epithelial cells or gingival fibroblasts, for example in dental implants; a bigger pore dimension will permit these rapidly developing cells to fill-up the deficiency gap and slow down the access and action of bone-developing cells (Heinze, 2004). A bigger pore dimension also provides an effortless passage for bacterial infection and operating to remove the infected membranes turn out to be complex due to the surplus inward growth of soft tissues (Zhang, 2004) and (Bartee & Carr, 1995). In case the holes or pores are smaller in dimension cell movement of all the cells is restricted and this situation results in added collagen deposits, the growth of tissues lacking blood vessels, and a lack of capillary development and permeation (Taylor & Smith, 1972). The dimensions of the pores also impact the ability of the substance to sustain the tissue. A pore of a bigger dimension will unavoidably reduce the ensuing exterior region of the substance that might restrict the significant first stages of cell bonding against the membrane (Yannas, 1992) and succeeding reduction of inward growth of blood vessel (Zhang, 2004).

5.2.4. Tissue integration

Tissue assimilation is the vital feature of the entire tissue renewal practices as it is necessary that the host tissue assimilates with the membrane. It is effectively ascertained that the constitutional quality of the membrane that acts as a barrier and the adequate adaptableness of its restrictions to the neighboring original bone comprise fundamentals for possible new bone development (Kostopoulos & Karring , 1994). Tissue assimilation evens out the curing injury procedure, and assists in creating a fastening between the bone and the substance, to stop fibrous connecting tissue assimilation in the deficiency area. Tissue assimilation between the membrane and the curves of the neighboring bone is dependent on the membrane gap-forming ability of the substance; a substance that is very firm may not be capable to adapt to the outline of the deficiency spot.

5.2.5. Clinical manageability

A membrane has to be convenient for medical procedure, especially for dental implants. A membrane which is complicated to utilize, like membranes that are very supple, can be exasperating and can mostly end in snags if it is not possible to utilize it in the same medical surroundings, especially the generally minute surroundings innate with dental works (Heinze, 2004). Alternatively, a membrane which is very rigid is difficult to be shaped without difficulty and the pointed ends might puncture the gingival tissue and succeeding uncovering of the membrane (Becker et al., 1996). One investigation revealed that barrier membranes which are non-resorbable offer a appropriate firmness compared to membranes that are resorbable for maximum bone thickness and height in GBR (Ito et al., 1998).

5.3. Types of barrier membranes, their basic characteristics and specific consideration

To-date research involving the development of an ‘ideal’ membrane for clinical application has been the need of the hour even though there are numerous non-resorbable and bioresorbable membranes. Some of the basic characteristics associated with the membranes are biocompatibility, cell-occlusiveness, space-making, tissue integration, and clinical manageability (Gottlow, 1993, Scantlebury, 1993).

Extensive research work has been made through the utilization of expanded-polytetrafuoroethylene (e-PTFE, Teflon) which is a non-resorbable membrane (Aaboe et al., 1995). They are highly stable and maintain their structural integrity during implantation. These membranes are associated with superior space maintaining properties and capacity for cell occlusion than degradable membranes as it might tend to collapse based on the size of the defect (Wiltfang et al., 1998). Other examples of the non-resorbable membranes include the titanium reinforced ePTFE, high density-PTFE or titanium mesh mainly in the oral and maxillofacial surgery (McAllister & Haghighat, 2007) (Table 1). With respect to bone regeneration, semipermeable ePTFE suits effectively than the high-density-PTFE (Marouf & El-Guindi, 2000). Satisfactory preliminary results have been obtained in the bone regeneration of large segment of bone defects through the utilization of titanium mesh cage used in the form of a scaffold (Ostermann et al., 2002). But the major disadvantage with regard to this method is that, it requires a second surgical procedure to be removed out. This factor has a potent risk factor that is associated with the newly regenerated tissues (Hardwick et al., 1995). Also membrane exposure is frequent that might increase the risk of secondary infection (Gielkens et al., 2008a; Gielkens et al., 2008b).

Table 1
Summary of the different types of barrier-membranes used for reconstruction of bone defects
Types of membranes
Bioresorbable membranes	Advantages	Disadvantages
Natural membranes	Collagen (different subtypes, predominantly type-I collagen, derived from different animals, (bovine or porcine) and from different sites (tendon or dermis) (Patino et al., 2002)	- highly biocompatible (no adverse effect to surrounding tissues during degradation) - it promotes wound healing(Bunyaratavej & Wang, 2001) - it allows good integration with connective tissue (fibrous encapsulation with differentiation of a periosteum-like tissue upon the external bony surface) (von Arx et al., 2005, Alpar et al., 2000) - osteoblasts and fibroblasts can attach to collagen membranes irrespective of its origin (Behring et al., 2008) - differently cross-linked collagen membranes can promote cell attachment and proliferation (Rothamel et al., 2004)	- degradation in vivo is too rapid to maintain the structural integrity necessary for bone regeneration(Behring et al., 2008) - different cross-linking techniques used to prolong degradation time (it varies from four weeks up to six months) (Patino et al., 2002, Bunyaratavej & Wang, 2001 , Tal et al., 2008) - differently cross-linked collagen membranes can also inhibit cell attachment and proliferation (Rothamel et al., 2004) - chemicals used for cross-linking have cytotoxic effects on the surrounding tissues leading to gap formation between the membrane and the connective tissue and facilitate microbial accumulation (Alpar et al., 2000) (to address this, a non-chemical ross-linking nanofibrous collagen membrane has been developed) (Lee et al., 2010) - variable mechanical properties among the different available membranes - risk of peri-operative rupture - moistening of the membrane (unavoidable in vivo) alters considerably the mechanical properties (Coïc et al., 2010) - possible disease transmission from animals to humans (Pitaru et al., 1988, Gielkens et al., 2008)
	Chitosan or chitosan-collagen hybrid	- non-toxic natural polymer (polysaccharide) - it enhances wound healing and bone formation (Gupta & Ravi Kumar, 2000) - it has hemostatic properties(Klokkevold et al., 1992) - excellent biocompatibility(Shin et al., 2005), osteogenic cells can proliferate and express osteogenic markers (Shin et al., 2005) - chitosan-hybrid membranes have superior mechanical properties(Lee et al., 2009, Kung et al., 2011)	- limited evidence from in vivo studies
Synthetic membranes	Aliphatic polyesters: PLLA, PLGA, polydioxanone and their co-polymers (Lee et al., 2009, Kung et al., 2011, Asikainen et al., 2006)	- the most commonly used and studied bioabsorbable polymer - commercially available and approved for clinical use - by changing the composition and the manufacturing procedure, resorption time, handling properties and mechanical durability can be adjusted to suit the clinical situation (Asikainen et al., 2006) - different chemical compositions did not affect on bone regeneration in vivo(Gogolewski et al., 2006) - slow-degrading membranes induce greater amounts of neovascularization and a thinner fibrous capsule versus fast degrading membranes (Kaushiva et al., 2007)	- they can induce host-tissue response and foreign body reactions during degradation (by non-enzymatic hydrolysis) (Meinig, 2010, Schmidmaier et al., 2006, von Arx et al. 2005, Kinoshita et al., 1996, Polimeni et al., 2008, Schliephake & Kracht, 1997) - the moderate cytotoxic reactions may reduce cellular adhesion (Alpar et al., 2000)
Non-resorbable membranes
Expanded polytetrafuoroethylene (e-PTFE) And others: titanium reinforced ePTFE, high-density-PTFE, or titanium mesh(McAllister & Haghighat, 2007)	- extensively studied (Aaboe et al., 1995) - biocompatible - they maintain their structural integrity during implantation and have superior space-maintaining properties and capacity for cell occlusion than degradable membranes - semipermeable ePTFE is more effective than the high-density ePTFE (Marouf & El-Guindi, 2000) - for large segmental bone defects, cylindrical titanium mesh cage used as a scaffold (Ostermann et al., 2002)	- a second surgical procedure is required for removal (additional potential risk to the newly regenerated tissues (Hardwick et al., 1995)) - membrane exposure is frequent, increasing the risk of secondary infection (Gielkens et al., 2008a, Gielkens et al., 2008b) - e-PTFE can induce slight to moderate cytotoxic reactions and reduce cellular adhesion
Alginate membrane	- close assimilation to bone surface - no inflammatory response(Ueyama et al., 2002) - easy handling with an alginate base self-setting barrier membrane versus a ready-made membrane (Ueyama et al., 2006) -more efficacious versus collagen membranes for mandibular and tibial defects (He et al., 2007, Jianqi et al., 2002)	- limited evidence from in vivo studies
Others (Kim et al., 2011, Zhang et al., 2010, Humber et al., 2010, Kothiwale et al., 2009, Wu et al., 2009 ): - degradable biopolymer poly (lactide-co-ε-caprolactone)(PLCL), - a nano-hydroxyapatite/ polyamide (nHA/PA66) composite - an in situ-formed polyethylene-glycol-hydrogel membrane - amniotic membranes - a bacterially-derived polymer - a hybrid membrane consisting of layers of collagen containing hydroxyapatite (HA) and chitosan (Teng et al., 2008) - polyethersulfone (PES) electrospun nanofibrous membranes (Shabani et al., 2011) - a biomimetic tubular calcium phosphate (CaP)-coated nanofiber mesh combined with platelet rich plasma-mediated delivery of BMP-7(Berner et al., 2012) - Latex (Ereno et al., 2010) - membranes with additional anti-bacterial properties or antimicrobial coating (Tokuda et al., 2009, Zhang et al., 2010, Chaturvedi et al., 2010)	- optimized properties for GBR - improved three-dimensional structure and osteogenic bioactivity - they can be loaded with cells to mimic natural bone - no foreign body inflammatory reaction or rejection and satisfactory bone formation - membranes with additional anti-bacterial properties or antimicrobial coating may reduce membrane-associated infections

Any difficulty of a secondary surgery is overcome through the usage of bioresorbable membranes that does not require any further surgeries. Usage of such membranes have been extensively studied not only in animals but also in humans affected with maxillofaciao, regenerative periodontal, and neuro-surgery (Retzepi & Donos, 2010; Kinoshita , 2004; Thomaidis et al., 2008; Needleman et al., 2006; Sculean et al., 2008; Nakajima et al., 2001; Schmidmaier et al., 2006). Currently commercially available bioresorbable membranes were introduced in the reconstruction of long bone defects in clinical settings. Such practices are followed at places where the bone defects are > 4 to 5 cm or with significant associated soft-tissue loss where autologous bone graft alone could not be a recommended due to the risk of resorption (Klaue et al., 1995), also securing a grafting material (Gielkens et al., 2008). Natural and synthetic membranes are the two broad categories that the biosorbable membranes are differentiated into. Collagen or chitosan forms the natural membranes whereas synthetic products are made up of aliphatic polyesters, primary poly (L-lactide) (PLLA) and poly (L-lactide-co-glocolide) (PLGA) co-polymers (McAllister & Haghighat et al., 2007).

The overall advantages of the resorbable membranes are that:

They follow a single step procedure.

The volume and the shape of the bone could be predefined / prefabricated.

Allows to be imaged as they are radiolucent

Potential effects of stress shielding the regenerated bone are as a result of bioresorption.

It should also be noted that variability in the membrane resorption differs according to the place in which it is being fixed. Based on the local pH and material composition the rate of membrane resorption differs. The details pertaining to the main characteristics, advantages and disadvantages of the bioresorbable membranes are presented in the table 1 (Meinig, 2010; Pitaru et al., 1988, Gielkens et al., 2008; Schmidmaier et al., 2006; Patino et al., 2002; Schliephake & Kracht, 1997). In the recent trend PLAA membrane is used for clinical use in orthopaedic surgery whereas PLLA, maxillofacial, dental and neuro surgery is taken care of by collagen and ePTFE membranes.

Though there are a number of membranes that are clinically used, development of novel membranes have aided in overcoming the limitations pertaining to the barrier membranes that are already being used. Some of these novel membranes include alginate membranes, new degradable co-polymers, hybrid or nanofibrous membranes, and amniotic membranes, which are summarized in table 1 (60-75). Current research trend it aimed at evaluating these novel membranes in establishing an ideal membrane for bone regeneration in terms of biocompatibility, space-making, tissue integration and clinical manageability for an efficient clinical safety.

5.3.1 Stability / degradation characteristics of GTR/GBR membranes

Gold standard non-resorbable (Table 1) membranes for GTR/GBR procedures in the markets (1) are high-density polytetrafluoroethyle, PTFE (e.g., Cytoplast® TXT-200, Osteogenics Biomedical, Lubbock, TX, USA) (Fig 3A) and (2) titanium-reinforced high-density polytetrafluoroethylene (e. g., Cytoplast® Ti-250, Osteogenics Biomedical, Lubbock, TX, USA) (Gentile et al., 2011).

One of the inert biocompatible membranes is the PTFE membrane that is inert in nature provides space and allows regeneration. It has been analyzed that there is a positive correlation between the space availability and bone regeneration (Polimeni et al., 2005). In a study it was found out that the titanium reinforcement of high-density PTFE membranes has led to a superior regenerative capacity of the soft tissue on comparing with the traditional expanded PTFE membranes. This is mainly because of the additional mechanical support provided by titanium frame against the compressive force exerted by soft tissue (Jovanovic & Nevins, 1995). The major disadvantage with that of the non-resorbable membrane is that it requires an additional surgical practice to remove it. This process not only adds pain to the body but is also not economically feasible.

Table 2. List of commercially available membranes for GTR/GBR applications.

Resorbability	Commercial name	Composition	Mechanical strength	Degradation rate	Biological properties
Non-resorbable	Cytoplast®TXT-200	High-density polytetrafluoroethylene (d-PTFE)	N/A	Non-degradable®	Biocompatible( Kasaj et al., 2008)
	Cytoplast®Ti-250	Titanium-reinforced high-density PTFE	N/A	Non-degradable®	Biocompatible( Kasaj et al., 2008)
Resorbable synthetic	Resolut LT®	Poly-DL-lactic/co-glycolic acid	11.7 MPa( Milella et al., 2001)	5–6 months( Donos et al., 2002)	Biocompatible( Donos et al., 2002)
	Vicryl®	Polyglactin 910 Polyglycolide/polylactide (9:1, w/w)	N/A	∼9 months( Klinge et al., 1999)	Biocompatible( Laurell & Gottlow, 1998)
	Atrisorb®	Poly-DL-lactide and solvent (N-methyl-2-pyrrolidone)	N/A	6–12 months( Coonts et al., 1998) and ( Hou et al., 2004)	Biocompatible( Coonts et al., 1998) and ( Hou et al., 2004)
Resorbable collagen-based	AlloDerm®	Collagen Type-I derived from cadaveric human skin	9.4–21.5 MPa( Bottino et al., 2009)	∼16 weeks( Owens & Yukna, 2001)	Biocompatible( Felipe et al., 2007) and ( Owens & Yukna, 2001)
	Bio-Gide®	Collagen derived from porcine skin (Types I and III)	7.75 MPa(103)	24 weeks( Bunyaratavej & Wang, 2001)	Biocompatible( Bunyaratavej & Wang, 2001)
	BioMend Extend®	Collagen Type-I derived from bovine tendon	3.5–22.5 MPa( Coic et al., 2010)	18 weeks( Bunyaratavej & Wang, 2001)	Biocompatible( Bunyaratavej & Wang, 2001)
	Cytoplast®RTM	Collagen Type-I derived from bovine tendon	N/A	26–38 weeks®	Biocompatible( Yamada et al., 2011)

To overcome this difficulty of the non-resorbable membrane, resorbable barrier membranes were developed (Sculean et al., 2008; Behring et al., 2008). Polymer based membranes were either processed using either melting (i.e., polymer heated above the glass transition or melting temperature) or solvent casting techniques for applications in GTR/GBR. Other methods involving the preparation of porous three-dimensional scafflolds or membranes for tissue engineering includes solvent casting / particulate-leaching (Park et al., 2000) and phase inversion (Li et al., 2009) techniques. In the case of solvent-casting / particulate-leaching method, the polymer solution is combined with an inorganic salt (eg., sodium chloride or organic sugars) (a leachable porogen) of a particular size in a mold. When the mold is solidified, the sugar and salt crystals would be dissolved in water leaving the pores in the polymer (Tessmar et al., 2006).

The pore size in the polymer could be tailored to required size based on the type of the component chemical and sugars used and its ratio respectively. But the negative impact in this particular technique comes from the usage of organic solvent, which might have a negative effect on the cells and tissues upon implantation (Tessmar et al., 2006).

As per the market, a synthetic resorbable membrane that is used in periodontal regeneration are either based on polyesters (e.g., poly (glycolic acid) (PGA), poly (lactic acid) (PLA), poly (ɛ-caprolactone) (PCL), and its copolymers (Donos et al., 2002; Klinge et al., 1999 and Laurell & Gottlow, 1998) or tissue derived collagens (Owens & Yukna, 2001; Coic et al., 2010 ; Yamada et al., 2011). These membranes of polyester origin are biocompatible, biodegradable, and are easier to handle on compared to PTFE membranes, which would allow tissue integration as well. The major factor that should be taken in to account about these membranes are their rate of resorption. This is because it takes around 4-6 weeks for a successful regeneration of periodontal system (Sculean et al., 2008; Piattelli et al., 1996).

One of the major alternatives to the synthetically derived membranes is through the naturally derived collagen component (Table 1). Collagen forms the major constituent of the natural extracellular matrix (ECM). The excellent cell affinity and biocompatibility nature of the collagen based membranes makes it an effective alternative to the synthetic polymers that is used in GTR/GBR procedures. Collagen based membranes are used in the tissue regeneration of human skin (Alloderm®, LifeCell, Branchburg, NJ, USA), bovine Achilles tendon (Cytoplast® RTM Collagen, City, State, USA) or porcine skin (Bio-Gide®, Osteohealth, Shirley, NY, USA) (Felipe et al., 2007; Santos et al., 2005 ; Gouk et al., 2008). The one disadvantage of the type-I collagen is its high cost factor and its poor commercial sources that would deteriorate its control over degradation and mechanical properties. One of the type-I collagen product that is mainly derived from the human cadaveric skin is Allodern ®. Alloderm is an acellular freeze dried dermal matrix graft (Bottino et al., 2009; Bottino et al., 2010; Gouk et al., 2008). In accordance with the manufacturer, the processing involved in the production of the membrane does not involve and damage the critical biochemical structural cues required to maintain the tissues’ regenerative properties. Apart from this the product also leaves behind a layer of extracellular collagenous matrix which would aid in structuring of the tissue and in the guidance of cellular functions (Livesey et al., 1995).

But in vivo studies have shown that collagen based membranes are poor performers as their degradation would be degraded (Behring et al., 2008). In addition to this details pertaining to the origin of the material (animal or human), and the risk associated with the transmission of diseases also paves another limitation along with the religious beliefs. With the introduction of ultraviolet radiation (UV), genipin (Gp), glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiiamide hydrochloride (EDC) could enhance the biomechanical properties of the collagen matrix through physical and chemical crosslinking. The introduction of Gp not only increases its stability through the crosslinking patters, but would also aid in the reduction of its antigencity. This reduces the chance of the membrane being eliminated through the host immunological response. Further the cross linking formed between the collagen membranes and the additive such as Gp increases the mechanical properties of the biological tissues (Bottino et al., 2010; Bhrany et al., 2008 ; Sundararaghavan et al., 2008).

The effect of additive such as Gp as a natural crosslinking agent into Alloderm® (rehydration protocol) and its effect in the mechanical properties of collagen matrix stability are studied through the work of Bottino et al., (2010). The impact in the enhancement of tensile strength was observed with an increase in the exposure time of Gp from 30 minutes to 6 hours. When this sample was analyzed through differential scanning calorimetry a significant shift in the denaturation temperature coincides with the increase in the enthalpy of denaturation. This shows that an effective crosslinking has taken place in sample that is amended with Gp (Bottino et al.,2010). This finding has indeed gone the way of previous investigations which has stated an increase in the stabilization of the collagenous matrices of biological origin (Bhrany et al., 2008; Sundararaghavan et al., 2008; Sung et al., 1999a; Sung et al., 1999b; Sung et al., 1999c).

The drawbacks of both the PTFE-based non-resorbable (e.g., second surgery) and resorbable membranes (e.g., insufficient mechanical properties, unpredictable degradation profiles) in relation to collagen have led researchers to find an alternative membrane. More research work have been carried out on the usage of membranes with functionally graded structure about its mechanical properties during service, degradation rate in vivo and bioactive properties (Erisken, Kalyon & Wang, 2008; Zamani, Morshed, Varshosaz & Jannesari, 2010). Utilization of calcium-phosphate based nanoparticles is involved in the induction of bone formation (e.g., BMP-2, TGF-β, among others) at the tissue/membrane interface. These nanoparticles also do possess antibacterial properties that inhibit the growth of organisms in these areas where tissue regeneration is under process (Erisken et al., 2008; Schliephake & Kracht, 1997).

5.3.2. Designed membranes for zone-dependent bioactivity

Many researches have been conducted to design and develop GTR/GBR periodontal membranes with essential functional properties with the inclusion of both natural and synthetic polymers. In these studies the membranes were prepared using different techniques including film casting (Park et al., 2000 ; Li et al., 2009) and dynamic filtration (Teng et al., 2009), and e-spinning (Bottino et al., 2011; Yang et al., 2009 ; Fujihara et al., 2005) using synthetic (eg., PCL) or natural polymers (eg., collagen, chitosan). These membranes were developed with or without the presence of therapeutic drugs (Park et al., 2000), growth factors (Fu et al., 2008) and calcium-phosphate nanoparticles (Kikuchi et al., 2004; Liao et al., 2005; Liao et al., 2007). A perfect material that has a good mechanical property, degradation rate and biological properties (absence of antigenic properties) for GTR/GBR is still being searched. But as a result of a high density (i.e., difficulty in handling) and heterogeneity factor (i.e., non-uniform degradation rate) majority of these methods, results in the development of membranes with low clinical potential. E-spinning technique has been illustrated as an effective processing technique for the development of membranes for periodontal regeneration (Zamani et al., 2010). This particular technique is highly important in the development of biomimetic nanoparticles that could be incorporated in the membranes for GTR/GBR applications.

5.4. Types of membrane:

5.4.1. Resorbable membranes

Resorbable substances which are utilized as membranes are from sets of real or man-made polymers. Out of them, aliphatic and collagen polyesters like polyglycolide or polylactide, are popular for its medicinal appropriateness (Hutmacher et al., 1996). Collagen is got from several sources and is dealt with in different means for membrane production. Polyglycolide or polylactide could be produced in huge amounts and the extensive variety of obtainable substances permits the making of a extensive range of membranes with diverse mechanical, physical and chemical characteristics (Fields, 2001).

As the term infers, resorbable materials have the benefit of being resorbed by the body, thereby doing away with the necessity for a second operation for removal. Thus, resorbable membranes are preferred by surgeons and patients, in bringing down the danger of illness, the threat of tissue destruction, and it is also less expensive. Generally, firm resorbable membranes support a related amount of bone renewal and bone development just as non-resorbable membranes (Chiapasco & Zaniboni, 2009; Imbronito et al., 2002). Furthermore, in circumstances where the bone fault limits are suitably preserved by the membrane, constructive outcomes were found (Hämmerle & Jung, 2003; De Macedo et al., 2008).

The shortcomings of resorbable membranes are that the extents of its capacity to be absorbed is not certain, consequently this could change the level of bone development (McGinnis et al., 1998). In the event of it being resorbed quickly, the substantial lack of firmness results in the necessity of more support (Gutta et al., 2009; Wang & Fenton, 1996). The other disadvantages are when aiming to shield huge particulate implants (von Arx et al., 1996). If the membranes lead to inflammation in the neighboring tissue, the enzymatic action of macrophages and neutrophils triggers the membrane to speedily degree, thus disturbing the constitutional reliability of the membrane and resulting in reduced barrier utility and lower bone renewal or bone fill; this can be specifically challenging when implanting in combination with embedded placement, as the implant is not stable (Monteiro et al., 2010). If the bone fault is not sustained by a physical barrier, bone renewal does not take place. Though membranes may seem to be appropriate in the beginning, they usually are unable to find support, cave into the gap and result in a renewal that is not complete (Zellin et al., 1995); for instance, when dealing with periodontal flaws, resorbable membrane might have a propensity to cave in (Warrer, 1989).

5.4.2. Non-resorbable membranes

Polytetrafluoroethylene (PTFE) and titanium mesh are two non-resorbable membranes. One disadvantage when utilizing this kind of membrane is the need to remove it with another operation. Nevertheless, this drawback might be outdone by the benefits on hand. These membranes offer an efficient barrier utility by means of biocompatibility (De Macedo et al., 2008), they can uphold the gap underneath the membrane for an adequate time frame, they are extra foreseeable in their functioning, and they have a decreased danger of long-term difficulties, and are easy to deal with during surgery (Zhang et al., 2010). Non-resorbable membranes have certain distinct features. Their configuration can be different with alterations in the pores if a better adjustable and tissue-compatible option and manifold patterns are obtainable in the market and could be additionally improved if wanted (Garg, 2011). Three main non-resorbable membranes, namely, the expanded PTFE or ‘e-PTFE’, the dense PTFE or‘d-PTFE’ and titanium mesh will be illustrated below.

5.4.3. e-PTFE membrane

In this formation, PTFE can be classified into two varieties, namely, expanded-PTFE or e-PTFE and high density-PTFE or d-PTFE. The Gore-Tex® membrane (Gore & Associates, Flagstaff, AZ, USA), which is made of e-PTFE, is extensively utilized in medical procedures and is considered the best possible option for tissue and bone renewal. It is moreover widely utilized for digestive, cerebral and cardio-vascular operations, and fundamental investigation has revealed its efficiency in tissue-guided restoration (Lee et al., 2010). Certainly, in a current restricted investigation (Antoun et al., 2001); it was revealed that a mixture of an e-PTFE membrane and autogenous bone graft at edentulous sites might restrict graft resorption, in consequence improving bone restoration.

There are two dissimilar micro-formations in e-PTFE membranes, namely the coronal border and an occlusive section. With an intermodal range of 25 μm, the coronal border has an open micro-structure collar that assists early clot development and collagen fiber addition to steady the membrane pending it being preset (Garg, 2011) and (Lee et al., 2010). The occlusive section has an internodal range lesser than 8 μm to permit nutrient passage even as stopping the permeation of other tissue cell kinds (Garg, 2011). e-PTFE is made up of several minute holes or pores, that help tissue cell connection that steadies the host-tissue interface. These minute pores also help in restricting the movement of epithelial cells (Herr, 2006). Nevertheless, this substance needs a second operation to remove it and that can result in bacterial infection of the membrane (von Arx et al., 1996). Additionally, e-PTFE has to be taken away without delay if there is any swelling. Currently, e-PTFE membrane is not utilized for surgical procedures and is unavailable for dental procedures; nevertheless other options are there.

5.4.4. d-PTFE membrane

The other option to e – PTFE is high density PTFE or d-PTFE membrane, namely, Cytoplast™ Regentex GBR-200 or TXT-200; Osteogenics Biomedical Inc., Lubbock, Texas, USA. This membrane was first introduced in the year 1993, and its accomplishment in bone and tissue renewal is duly recorded (Bartee, 1998; Bartee, 1995). This membrane is composed of a high-density PTFE, with a submicron (0.2 μm) pore dimension. Due to the quality of high density and minute pore dimension, bacterial infection of the bone expansion area is not found, thus protecting the fundamental graft substance and the implant. Moreover, principal soft tissue closing is not necessary (Bartee & Carr, 1995; Bartee, 1995). Preceding researchers have revealed that d-PTFE totally obstructs the infiltration of food and bacteria, and therefore, in spite of being open to oral cavities, it will yet be a suitable membrane barrier (Rominger & Triplett, 1994; Barber et al., 2007). Fascinatingly, one of the substances, Cytoplast™, has no pores in its formation and its connection to tissues is feeble. Consequently, it is possible to be taken off effortlessly by tugging on the membrane and it is not necessary to lift the mucosal flap. Additionally, the danger of it being infected, even if exposed, is comparatively less than e-PTFE (Lee et al., 2010).

5.4.5. Titanium mesh

Other than the two PTFE membranes, titanium is a non-resorbable substance appropriate for dental bone restoration. In the year 1969, Boyne et al., installed a mesh from titanium for the renewal of big discontinuity osseous faults (Boyne et al., 1985). Titanium has been utilized expansively in several operations due to its great strength and firmness, its lower density level and consequent lower mass, its capability to endure higher temperatures and its ability to resist being corroded (Wang & Fenton, 1996; Zablotsky et al., 1991; Degidi et al., 2003). This metal is extremely reactive, and can be willingly made to form a protecting oxide coating, that is accountable for its great ability to resist corrosion (ADA Council on Scientific Affairs, 2003). The lower density of titanium offers both greater -strength and lower weight dental substances (ADA Council on Scientific Affairs, 2003).

5.4.6. Electron spin membrane

To-date a number of resorbable membranes (i.e. ones that do not require a secondary surgical procedure for removal) have been developed as an alternative to the non-resorbable membranes (e.g. expanded polytetrafluoroethylene, e-PTFE) that were used before. These membranes are based on membranes such as poly (lactic acid) (PLA), poly (glocolic acid) (PGA), poly (caprolactone) (PCL) and its co-polymers (Piattelli et al., 1996; Geurs et al., 2008, Gielkens et al., 2008 ; Milella et al., 2001) and or tissue derived collage (Felipe et al., 2007; Behring et al., 2008; Bottino et al., 2009). These polyester based membranes are easier to be handled during surgical procedures would also allow easy tissue integration on compared to the conventional non-resorbable membranes. But a lack of cell response for these polyester-bases membranes is their major drawback (Behring et al., 2008; Kikuchi et al., 2004). On the other hand the collagen membranes have shown excellent regenerative properties due to their high cell affinity and biocompatibility nature. But their easy degradation property along with the associated poor mechanical and dimensional stability nature makes them fallout (Behring et al., 2008).

These studies would surely have shown that both e-PTFE (e.g. second surgery) and collagen-based resorbable membranes (e.g. insufficient mechanical properties) were not the ideal periodontal membrane and the search for an effective membrane is still on. The membrane should be possessing sufficient mechanical and physical properties in order for them to be placed in vivo avoiding membrane collapse. There should be a proportional ratio between the membrane degraded and the tissue newly formed. It should be noted that for a successful regeneration of the periodontal system, these membranes should be inside the human system eliciting its function for about 4-6 weeks without being degraded. (Karring, 2000; Liao et al., 2007)

Research work for more than a decade has been carried out in developing an effective periodontal membrane that possesses the requisite features and properties. Membranes were prepared using different techniques including film casting (Liao et al., 2007) and dynamic filtration (Teng et al., 2009), and e-spinning (Fujihara et al., 2005) using synthetic (eg., PCL) or natural polymers (eg., collagen). Few of these membranes were amended with therapeutic drugs (Part et al., 2000), growth factors (Milella et al., 2001) and calcium-phosphate nanoparticles (Liao et al., 2007, Kikuchi et al., 2004; Yang et al., 2009) eliciting additional properties. The addition of these components are sometimes complex (e.g. nanoparticles might become toxic over a higher concentration and their dissolution rates (α-TCP > β-TCP >> HAp)) and hence are not universally accepted (LeGeros, 1993; LeGeros, 2002). This shows that a control should be kept in place for the calcium phosphate release rates and its particulate size has to be optimized. These practices have indeed induced the concern about the development of carcinomas, though there has been no scientific or clinical evidence to prove the fact (Thawani et al., 2010).

The very less cost in generating electrospun scaffolds with a matrix structure that have a functionally graded matrix structure with distinct chemical composition and modified mechanical and degradation characteristics has been shown (Thomas et al., 2007; Erisken et al., 2008; Thomas et al., 2009; Zhang et al., 2009; McClure et al., 2010). Recently a trilayered electrospun tubular vascular graft in combination with bio-artificial polymers (i.e. polyglyconate-Maxon®, gelatin and elastin) that imitated not only the dimensions of the extracellular matrix (ECM) but also controlled degradation behaviour (Zhang et al., 2009). The addition of biopolymers such as collagen, gelatin or elastin donated the membrane with improved hydrophilicity and with active bioactive properties, hence translating into better cell adhesion (Kwon and Matsuda 2005; Lee et al., 2007; Dong et al., 2008), growth and function (Zhang et al., 2010).

The fabrication and design of a functionally graded periodonatal membrane (FGM) through electrospinning holds promise as an interface between alveolar bone and epithelial tissue. To meet the local functional requirements, the implant needs to utilize a graded structure with compositional gradients and sub-compartments (Pompe et al., 2003; Leong et al., 2008). With the help of sequential electrospinning, this work has fabricated a novel FGM with a spatially designed layered structure. To permit periodontal regeneration, one can tailor the properties of different layers to design a membrane that will retain its structural, dimensional and mechanical integrity, the rationale of having a periodontal membrane with a graded structure relies on this idea. To enhance bone formation, hydroxyapatite nanoparticles (n-HAp) were incorporated on the surface layer facing the bone defect and metronidazole benzoate (MET). MET is an antibiotic mainly used in treating periodontitis (Al-Mubarak et al., 2000). The antibiotic is added to avoid bacterial growth on the surface layer facing towards the epithelial tissue. The research also gives reports on electrospun composite fibers of poly (DL-lactide-co-ε-caprolactone) (PLCL) and PLA and for the first time, gelatin is loaded with n-HAp or MET.

5.4.7 Functionally graded membrane design

The membrane of electrospun was designed to have a functionally graded layer structure that comprises a CL and two functional SLs of composite fibers (Fig. 1). On the basis of mechanical characteristics, degradation properties and biocompatibility, to different synthetic polyesters, PLCL and PLA, were selected (Kwon & Matsuda, 2005; Lee et al., 2007), (Dong et al., 2008), (Jeong et al., 2008; Meek et al., 2004). To provide the required mechanical properties, the CL included a neat PLCL layer that is surrounded by polymer (PLCL: PLA: GEL) ternary blend layers (Fig. 1). To enhance the bioactivity, gelatin was being used, i.e. it increases cell response (Zeugolis et al., 2008; Sell et al., 2009). On the basis of tissue with which each would interface, the SLs were selected. For example, PLA: GEL + n-Hap, was formulated to imitate the collagen–HAp matrix of bone, this layer was designed to be linked with the bone defect. On contrary, the layer that is designed to abut the epithelial tissue (PLA: GEL + MET) was loaded with 25 wt.% MET. A step-wise grading was seen in the chemical composition of CL to the SLs, specifically the polymer content decreased and protein content increased (Fig. 1).

Fig. 1: Schematic illustration of the spatially designed and functionally graded periodontal membrane. (A) Membrane placed in a guided bone regeneration scenario. (B) Details of the core layer (CL) and the functional surface layers (SLs) interface (Marco et al., 2011)

Among others, the fabrication of FGMs for GTR/GBR has been explored by different technique that includes layer by layer filtration and casting (Liao et al., 2005; Teng et al., 2009; Xianmiao et al., 2009). To generate a graded membrane with a unique design, properties and structural characteristics, multilayered electrospinning was used in this study (Fig. 1). To fabricate nano and micro-scale fibers, the method of electrospinning is used, which is a simple, different and well-documented method (Cooper et al., 2006; Thomas et al., 2006). During processing, the chemical characteristics of the polymers or blends are not used; this made investigators to incorporate drugs like tetracycline and metronidazole into unique polymers and to develop a material with therapeutic properties (Kenawy et al., 2002; Zeng et al., 2003; Kim et al., 2004).

5.5. Specific considerations for orthopedic surgery

In humans, for oral and maxillofacial surgery, bioresorbable membranes are currently used mainly for bone regeneration. A field of interest is represented by their use in various orthopedic conditions, mainly since the number of revision surgeries (Pedersen et al., 2005; Ulrich et al., 2008) and limb salvage procedures are increasing (Mavrogenis et al., 2011; Sampo et al., 2011). For example, to preserve the continuity of the diaphysis for the repair of large diaphyseal bone defects (Aldini et al., 2005), bioresorbable membranes can be shaped as tubular chambers. Using two concentric perforated membranes by forming a 'tube-in-tube implant' in combination with cancellous bone-graft, for the treatment of segmental diaphyseal defects in sheep tibiae (Gugala & Gogolewski, 2002), the reconstitution of the neocortex with well-defined thickness was possible. To prevent significant absorption of the bone graft, barrier membranes can be used which is estimated to be up to 40% to 50% at four weeks (Gugala & Gogolewski, 2002) and seems to be due to absorption of bone that is not mechanically functioning (Jaroma & Ritsilä, 1988). As the bioresorsable membranes are radiolucent, the bone formation assessment is done with conventional radiographs, CT or MRI (Meinig, 2010), that play an important role in monitoring the regeneration process.

The evidence on the efficiency for cortical perforation (decortication) during GBR procedures remains controversial in an effort to increase bone formation (Greenstein et al., 2009). The blood supply is improved by cortical perforations as shown in various studies, it facilitates angiogenesis, and progenitor cells access can be moved into chamber from the bone marrow (Greenstein et al., 2009). In contrast, other studies proved that increase in bone formation is not due to perforations (Schliephake & Kracht, 1997; Gutta et al., 2009) and it also showed that from a non-injured cortical bone surface the formation of bone has occurred. For orthopedic GBR applications, recommendations for additional bone decortication cannot be made. Local vascularity is superior to long bones, since there are no relevant human clinical studies and animal studies that represent mandibular defects (Greenstein et al., 2009).

In the case of bone defects, membrane barriers can be used in combination with bone grafting to augment osseointegration of orthopedic implants (Guerra et al., 2011). As the preliminary results are encouraging, they may also be used for regeneration of other tissues with potential orthopedic applications, including tendon regeneration in rotator cuff repair, and post-traumatic nerve regeneration (Yokoya et al., 2008; Amado et al., 2008).

5.6. Enhancement of bone regeneration and future research

5.6.1 Biological augmentation of GBR with growth factors

Researchers combined the membrane technique with osteoinductive or growth factors due to their interest in accelerating bone formation. Results are often controversial since the growth factors that increases proliferation, chemotaxis, and differentiation of osteogenic cells enhance the concept of additional biological enhancement of bone formation. While evaluating the long-term outcome of oral implants in a study that are placed in bone augmented with an allograft and a collagen membrane with or without the addition of recombinant-human bone morphogenetic protein-2 (rhBMP-2), on clinical and radiological outcomes, no statistically significant differences were observed (Jung et al., 2009). A number of in vivo and in vitro studies have been demonstrated in contrast to improve bone formation when platelet-derived growth factor (PDGF-BB) (Lee et al., 2001 ), basic fibroblast growth factor (FGF2) (Hong et al., 2010), and rhBMP-2 when loaded with barrier membranes (Linde & Hedner, 1995 ; Jung et al., 2009; Zellin & Linde, 1997).

To maintain the therapeutic concentration of growth factors within bone defects, the evidence that are opposite may be considered secondary due to rapid c.learance and with the help of unique delivery methods with supraphysiological non-standarized doses to obtain efficiency in therapeutics (Lee et al., 2001). The research work carried out at present moreover evaluates growth factors that are single or in combinations that does not revert back the complex physiological process of bone formation. Recent research has developed novel membranes and scaffolds that have improved growth factor delivery systems to increase bone regeneration of critically-sized segmental bone defects with good preliminary results (Kolambkar et al., 2011). Growth factors with a controlled spatiotemporal delivery, for optimal regenerative efficacy, excess of local protein concentration can be improved and maintained, the concomitant adverse effects and by avoiding the supraphysiologic doses that are used recently (Kolambkar et al., 2011). At the end, the optimal ‘combination’ of growth factors to be delivered was also needed to be established.

5.6.2 Other strategies to improve bone regeneration

Additional strategies have been scrutinized in relation with membrane barriers and grafting, this is done with an aim to accelerate or maximize bone formation. The potential advantage of low-level laser therapy (LLLT) has been assessed as an aide in regeneration of long bone defects that is done in animal studies showed positive results (Gerbi et al., 2005; Pinheiro et al., 2003). Synergistic regenerative effects in the past have shown additional treatment with hyperbaric oxygen (Dahlin et al., 1993). In addition, preliminary results have shown that systemic administration of synthetic salmon calcitonin increases bone regeneration defects (Arisawa et al., 2008).

To enhance bone regeneration, research is carried out to evaluate other methods with promising preliminary results, such as administration of parathyroid hormone locally (PTH (1-34)) (Jung et al., 2007) and other growth factors. To enhance bone formation at the cellular and molecular level, various methods have also been investigated to optimize surface microtopography of the membranes (Donos et al., 2011). As part of the bone-tissue engineering approach combined with osteoprogenitor cells or osteopromotive factors or even gene therapy, improved barrier membranes can be used finally in the future with an aim to produce composite grafts (Dimitriou et al., 2011). The research done in the preliminary stage is promising. For example, adeno-associated virus encoding BMP2 is coated over a novel three-dimensional porous polymer poly (ε-caprolactone) (PCL) scaffold, using both ex vivo or in vivo gene therapy; this led to accelerated bone ingrowth with increased mechanical properties in a rat femoral defect model (Dupont et al., 2012).

Chapter VI

6.1. Barrier membrane applications:

6.1.1. Use of barrier membranes in anti adhesion and anti ossification of trauma orthopedic surgery:

In this section we are going to discuss about the role of biodegradable films in preventing adhesions and ossifications. During the initial stages of healing, the biodegradable films were used for the separation of tissue layers. Such films were developed in the 1980s where these films were used to prevent adhesion in burn wound dressing during early 1990s (Wolf, Capozzi & Pennisi, 1980). It is necessary to achieve some specific characteristics while using these films clinically. These characteristics include physical parameters such as flexibility and durability as well as the following biological characteristics.

• 1. The material should be harmless and it should not elicit the immune system.
• 2. The material should have the ability to bare the sterilization process and the inoculation process should be conducted properly.
• 3. The material should have a certain level of shelf-time.
• 4. There should be a realistic product pricing for the material.

Till date, three anti-adhesion or anti-ossification films are found to satisfy the aforementioned requirements. They are:

Interceed

This absorbable, oxidized, regenerated cellulose is the product of Johnson & Johnson Medical Inc. This product was introduced in the year 1989 (INTERCEED, 1989). These films are useful in surgical procedures on the bowel and obstrectical operations. There have been so many reports on the efficacy of these films in reducing adhesions at post operative stages. They are also useful in the treatment of strabismus (Hwang, & Chang, 1999), or urology (orchidopexy (Genc et al, 2004)). They have also been proved to be useful in treating orthopedic trauma.

There has also been a study on their efficacy in reducing tendon adhesion in rabbit model. Tendon adhesion is the major problem in tendon surgery where a healing tendon adheres with the surrounding areas including bone, muscles, skin, tendon sheath or other tendons through scar tissue (Meislin et al., 1990). However, tendon adhesion can be considerably reduced with the use of Interceed (TC7). Further, no immune response was observed while using this product.

In addition, Interceed is helpful in preventing neural adhesions. In Rabbit model, adhesions were induced by exposing at the middle of the thigh and coagulation was induced using bipolar electrocauter. In controls, neural adhesions and intraneural fibrosis were observed when there was only little adhesion found with Interceed (Ikeda, Yamauchi & Tomita, 2001).

There has been a study on the efficacy of Interceed in 9 carpal-tunnel syndrome patients who undergone division of the flexor retinaculum as well as epineurotomy or synovialectomy (Loick, Joosten, & Lucke, 1997). The median nerve was covered with a monolayer of Interceed. From this study it is understood that recurrence rate can be reduced carpal tunnel surgery with the use of Interceed.

Seprafilm

This bioresorbable membrane of carboxymethyl cellulose and hyaluronic acid was introduced by Genzyme Corp., in 1996. It is found be useful in preventing adhesions of small bowel operations in abdominal surgeries and also in gynecological procedures. Similar to Interceed, Seprafilm is also useful in strabism surgery (Ozkan et al, 2004).

The advantages of Seprafilm whwn compared to Goretex® in preventing peridural fibrosis have been proved in rabbits (Topsakal et al., 2004). They can also be useful in preventing adhesion prophylaxis in cardiac surgery (Walther et al., 2005) and also for repairing subtotal tympanic membrane perforations. While testing the efficacy of Seprafilm in animals, Kessler–Tajima suture-technique was used to repair the damage caused by the removal of the deep flexor tendons of the first finger of the back foot of rabbits (Menderes et al., 2004). Following the repair, one group of rabbits were wrapped with Saprafilm and other group with sodium hyaluronate gel. Better movements as well as lesser histopathological adhesions were observed in both of these groups than controls.

There has also been a study on the efficacy of Seprafilm in preventing adhesion of tendons in chicken flexor tendons (Karakurum et al., 2003). Great average gliding excursion had been achieved in this study. It is understood from this study that adhesions can be prevented using Seprafilm after tendolysis.

Mesofol®

It is a lactid caprolacton film. Its efficacy in preventing adhesion and ossification has been observed in a prospective study using 10 forearm synostoses and 20 arthrolyses of the elbow joint with and without heterotope ossifications (Grimme et al., 2004). No complications and low pain score was observed in this study. There has been a better range of motion (ROM) for pro- and supination of 85 ± 15° following the application of Mesofol® . However, control group showed a pro- and supination of 30 ± 14°. There has also been an improvement in the range of motion for flexion/extension has been observed following the surgical arthrolysis of the elbow joints with removal of heterotopic ossifications and the use of Mesofol®. No Re-occurrance of synostoses or heterotopic ossifications has been observed in the follow-up period of 6.5 months. In the earlier studies on postoperative treatment those were used after removal of synostoses and heterotopic ossifications irradiation was used]. With the arrival of biodegradable films, the use of irradiation was reduced. However, these films are found to produce better and effective ROM even in the absence of irradiation.

6.1.2. Induced membrane technique for reconstruction after bone tumour resection

Complex reconstructive surgery is the only remedy for the trauma or infection induced segmental long-bone defect. There have been various studies (Marsh et al., 1994; Abudu et al., 1996; Muscolo et al., 2004; Capana et al., 2007) on these surgical procedures.

Masquelet technique, a two staged technique, also called as the French technique of bone-grafting within induced membranes (Masquelet et al., 2000; Pelissier et al., 2002; Masquelet, 2003) can be used in place of complex reconstructive surgery (Biau et al., 2009; Villemagne et al., 2011; Masquelet & Begue, 2010;Vieateau et al., 2010). At the initial stage, bone is stabilized after bone resection and then soft tissue repair will be induced to allow the insertion of cement spacer. After few weeks, spacer will be removed along with bone decortications and then cancellous bone graft will be inserted into biological induced membrane. Recent studies (Pelissier et al., 2004;Viateau et al., 2006;Zwetyenga et al., 2009) proved the presence of rich capillarity network as well as growth and osteoinductive factors in these membranes. Reports (Masquelet, 2003;Viateau et al., 2010;Apard et al., 2010) are available on the efficacy of these membranes in treating post-traumatic or post-infection diaphyseal defects.

These membranes can be used to treat diaphyseal bone defect in children (Biau et al., 2009).In a recent study the same effect has been studied in 12 patients for the follow-up period of 6.2 years (Villemagne et al., 2011). In another study, the efficacy of Masquelet technique in treating metaphyseal or meta-diaphyseal bone tumors of 8 children was observed (Chotel et al., 2012). from this study it is understood that this induced membrane technique can minimize the infection rate and complications in donor site. These membranes are also used even after major bone resection. In addition, while performing intercalary resection of the tibia in patients with Ewing sarcoma, these membranes can be used.

6.1.3. Role of Resorbable membrane in post operative peridural adhesion:

Fibrosis is the common post operative condition of surgical wound healing. In a study [29] The source of fibrotic tissue after spinal surgery was originally thought to be the disrupted intervertebral disc. However, in another study [31], the disrupted epaxial muscles found around the surgical areas were found to develop fibroblasts. Though there have been different views on the importance of peridural fibrosis in failed-back syndrome,[17], it has been considered as a non-effective treatment [1]. Recently, a technique called magnetic resonance imaging is used to find out recurrent intervertebral disc extrusion exactly using a contrast material known as Gd-diethylenetriamine pentaacetic acid[15] . As it is very difficult to locate the neural structures those are located closer to scar tissue, we could not get better outcomes, if we do repeated surgery to remove scar tissue .[5] As a result, various studies have been conducted to prevent the scar tissue adhesion nearer to the dura mater and nerve roots.

Peridural adhesion and fibrosis prevention agent should ideally have the following characteristics: 1). it should be able to deter adhesion of scar tissue to dural tissues, 2). it should prevent leptomeningeal arachnoiditis, 3). should curtail possibilities of damaging dural healing post CSF leakage and tearing, 4). prevent chances of inflammation about neural tissues. Some of the well studied and documented materials and methods used are autografts; biomaterials synthetically produced like polytetrafluoroethylene, Gelfoam, TachoComb etc; biochemicals administered to minimise fibroblast activity and infiltration, for example, urokinase, hyaluronic acid and glucocorticoids; use of CO2 laser therapy intra-operatively or localized treatment using external-beam radiation. These methods’ efficiency has however not been acknowledged and fully accepted in the medical field. Free fat autografts is believed to be the most commonly and frequently used interposition material used in spinal cord operation done in humans but some studies have revealed that this procedure has drawbacks arising due to herniation of the fat graft followed by neural impingement. However, earlier studies have shown a successful method employing ADCON-L which has shown consistently positive results. But usage of this material later generated negative feedbacks since reports published evidenced impaired dural healing and prolonged CSF leakage in humans. In spite of previous reported studies, ADCON-L mediated complications such as dural tearing that occurred went unrecognized.

Initial examinations carried out in rat displayed appreciable decrease in peridural scarring with the use of polylactide resorbable film. A previous study was conducted to analyse the efficacy of 0.02-mm-thick polylactide resorbable membrane as anti-adhesion barrier in canine and ovine small laminotomy models. The study presented optimal results the ovine laminotomy model. Later, another more comprehensive large scale clinical trial was carried out using polylactide barrier employing dorsal (posterior) laminectomy and durotomy procedures in ovine model. The study ensured that a premortem myelographic and postmortem gross examination was performed in order to analyse the tenacity and volume of peridural scar adhesion. The result of this study was consistent with previous studies. The membrane material reduced posterior dural adhesions in the spinal cord.

6.1.4. Animal studies on use of barrier membranes for long bone defects

Jégoux et al. (2010) conducted a study in dogs to evaluate the efficacy of calcium phosphate ceramics and collagen usage in mandibulectomy following delayed bone marrow grafting technique. Based on SEM (Scanning Electronic Microscopy) and microtomographic analysis it was concluded that the materials used for grafting had successfully established osseointegration, correcting the mandibular defect and formation of a new bone was evident. The bone marrow graft had successfully osteoinducted and the membrane had formed a scaffold which shows the healing effect. Another study involved comparing a new Acellular Dermal Matrix (ADM) with traditional bioabsorbable synthetic membrane in mandibular bone grafting, with dogs as the animal model. The test group was integrated with ADM while the control group was integrated with a copolymer of glycolide and lactide as bioabsorbable membrane. Post surgical examination was conducted at 8 and 16 weeks after surgery by radiological methods. Also, after 16 weeks, a physical evaluation of the width and thickness of keratinized tissue and histomorphometric analysis was performed. Evaluation studies revealed that ADM acted as a barrier in guided bone regeneration and similar observation was made with bioabsorbabale membrane as well.

Sverzut et al. (2008) conducted another study also in Dogs to study the efficiency of using poly L/DL-lactide 80/20% membrane with different permeability patterns. The material was used to correct 10mm segmental defects using mechanical stabilization methods with microporous bone grafts in 6 test groups including control, bone graft, microporous membrane, Mi plus bone graft, microporous laser-perforated (15 cm2 ratio) membrane (Mip) and Mip plus bone graft. The outcome of the surgery was evaluated at 6 months post operation using histological, histomorphometry and fluorescence microscopy methods. The evaluation with respect to each group showed: bone graft protected by Mip regenerated larger bone quantity; no difference was observed between Mip group and bone graft alone group; bone graft with Mi alone osseointegrated the lowest bone area and reduced the bone quantity to control levels. In the bone graft group, bone was in the developing stage regardless of whether or not it was protected by a membrane at 3 months. Also, Mip group showed increased bone formation at 3 months. These observations inferred that Mip alone can be used in place of bone graft. In addition, Mip and bone graft membrane in combination increased bone quantity efficiently especially in segmental defects.

Two different types of bioabsorbable membranes – collagen membranes and cross-linked collagen membrane were used to correct three standardised mandibular defects in dogs. Bone chips and deproteinized bovine bone mineral (DBBM) were used for filling and 3 groups were studied. 1. Control without membrane, 2. Test group 1 with collagen membrane and 3. Test group 3 with cross linked membrane (CCM). Each side of the mandibles were either tested at 8 or 16 weeks post grafting using histomorphometric analysis. Each group showed increased height in week 16 compared to week 8, however, the CCM group showed significant increase and highest value of treatment modality in week 16 with optimal healing capacity. However, in membrane protected defects, CCM group showed only limited bone regeneration and healing (Bornstein et al., 2007).

Zubery et al.(2007) performed a study to analyse the efficacy of type I collagen membrane employing a novel cross-linking technology (GLYM) and type I and III collagen membrane (BCM) employing a non-cross linking technology. The materials were used to correct mandibular bilateral critical size defects. Five groups were studied: GLYM and bone mineral (BBM), BCM and BBM, BBM alone sham-operated, or GLYM alone. Regular follow ups were done at 8, 16 and 24 weeks using qualitative, semi-quantitative and quantitative light microscopic examination. Optimal bone growth was observed between BBM particles with almost complete bone regeneration with natural ridge morphology that augmented till week 16 and thereafter remained constant which was confirmed at week 24. Both the membranes progressively degraded between 16 and 24 weeks. In addition all GLYM sites and one BCM site showed optimal ossification which also improved until 24 weeks. At the point of contact of GLYM membrane with the bone, an increased bone volume was observed.

Fritz et al. (2000) conducted a study to evaluate the efficiency of reinforced ePTFE membranes in correcting Standardized 8 × 19 mm mandibular defects using Macaca mulatta monkeys as model animal. Reinforced ePTFE membranes were fixed using mini screws and by suturing and allowed to regenerate for 1 to 12 months. These model animals were analysed using digital subtraction radiology and tetracycline and histomorphometry (fluorescent dyes) labelling. Bone development was observed periodically from 1 to 12 months. It was seen that membranes held in place for 1 month or less showed lesser bone growth than between 2 to 12 months. Histochemical staining methods revealed that bone that formed after 2 months of membrane fixture was mature.

The effect of standard and prototype reinforced e-PTFE membranes was analysed in treating mandibuar defects introduced in dogs. The test group was divided into two to evaluate both standard and prototype reinforced e-PTFE membrane and the control group with no membrane placement. Post operative follow up was done at 2 and 4 months using histological techniques. As expected, controls without membrane displayed uneventful bone restoration and healing. Although test sites exhibited bone regeneration, it was not complete until 4 months. More clear evidence was produced by histological studies showing that once bone regeneration was initiated, a progressive bone growth was observed which was similar to the actual pattern of bone growth and development in nature (Schenk et al., 1994).

6.1.5. Clinical studies with bioresorbable membranes for reconstruction of segmental mandibular defects

Based on a series of case studies conducted from 1995 to 2001, Kinoshita (2004) summarised the results of using bioreabsorbable membrane, PLLA mesh to treat segmental defects mandibulectomy and semi-mandibulectomy. 62 patients divided into 6 test groups were subjected to bone reconstruction and the results were examined. The test groups consisted of patients with malignancy, benign tumors, cysts, osteomyelitis, alveolar atrophy and trauma. Autologous cancellous bone graft in combination with bone marrow was used and stainless steel wires were used as fixtures. Post operative examination at 6 months showed 56.5% excellent bone growth, 27.4% effective bone regeneration and a minor percentage 16.1% showed poor regeneration. A similar series of case studies conducted between 1995 and 1998 to study the effect of membrane in correcting segmental defect or large partial defects mandibulectomy. Two groups with 19 patients suffering from malignancy and 22 patients with benign tumors were subjected to the treatment. 46.3% displayed excellent bone regeneration, a close 31.7% showed good regeneration and 22.2% exhibited poor regeneration. Until 5 years, PLLA mesh was stable with good osseointegration (Kinoshita et al., 2000). In addition, 2 subjects with segmental defects and tumor were analysed for bone regeneration efficiency with bioabsorbable membrane PLLA mesh and showed full bone regeneration at 3 months (Kinoshita et al., 1996).

6.2. Bone graft applications:

This section discusses in detail about the applications of bone graft materials in various bone replacement surgical procedures.

6.2.1. Bone graft in joint replacement:

Combining bone graft substitutes with allograft is common these days, but it may result in insufficient supply of allograft material (McNamara, 2010). This combination may alter the advantages in relation to better mechanical properties and reduced instances of infection. When autograft or allograft bone was compared, the possible disadvantages of bone graft substitutes include poor integration with remaining bone stock and foreign body reaction.

Previous studies on the usage of bone graft substitutes with the help of hydroxyapatite in combination with autologous bone in the moderate load areas showed a promising effect in the treatment of patients affected by bone deficiencies such as scoliosis, bone cysts, tumours, trauma and other indications (Heise et al., 1990; Shibuya et al., 2005). The study by Heise and colleagues found similar outcomes at the end of 22 months in comparison with those studies using autologous bone graft alone (Heise et al., 1990). Likewise, hydroxyapatite was used in the treatment to fill the diseased and donor sites in the patients affected by benign bone tumours and tumour-like diseases. Shibuya and colleagues found no unfavourable events and good radiological outcomes were recorded after a period of 8.6 years for both autograft and allograft in combination with hydroxyapatite, or substitute alone (Shibuya et al., 2005)

From our earlier studies (Blom et al., 2002), it was learned that the major requisite for a ideal bone graft substitute in the treatment of hip revision surgery was that it should provide structural stability with neo-ossification through osseoconduction, osseoinduction and substitution. Using ideal bone graft substitute will not result in spreading of infection or provocation of a detrimental host response. Ideal bone graft is easily available and its cost is relatively effective when compared to the allograft.

Aulakh et al. (2009) carried out a study in UK from 1994 to 1999. The objective was to study the efficiency of revision arthroplasty by treating femoral and acetabular osteolytic lesions. The bone graft substitute was prepared with 50/50 allograft and Allogran-N solid particulate hydroxyapatite or pure morsellised allograft. One group received bone substitute mix (N = 23), and another received pure allograft (N = 42). The decision was based on the availability of allograft bone, including femoral (n = 27), acetabular (n = 9) and both (n = 29). The period of follow-up was 13 years. It was stated that the long-term prosthesis survival and function with a 50/50 mixture of allograft and hydroxyapatite were similar to allograft alone. The allograft survival in the bone substitute mix group was 82% when compared with 84% in the pure allograft patients.

Another study was carried out by Fujishiro et al. (2008) in Japan in 1996 to study femoral revision presented with bone loss. The bone graft substitute was prepared with 50:50 mixture of morsellised allograft and hydroxyapatite-tricalcium phosphate. The study group contained 15 people. The duration of follow up was not specified. From the results, it was evident that clinical hip scores improved postoperatively for the majority of patients. Only one patient underwent re-revision for loosening and one patient underwent re-revision after a fracture.

Müller and Stangl (2006) from Germany studied the revision arthroplasty with acetabular reconstruction. At the end of cancellous bone grafting, the bone defects were filled with osteoconductive calcium phosphate cement (Norian SRS), if needed to prepare bone grafts substitutes. Six people were included in the study. Five out of six were followed up. The follow up period involved 16 months. Harris and Merle d’Aubigné scores were used to determine patient satisfaction. Four out of five patients in the evaluation of group achieved good or very good functional results. Better functional outcome was associated with greater patient satisfaction.

McNamara et al. (2010) from UK studied hip replacement requiring acetabular reconstruction in 2002. 1:1 mixture of frozen, ground irradiated bone graft and porous synthetic hydroxyapatite bone substitute (Apapore 60) was used to prepare bone graft substitute. The size of the study population was N = 48 (50 hips: 37 revision, 13 complex primary). The period of mean follow up was 60 months (range 41–91). The results showed 100% clinical survival, evidence of incorporation was 60%, radiolucent lines 20%, and migration of acetabular component was 4%. The Harris hip score showed improvements in pain.

6.2.2. Bone graft application for treatment of fractures:

The gold standard method for achieving dead space filling, stable fixation, structural support and uneventful bony union is the bone graft augmentation in the presence of bone defects. This has been extensively used particularly in the metaphyseal areas of load bearing bones, such as the tibial plateau, autologous bone graft and/or a wide spectrum of biologic and synthetic substitutes. Nevertheless, the “ideal” bone graft substitute, which was described as osteoconductive, osteoinductive, biocompatible, structurally similar to bone and cost effective (Greenwald et al., 2001) does not exist at present. However, discussions on some latest studies on the application of synthetic bone grafts were as follows.

One of the objectives of study conducted by Ong et al. (2012) was to study the fracture stability and functional outcome of synthetic bone graft and natural bone graft with internal fixation of tibia plateau metaphyseal defects and thereby determine their differences. In the 14 patients (six males and eight females), hydroxyapatite calcium carbonate synthetic bone graft was used. In the remaining 10 patients (six males and four females), allograft/autograft was used.

Clinical, radiological and subjective functional score assessments were performed in all of the 24 patients. No significant statistical difference was found between the groups for post-operative articular reduction, long-term subsidence and WOMAC scores. Age or fracture severity was found to be not related to the degree of subsidence. Allograft/autograft group (p = 0.048) was found to have a better maintenance of knee flexion when the groups were compared. On comparing graft type, fracture severity, post-operative reduction, subsidence rate, range of movement and WOMAC score using multivariate analysis, it was found that there was only a statistical significant associated with the graft type related to the 6-month range of movement figures.

The important objective of the study (2012) conducted by Yin et al. was to examine the histological changes in the bone morphology after the use of calcium phosphate cement in replacing autologous bone grafting in the surgical treatment of tibial plateau fractures. Open reduction, internal fixation, and calcium phosphate cement were used in the treatment of 42 patients affected by tibial plateau fractures. Open reduction and internal fixation were done in 34 control patients. During the surgery, bone samples were obtained for histology, and in addition bone healing and functional recovery were assessed. When compared to the first surgery (45.4, p < 0.001), bone cell counts were significantly higher in the samples obtained from the second surgery (81.2). After surgery, bone healing scores significantly increased with time (p < 0.001) and for both the calcium phosphate cement group (82.3) and control group (79.4) the mean Hospital for Special Surgery knee scores were rated "good" at 12 months, and no significant difference was found between the groups. Well-arranged trabeculae, along with new bone and blood vessel formation were the significant results obtained during the histological examination of samples from the second surgery. From these histological, radiological, and functional findings, it was found that calcium phosphate cement acts as a promising substitute for autologous bone grafting in the treatment of tibial plateau fractures.

The main aim of study carried out by Hiikkila et al. (2011) was to compare the efficiency of bioactive glass and autogenous bone as a bone substitute material in the treatment of tibial plateau fractures. A prospective, randomized study was designed using 25 consecutive operatively treated patients with depressed unilateral tibial comminute plateau fracture (AO classification 41 B2 and B3). The bioglass group (BG) consisted of 14 patients (7 females, 7 males, mean age 57 years, range 25-82) and autogenous bone control group (AB) included 11 patients (6 females, 5 males, mean age 50 years, range 31-82). Clinical examination was done in patients at 3 and 12 months, and at 12 months patients' subjective and functional results were evaluated. Likewise, periodical radiological analysis was done preoperatively, immediately postoperatively and at 3 and 12 months. For both studied groups, the postoperative re-depression was 1 mm up to 3 months and was the same till 12 months. After 1 year follow-up, the subjective evaluation, functional tests and clinical examination revealed no significant differences in between the two groups. Thus, it is well understood that the bioactive glass granules are effective in replacing autogenous bone as a filler material in the treatment of lateral tibial plateau compression fractures.

Chapter VII

7.1 Comparison of effectiveness of bone draft and graft material

The advantages of different approaches with respect to the treatment of infrabony defects have been dealt with in several studies. Among them, the study by Orsini et al (2008) was one of the significant studies which aimed at evaluating the results of experimenting the combination of autogenous bone graft with calcium sulphate and to cross-examine these results with outcome of amalgamation of autogenous bone grafts with bioabsorbable membrane. The samples of 12 participants were examined in this split-mouth trial. Among them, twelve with 2 or 3 periodontal faults were treated with autogenous bone grafting and calcium sulphate combination. Such subjects were compared with 12 subjects who were treated with a combination of autogenous bone grafting and bioabsorbable membrane (Control). Before subjected to surgery, all the participants were initially explained about oral hygienic techniques, scaling and root planning. The procedural method was common for both the groups apart from the regenerative materials. After surgery, probing depth, clinical attachment level and bleeding on probes were noted down at baseline, 6 months, and 6 years. At baseline, no significant changes were noted between the two groups. At 6 months, probing depth reduction was noted at a rate of 4.3 ± 1.0 mm in the control group and 4.4 ± 1.1 mm in the test group. As a result, there was a clinical attachment gain of 3.5 ± 1.1 mm in control group and 3.6 ± 1.0 in test group. At 6 years, there was a PD reduction of 3.3 ± 1.0 mm in controlled group and 4.2 ± 1.2 in test group. As a result, there has been more clinical attachment gain of 2.6 ± 1.2 mm in the control group and 2.4 ± 1.1 mm in test group. From this, the author concluded that there was no significant change noted between the two groups at 6 months or 6 years.

Study by Camargo et al. (2000) was aimed at determining the effect of bovine porous bone mineral in the treatment of periodontal regeneration in intrabony defects in humans. The samples of 22 paired intrabony defects were treated and surgically re-entered 6 months after treatment. The technique of split-mouth design was employed in this study to compare the patients treated with bovine porous bone mineral and patients treated with an open flap debridement (control). Both groups were treated with similar procedures and recorded pre-operative pocket depths, attachment levels and trans-operative bone measurement. Post-operative measurements showed the pocket depth reduction of 1.89±0.31 mm on bucca, 0.88±0.27 mm on lingual measurements and more experimental gain of clinical attachment of 1.51±0.33 mm on buccal and 1.50±0.35 mm on lingual measurements in tested groups. Surgical re-entry of the treated defects revealed significant improvement in defect fill in experimental sites (2.67±0.91 mm on buccal and 2.54±0.87 mm on lingual measurements). Accordingly, the final outcome of the study concluded that effective treatment for intrabonv defeats can be achieved through the combination of bovine porous bone mineral with an absorbable porcine derived collagen membrane while employing a technique based on guided tissue regeneration. To confirm the presence of new attachment, it is essential to evaluate the nature of attachment between newly regenerated tissue and the root surface.

Following traumatic injury or resection, a functional restoration to the mandible mainly depends on the effectiveness of bone reconstruction to heal primary as well as support endosseous implants. Both vascularised and non-vascularised bone grafts are highly used to construct the mandible but still indications for both the process remain unclear. Thus, the study of Foster et al., (1999) was aimed at evaluating bone graft/flap healing and success of implants in patients treated with VBF against NVBG. Over the last ten years, more than 75 consecutive mandibular reconstructions have been performed, of these, 26 were free bone grafts and 49 were vascularised bone flaps. Patient’s history of the defects such as bone defect size, past irradiations, number of operations, graft/flaps success and implant success were determined and analysed. The success of bone graft/flap has been determined from complete bony union in the body. Similarly, implant success has been determined from the complete integration of living bone and implanted bone. The average follow-up duration was up to 3 years. Generally, free flaps were preliminarily used for the treatment of malignant diseases (78%, 38/49). Similarly, bone graft was earlier used for the treatment of benign diseases (88%, 23/26). On comparing the implants success between NVBG and VBF, the following results were observed: Etiology of irradiation: 11% (3/26) NVBG vs 45% VBF (22/49). Length of body defect (mean): 8.1 cm NVBG vs 9.4 cm VBF. Successful body union, any size defect: 69% (18/26) NVBG vs 96% (47/49) VBF (p<.0005); lateral defects only: 75% (15/20) NVBG versus 100% (17/17) VBF (p < .05). Number of operations to achieve bony union (mean), any size defect: 2.3 NVBG versus 1.1 VBF (p < .001); lateral defects only: 1.9 NVBG versus 1.0 VBF (p < .005). Over 22 patients had been treated with 104 endosseous implants of which, 8 patients received 33 implants with NVBF and 14 patients received 71 implants with VBF. Immediate implant placed: 0/33 NVBG versus 54% (38/71) VBF. At the end of implant transformation, around 82% (27/33) of patients showed successful implants transformed with NVBF vs 99% (7071) with VBF (p<. 0001). The overall implant success in VBF patients with a history of RT is found to be 100% (15/15). In spite of such facts, patients who are treated with VBFs were found to be very old and they had larger defects and were treated for malignant diseases and thus had a larger frequency of irradiation when compared to patients treated with NVBGs. But at the same time, occurrence of bony union was very high through fewer operation procedures in VBF. Moreover, the success rate of implant transformation was significantly high in VBF than NVBG. The final outcome of the study indicated that the VBF are commonly used for mandibular reconstruction. However, NVBF can also be so effective for short bony union (<5-6 cm) in non-irradiated tissues, and/or in patients who are found to be more medically compromised to bear the extra times required for free-flap reconstruction.

Traditionally, lateral ride augmentation can be performed by using autogenous bone grafts to support membranes for guided bone regeneration (GBR). We can measure the excellence of bone-harvesting procedure through considerable morbidity of patients. Study by Hammerle et al., (2008) aimed to study whether the resorbable membrane and bone substitutes will cause successful implantation of horizontal ridge augmentation in standard condition. The samples of 12 patients were selected for this study to identify the competency of resorbable membrane and bone substitutes for successful implantation. Such patients were identified with bone deficits in the area intended for implantation. First, soft tissue flaps were raised carefully followed by insertion of blocks or particles of deprotenized bovine bone minerals (DBBM) (Bio-Oss®) in the defect area. To cover DBBM, a collegenous membrane was applied along with poly-lactic acid pins to fix the surrounding bone area. Then, the flaps were stitched to allow for healing purposes. Eleven sites in twelve patients healed unsuccessfully as per the procedure. No significant flaps dehiscences and no exposure to membranes were observed. After 9 to 10 months of augmentation surgery, flaps were raised to observe the consequences of augmentation. At the same time, the integration of DBBM particles with newly formed bone was observed. However, some pieces of grafting particles on the surface of new bone were found to be partly integrated with the bone. However, these tissues were not enclosed by connective tissues but rather integrated into newly formed bones. In most of the cases, the volume of bones after regeneration has found to be more adequate for implant placements. The average crestal bone width before regeneration was found to be 3.2 mm and it was raised up to 6.9 mm at the time of implant placement. The significance of such difference was statistically identified from Wilcoxon's matched pairs signed-rank test (P<.0.05). After 9-10 months of healing period, it was identified that the integration of DBBM material with a collagen membrane is an effective treatment for horizontal bone augmentation graft before implant placement.

The effect of resorbable membrane, non-resorbable membrane and no coverage with reference to effective healing of onlay bone graft in a dog was studied by Dongieux et al., (1998). The samples of four dogs with six corticocancellous blocks of ramus bone was laid bilateral to the inferior lateral aspect of the mandible as onlay graft. Placed on each side of six blocks, one was covered with a collagen membrane, one left uncovered and one was covered with an expandable PTFE (Poly Tetra Fluoro Ethylene) membrane. At the time of graft placement, the bone volume was determined, as the animals were killed. The final outcome of the study indicates no significant differences in bone volume and histologic outcome between three groups.

The clinical outcome of sinus bone resorption and marginal bone loss after sinus bone implantation was reported by Kim et al., (2008). In their study, the initial osseointegration failed in 3 patients even though they underwent prosthodontic treatment after implant placement. Before surgery, the mean height of remaining alveolar bone in group I (Xenograft + minimal amount of autogenous bone) was found to be 4.9 mm and 19.0 mm after the surgery and 17.2 mm, after 1 year of surgery. Similarly, the mean height of remaining alveolar bone in group II (allograft + xenograft + minimal amount of autogenous bone) was found to be 4.0 mm, 19.2 mm and 17.8 mm before operation, post operation and 1 year after operation respectively. In group I, the minimal bone loss after 1 year of prosthodontic loading and after 20.8 months follow up was found to be 0.6 mm and 0.7 mm respectively. Three implants of group I showed bone resorption of >1.5 mm within 1 year of prosthodontic loading. So the success rate of group I was 93.9%. Subsequently, the similar observation was made in group II and it was identified as 0.7 mm and 1.0 mm respectively. Four implants of group II showed bone resorption >1.5 mm within 1 year of prosthodontic loading and thus the success rate was 83.3%.

7.2 Treatment outcomes: expectations based on evidence-based reviews

7.2.1 Intrabony Defects

From the systematic reviews of randomized controlled trials, it was clear that bone replacement grafts improve the clinical parameters, including probing depth, clinical attachment level, and defect fill, as compared to open flap debridement alone in intrabony defects (Table 1) (Reynolds, Aichelmann & Branch-Mays, 2003). According to the literature, it was estimated that bone fill ranges from 2.3 to 3.0 mm or 60% of the defect was due to grafting of intrabony defects. Crestal bone loss was found to be decreased in the presence of particulate bone grafting materials. On the contrary, gingival margin recession was found to be increased in the presence of barrier materials (Scannapieco, 2010). When compared with graft alone, particulate graft in combination with a barrier provides modest but non significant improvement in bone levels in addition to significant attachment level gain and probing depth reduction.

Table 1: Mean change in clinical measures following bone replacement graft therapy

Bone Replacement Graft	N	Clinical Attachment Level (mm)	N	Probing Depth (mm)	N	Defect Fill (mm)
Allograft	12	1.6 ± 1.3	10	2.9 ± 1.6	13	2.2 ± 1.4
Autograft	3	2.6 ± 1.2	1	1.4 ± 1.0	2	2.2 ± 1.2
Synthetic hydroxyapatite	4	2.8 ± 1.7	7	3.2 ± 1.4	6	2.1 ± 1.3
Coralline hydroxyapatite	4	2.3 ± 1.4	4	3.2 ± 1.5	3	2.8 ± 1.2
Bioactive glass	4	2.3 ± 1.8	4	3.4 ± 1.8	4	2.6 ± 1.0
PepGen P-15	1	1.3 ± 1.9	1	2.4 ± 1.5	1	2.8 ± 1.2
GEM 21S	1	3.7 ± 0.2	1	4.4 ± 0.2	1	2.5 ± 0.3a

Comparisons obtained from different studies should be made with caution especially in the treatment outcomes like the defect fill. All observational and controlled studies on the surgical treatment of intrabony defects with open flap debridement, GTR, and bone grafts published during the period of 20 years were reviewed by Laurell and colleagues (1998).

Defect fill was found to be in positive correlation with initial defect depth followed by open flap debridement and grafting procedures, as stated by others. It is difficult to evaluate and tcompare improvements (effect sizes) across the studies quantitatively because of the differences in mean defect fill parallel initial differences in the mean pretreatment defect depth and is prone to confusion.

For the autogenous bone and DFDBA, histologic evidence suggests periodontal regeneration especially in intraosseous defects, in spite of the case reports which provide proof-of-principle of regeneration for the other bone grafting materials, such as PepGen P-15, (Yukna, Salinas & Carr, 2002), Bio-Oss Collagen (an organic bovine-derived bone matrix and collagen), (Hartman, Arnold, Mills, David & James, 2004) and GEM 21S.87. On the contrary, primary periodontal repair, which is characterized by the formation of a long junctional epithelium was supported by open flap debridement and alloplastic grafts.

7.2.2 Furcation Defects

It was strongly evident from the systematic reviews that Class II furcations show positive and predictable response to a combinatorial approach using GTR along with a bone grafting material (Murphy & Gunsolley, 2003). Bowers and colleagues with the help of the combination therapy found out that the highest rate of complete furcation closure in the mandibular molar Class II facial defects was achieved at the horizontal probing depths of 5 mm or less and interproximal bone at or above the level of the furcation entrance. Generally, the furcation defect should be less advanced in order to have a favorable prognosis for complete periodontal regeneration and furcation closure. Similarly, the earlier the treatment of furcation defects, the higher the rate of improving overall prognosis.

7.2.3 Factors impacting treatment outcome

A number of factors in relation to patient, periodontal defect, and surgical management affect the success of regenerative therapy that result in variability in clinical outcome following the regenerative therapy (Wang & Cooke, 2005). Surgical access and containment of the bone replacement graft were affected by defect morphology and root configuration and eventually influencing treatment outcome. In order to achieve an optimal regenerative outcome and long-term therapeutic success, patients are requested to be compliant with the recommended oral hygiene procedures and supportive periodontal maintenance programs. Lastly, smoking is prohibited as it adversely affects wound healing, which includes periodontal regeneration, and thereby resulting in the increased risk for periodontal breakdown at the end of the treatment (Johnson & Hill, 2004).

7.3 Conclusion

From the controlled clinical trials, it was strongly evident that bone replacement grafts were highly effective in treating the periodontal defects. Some of the selected bone replacement grafts such as DBM shows high potential in supporting the true periodontal regeneration like new bone, cementum and periodontal ligament as suggested by the histological studies of humans. Most of the present bone grafting materials act as passive matrices for the ingrowth of osteoprogenitor tissue and thus behave like osteoconductive materials (Scannapieco, 2010). It was possible to improve the regenerative outcomes to a great extent by integrating biomimetic and biologic components into the composite grafting materials. The upcoming bone grafting materials are expected to be constructed on innovative polymeric and ceramic platforms together with controlled biophysical properties that help in the targeted delivery of drugs, biologics and cells, eventually enhancing the degree as well as predictability of periodontal regeneration.

The literature data suggest that GBR procedures serve as a trustworthy method for bone augmentation in the treatment of patients affected by dental problems (Clementini, Morlupi, Agrestini & Ottria, 2011). From the data, it was clear that it would be impossible to achieve bone formation and dental implants placements without the help of GBR and thus it acts as a credible technique.

Although it is impossible to report the mean bone gain/resorption values, these findings were confirmed from the periodical analysis of ridge dimension changes as stated by the included studies. It is possible to have errors during the outcome evaluation due to the absence of sufficient standardized radiographs and well-performed radiographs or the immediate clinical measurements after implant placement. The study involves various types of measurements carried out at various times reporting either augmented bone resorption over time or the desired bone volume values or bone gain values. By analyzing the implants success rate laid in these augmented areas, data in relation to the success of GBR were obtained. It is necessary to determine the difference between the concepts of survival and success rate in order to examine the present systematic review. At times, implants which do not satisfy the requirements of success rate are considered to be ‘survived’. An implant’s survival rate is described as the existence of implant in the bone inside the mouth (Clementini et al., 2011). Survival rate was defined by Van Steenberghe (1999) as ‘the proportion of implants still in place in a specific time, even if they do not have any function’. A survived implant is described as an implant which does not function well or an implant along with a notable bone loss or with radiolucency signs and/or inflammation. This may be a limiting factor in determining the effectiveness of GBR because the success of GBR technique may not be related to high survival rate of implants, since there was a possibility for an implant to remain stable and osseointegrated even though the total regenerated tissue has been resorbed after the GBR procedure.

From the outcomes of the present review, it was suggested that GBR gives a high implant success rate and hence it is a fairly trustworthy ridge augmentation method. These studies involve considerable heterogeneity of the observed parameters, and hence meta-analysis was not carried out and the data were synthesized from only the evidence table. Since there was a greater variability in the success rate of dental implants (when reported) kept in GBR augmented sites, it may be considered that the surgeon’s ability is one of the important factors that affect the outcome of this technique.

From the analysis of available publications, it is identified that there is no adequate amount of universally established success criteria for implant inclusion (Clementini et al., 2011). Subsequently, the analysis of quality assessment of studies included in this review demonstrated that, out of eight, four studies are considered to be at high risk, three at modest risk and one at low risk. Such observation emphasis the need of providing well-defined studies to attain strong evidence based results. Though these considerations confine the results of this review, it focuses the importance of literatures studies that reported well-defined implant success rate with a minimum five years of post loading follow up including a controlled group as well.

In brief, it was concluded that GBR is a reliable technique as it allows the placement of implants in atrophic areas. However, studies with clear implant success criteria after prolonged follow up are yet to be developed (Clementini et al., 2011).

In brief, it was concluded that GBR is a trustworthy technique as it allow the placement of implants in atrophic areas.

7.4 Summary and Future outlook

Periodontitis is a kind of inflammation pathology that develops infection around the teeth and ultimately leads to tooth loss. So far, flap debridement and periodontal regenerative treatment with membranes and bone grafting materials have been used as an effective treatment to medicate peridontitis (Bottino et al., 2012). Currently, resorbable and non-reosorbable membrane coverage can be used to prevent the proliferation of defect in connective as well as epithelial tissues. However, this membranous coverage has many structural, mechanical and bio-functional limitations. From this, it is clearly understood that the ideal membrane for the treatment of periodontitis is yet to be developed. On the basis of graded-biomaterials approach, researchers have framed nanofibrous materials which is structurally, mechanically and biologically functional material that imitate the native extra cellular matrix, could achieve as the next generation of guided bone regeneration or membrane for periodontal tissue regeneration.

Though the aforementioned approach can be used to develop efficient polymeric biomaterials for periodontal tissues, however, the one which is based on graded approach for instance, hydorgels can produce greater flexibility in design and process as well. There are several hydrogel systems have been examined in the past for tissue engineering purposes. Such technology can also be used to perform periodontal tissue regeneration purposes (Zhu, 2012). Hydrogel with synergistic combination specific for periodontal tissue regeneration is highly beneficial in these applications. Such hydrogel system can be designed with specific chemical, physical and bio-chemical properties. For instance, physical, chemical and mechanical properties of hydrogel can be altered by either integrating or incorporating with other scaffold materials so as to control degradation trait, sustained drug release and protein /cell adhesion (Bottino et al, 2012). Besides, hydrogels tend to be more conductive to the passage of the cells within the mass of the scaffolds, to change the biological properties (bioactivity) and incorporate biomolecules (growth factors) to enhance or prevent cell adhesion and growth (Zhu, 2012).

Several researchers have made an attempt to explore the ways to control and enhance the mechanical properties of hydrogels; however, regulating mechanical strength during initial degradation is uncertain. This degradation will influence mechanical properties as well as the mechanical surface or bulk degradation. Through the generation of interpenetrating or nanocomposite hydrogel networks, one can enhance mechanical strength of hydrogels (Gaharwar, dammu, Canter, Wu & Schmidt, 2011). These elastomeric hydrogels can have advanced mechanical properties as compared to their polymer controls. More often, a fusion of several cross linking mechanisms can facilitate the development of mechanical strength, resilience and self-heating characteristics, which in turn acts as suitable parameters for successful periodontal tissue engineering. Development of such hydrogels with renowned chemists will definitely help to reach target values with respect to mechanical strength as well as rate of degradation (Raeber, Lutolf & Hubbell, 2005). In changing physiological condition, it is very critical to maintain or control the shrinking of hydrogel networks over time. In order to minimise this problem, it is essential to adopt or formulate hydrogels with biomolecules, salt or nanoparticles (Gaharwar et al., 2011).

Thus, in the development of hydrogel networks, maintenance of biocompatibility is highly imperative during degradation. As mentioned before, hydrogels and their by-product degradation must be biocompatible and the degradation trait must be tailored before being employed in periodontal regeneration process. As it is not possible to compare in vitro and in vivo, flexibility is needed in tailoring degradation time, if deemed necessary (Bakota, Aulisa, Galler & Hartgerink, 2011). Hydrogel system provides such flexibility in tailoring degradation time through polymer of networks that incorporates enzymatically degradable cross linkers within the polymer backbone.

Certain potential measures that are taken in the domain of periodontal regeneration have been found to be left untouched beyond a point. For instance, roughened titanium plates layered with growth factors are available in current market place. Such plates can enhance peri-implants connective tissue attachment. This development has enormous scope and value for innovation but no further studies has been performed on this basis. Moreover, the clinical trials and outcomes regarding new implants are not studied clearly so far. For example, the clinical studies regarding the effect of ‘Chochrane’ have not been properly studied and reviewed (Esposito et al., 2005). Thus, by using available resources, it is imperative to entirely characterise the chemical composition and surface coating implants that are clinically used (Lausmaa, 1996).

Future biosynthetic bone implants may diminish the process of autologous bone grafts. Researchers have showed much interest in the fusion of osteoconductive protein with osteoconductive carrier medium to aid timely release of a material scaffold for bone regeneration. In addition, progress in the area of tissue engineering with respect to the integration of physical, chemical, biological and engineering sciences will facilitate newer notion to repair, regenerate and restore tissues in its functional status (Nandi, Roy, Mukerrjee, Kundu, De & Basu, 2010). Such constructs are likely to include growth factors, evolving biological scaffolds and incorporation of mesenchymal stem cells. Finally, the development of ex vivo bioreactors with apt bio-chemical composition will be capable of providing tissue engineered constructs for direct use in the bones.

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