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Treatment of large bone defects represents a great challenge in orthopedic and craniomaxillofacial surgery. Although there are several methods for bone reconstruction, they all have specific indications and limitations. The concept of using barrier membranes for restoration of bone defects has been developed in an effort to simplify their treatment by offering a sinlge-staged procedure. Research on this field of bone regeneration is ongoing, with evidence being mainly attained from preclinical studies. The purpose of this review is to summarize the current experimental and clinical evidence on the use of barrier membranes for restoration of bone defects in maxillofacial and orthopedic surgery. Although there are a few promising preliminary human studies, before clinical applications can be recommended, future research should aim to establish the 'ideal' barrier membrane and delineate the need for additional bone grafting materials aiming to 'mimic' or even accelerate the normal process of bone formation. Reproducible results and long-term observations with barrier membranes in animal studies, and particularly in large animal models, are required as well as well-designed clinical studies to evaluate their safety, efficacy and cost-effectiveness.
Treatment of large bone defects represents a great challenge, as bone regeneration is required in large quantity and may be beyond the potential for self-healing. Large bone defects include segmental or large cortical defects created by trauma, infection, tumor resection, aseptic loosening around implants and skeletal abnormalities [1,2]. Critical size defect (CSD) is defined as the defect with the minimum length that cannot be spontaneously bridged leading to non-union [2,3]. Such defects are generally accepted to be ≥ 1.5 to 2 times the diameter of the long bone diaphysis, but they vary according to the host and the bone .
Although many methods for bone reconstruction exist, they all have specific indications and limitations. Established methods are distraction osteogenesis and bone transport, or bone grafting, including autologous bone grafts, bone marrow aspirate, allografts, bone substitutes or growth factors [4-8]. Furthermore, the concept of an induced-membrane represents another strategy for bone regeneration and particularly in cases of large bone defects secondary to trauma, infection or tumor excision. This method involves a two-stage procedure, where a 'biological' membrane is induced as a foreign body response after application of a cement spacer at the first stage, acting as a 'chamber' for the insertion of autologous bone-graft at the second stage [9-11]. It has been shown that this induced membrane possesses osteoinductive, osteogenic and angiogenic properties and several clinical studies have demonstrated satisfactory results [9,12]. Finally, the concept of Guided Bone Regeneration (GBR) using a bioabsorbable or non-resorbable membrane that acts as a barrier to prevent soft-tissue invasion into the defect and forms a 'chamber' to 'guide' the bone regeneration process [13-15] is also used for bone reconstruction.
Historically, the concept of GBR has been used in experimental reconstructive surgery since the mid-1950s, for spinal fusion  and maxillofacial reconstruction [17,18]. The initial hypothesis was that different cellular components in the tissue have varying rates of migration into a wound area during healing and that a mechanical hindrance would exclude the invasion of inhibiting substances, such as fibroblasts . Preliminary studies showed that the use of a non-resorbable membrane as a mechanical barrier resulted in complete healing of the bone defect in vivo , and collagen membranes prevented the apical migration of epithelium and supported new connective tissue attachment and tissue regeneration . The regeneration process occurring within the barrier membrane involves angiogenesis and migration of osteogenic cells from the periphery towards the center to create a well-vascularized granulation tissue. Initial organization of the blood clot is followed by vascular ingrowth and woven bone deposition, subsequent lamellar bone formation and finally remodeling, resembling bone growth [22,23]. When ingrowth of bone marrow into the bone defect was hindered or delayed, regeneration of mineralized bone was also delayed . However, in large defects, bone formation occurs only to the marginal stable zone with a central zone of disorganized loose connective tissue, and, therefore, additional use of bone-graft materials is required in these cases, with the graft acting as a scaffold for osteoconduction and as a source of osteogenic and osteoinductive substances for lamellar bone formation .
Although different non-resorbable and bioresorbable barrier membranes have been developed and their use has been extensively investigated, research is ongoing to develop the 'ideal' membrane for clinical applications. The basic characteristics of these membranes are biocompatibility, cell-occlusiveness, space-making, tissue integration, and clinical manageability [15,25].
Non-resorbable membranes and especially expanded-polytetrafuoroethylene (e-PTFE, Teflon) have been extensively studied . They are biocompatible and maintain their structural integrity during implantation. They have superior space-maintaining properties and capacity for cell occlusion than degradable membranes, as the latter tend to collapse depending on the size of the defect . Other non-resorbable membranes are titanium reinforced ePTFE, high-density-PTFE, or titanium mesh mainly used in oral and maxillofacial surgery  (Table (Table1).1). Semipermeable ePTFE is more effective than the high-density ePTFE with respect to bone regeneration . For bone regeneration of large segmental bone defects, the cylindrical titanium mesh cage has been used as a scaffold with satisfactory preliminary results . However, a second surgical procedure is required for removal, which represents a limitation and involves a potential risk to the newly regenerated tissues . Finally, membrane exposure is frequent, increasing the risk of secondary infection [31,32].
Bioresorbable membranes have been developed to avoid the need for surgical removal. Such membranes have been extensively studied, mainly in animals but also in humans in maxillofacial, regenerative periodontal, and neuro-surgery [14,33-38]. Recently, commercially available bioresorbable membranes have also been used for reconstruction of long bone defects in the clinical setting. It has been shown that they enhance bone healing, especially in cases with bone defects > 4 to 5 cm or with significant associated soft-tissue loss, where autologous bone grafting alone is not recommended due to risk of resorption , and they also secure the grafting material . There are two broad categories of bioresorbable membranes: the natural and the synthetic membranes. Natural membranes are made of collagen or chitosan, whereas synthetic products are made of aliphatic polyesters, primarily poly(L-lactide) (PLLA) and poly(L-lactide-co-glycolide) (PLGA) co-polymers . Overall, their advantages are: 1) they allow for a single-step procedure, 2) the shape and volume of the regenerated bone can be predefined-prefabricated, 3) they are radiolucent allowing imaging, and 4) their bioresorption eliminates potential effects of stress shielding of the regenerated bone. Conversely, there is variability and lack of control over the rate of membrane resorption, which is influenced by factors such as the local pH and material composition. A summary of the main characteristics, advantages and disadvantages of the different bioresorbable membranes is presented in Table Table11[13,21,31,38,40-59]. Currently, mainly PLLA membranes are available for clinical use in orthopedic surgery; whereas PLLA, collagen and ePTFE membranes are used for GBR in maxillofacial, dental and neuro- surgery.
Although a number of barrier membranes are already being used in clinical practice, novel membranes have been developed in an effort to overcome the limitations of the currently used membranes. Such novel membranes include alginate membranes, new degradable co-polymers, hybrid or nanofibrous membranes, as well as amniotic membranes. They are summarized in Table Table11[60-75]. Ongoing research is evaluating these novel membranes, aiming to establish an 'ideal' membrane for bone regeneration with optimized characteristics in terms of biocompatibility, space-making, tissue integration and clinical manageability for maximum clinical efficacy and safety.
The pore size of the barrier membrane is very important in order to prevent excessive penetration of fibrous tissue into the bone defect (soft tissue ingrowth) but to allow neovascularization and bone formation. Differences in the intensity of bone regeneration were observed depending on the pore size . Pores in excess of 100 μm are required for the rapid penetration of highly vascular connective tissue, and small pores tend to become filled with more avascular tissue , as they are inadequate for penetration of capillaries . A pore size of 50 to 100 μm allows bone ingrowth, but size greater than 150 μm is required for osteon formation [79,80]. A recent animal study showed that macroporous membranes facilitated greater bone regeneration compared to microporous membranes and prevented significant soft-tissue ingrowth . Further research should be directed to identify the critical pore size, since an increase in pore size may result in decreased mechanical properties. A multilayer scaffold has been suggested to achieve suitable mechanical properties and porosity and mimic the structure of cancellous and cortical bone . In addition to the porosity, the tri-dimensional topography of the membrane with interconnecting pores and channels is also important, as it can alter the cell occlusion properties and the biologic response of different cell types to the membrane .
Although barrier membranes are used to prevent soft-tissue invasion, a thin layer of soft-tissue ingrowth (up to 1 mm thickness) can be formed under the membrane, overlying the regenerated bone [84-86]. This may be secondary to shrinkage of the initial blood clot under the membrane, entrapment of air or membrane micromovements. Currently, it is not known if this soft-tissue layer under the membrane undergoes mineralization if left for a long period. Some studies reported this tissue-layer was a periosteum-like tissue, and others reported it to be fibrous tissue  but its clinical implications are unknown.
It is known that micromovements between bone and any implanted material prevent bone formation, resulting in the development of fibrous tissue [87,88]. Adequate stability and minimal stress are required to allow the early tissue that infiltrates through the pores to differentiate into bone by direct or appositional bone formation . Bone formation can occur within porous materials even with limited initial movement provided the site is highly vascular and local inflammatory reaction is minimal . New vascular network formation, which is a prerequisite for bone formation, is also highly sensitive to mechanical conditions with delayed mechanical loading significantly enhancing bone formation and stimulating vascular remodelling by increasing the number of large vessels and decreasing the number of small vessels . Therefore, optimal stability should be provided in terms of the attachment of the membrane itself, since most bioresorbable membranes are flexible and they cannot be applied without additional fixation as well as the type of fixation of the bone defect . To maximize stability of the membrane, the use of membrane-fixing pins has been suggested. It has been observed that bone formation is significantly enhanced when the resorbable membrane is tightly attached and immobilized to the bone surface . Regarding the effect of the type of additional fixation to the process of bone formation, it is known that intermediate tissues, such as fibrous tissue, cartilage and woven bone, precede final bone formation, with the mechanical loading affecting the regeneration process and different stress distribution favoring or inhibiting differentiation of particular tissue phenotypes . High shear strain and fluid flows stimulate fibrous tissue formation, whereas lower levels stimulate formation of cartilage, and even lower levels favor ossification. It has been demonstrated in vivo that there is more rapid and more organized new bone formation in rigidly fixed defects with plate osteosynthesis, covered with a resorbable collagen membrane, compared to non-rigidly fixed defects .
As research on the field of bone regeneration is ongoing and the evidence is expanding, we aimed to summarize the current experimental and clinical research on the use of barrier membranes for restoration of bone defects and focus on maxillofacial and orthopedic applications. We searched the PubMed Medline and Ovid Medline databases, from 1991 to 2011, to retrieve all relevant articles reporting on the use of absorbable and/or non-absorbable membranes for bone regeneration in animal and clinical studies. Different combinations of searching terms were used including: membrane/bone regeneration/long bone/bone defect/segmental bone defect/segmental mandibular defect/mandibular defect. The search was restricted to studies published in English. We analyzed all preclinical studies using established animal models to evaluate barrier membranes for bone regeneration of segmental, large and critical-sized mandibular or long-bone defects, in which bone regeneration was documented and assessed using radiological or biomechanical and/or histological analysis. Regarding the clinical studies, all papers reporting on the clinical use of barrier membranes were analyzed.
Tables Tables22 to to55 summarize the preclinical studies with non-absorbable or bioabsorbable membranes. There were 23 animal studies reporting on the use of membranes in maxillo-facial surgery for reconstruction of segmental or critical mandibular defects using small or large animal models (Table (Table2,2, 15 studies [31,34,54,63,64,94-103] and Table Table3,3, 8 studies [22,104-110], respectively). Overall, the membrane-treated groups showed improved bone formation within the mandibular defects compared to the non-treated animals [22,96,98]. Differences in the rate of bone regeneration and the inflammatory response in the surrounding soft tissues were observed with different types of membranes [31,97,100].
A total of 27 animal studies reported on the use of membranes for reconstruction of long bone defects. There were 21 studies using a small animal model (Table (Table4)4) [55,60,62,76,82,111-126], and only six studies using a large animal model (Table (Table5)5) [127-132]. As in maxillofacial animal studies, superior bone healing has also been observed in long bones treated with a barrier-membrane compared to the non-treated defects using bioabsorbable as well as non-resorbable membranes [111,117,118,121]. Bone defects treated with improved bilayer membranes displayed better regeneration of cortical bone tissue , whereas novel composite membranes displayed affluent neovascularization and bone formation with little fibrous tissue formation . The differences in chemical composition of the polylactide membranes did not seem to have an evident effect on bone healing in a small animal model , but different pore sizes resulted in differences in the intensity of the bone regeneration process . Large animal studies also showed promising results for restoration of long bone defects but only when combined with additional bone grafting material [131,132]. When two concentric perforated membranes (the tube-in-tube implant) were used in combination with cancellous bone graft in segmental diaphyseal defects, a 'neocortex' was reconstituted with well-defined thickness .
Tables Tables66 and and77 summarize the clinical studies, in which absorbable membranes were used for bone regeneration of the mandible and the long bones, respectively. The absorbable membranes used were either experimental materials [57,133], similar to the ones used in the animal studies, or commercially available material manufactured for other purposes [13,134].
There are only three studies in humans where bioabsorbable membranes have been used for reconstruction of segmental or large mandibular bone defects using bioresorbable PLLA barrier membrane (mesh) in combination with autologous bone graft (Table (Table6)6) [33,57,133]. The majority of the bone defects were secondary to benign or malignant tumors of the mandible, but other causes included infection, alveolar atrophy and trauma. Overall, the preliminary clinical results were satisfactory (rated as excellent and good in 56.5% and 27.4%, respectively). Radiologically, a certain degree of bone absorption was noted in more than half of the cases; nevertheless, only in one case was the absorption significant (up to 30%).
Finally, regarding the use of bioabsorbable membranes in long bone defects, there are only two clinical studies reporting on the clinical results in a total of 16 patients (Table (Table7)7) [13,134]. Long bone defects were mainly posttraumatic, but there were also a few cases of osteomyelitis and benign tumor resection. The bioresorbable PLLA synthetic membrane used was used in combination with autologous cancellous bone graft or bone marrow, and long bone fixation. Preliminary results showed healing of the defects and satisfactory function in all cases, except one which required further intervention.
Barrier membranes are among the most widely studied scaffolds for tissue regeneration, including bone, and the choice of type of membrane depends largely on the required duration of membrane function . Regarding bone regeneration, their use is mainly indicated for bone regeneration in sites where limited mechanical loading exists, such as in cranial, oral and maxillofacial applications. Even though there is extensive research on barrier membranes in animals, human studies are still few. Therefore, the most reliable current evidence originates mainly from studies in animals of higher phylogenetic scale which are still limited in number. Findings from the experimental setting indicate that GBR follows the same course of steps regardless of the animal. Bone quality though is highly dependent on the species (evolution hierarchy), bone healing potential (age, general nutritional status), the membrane used, local conditions (vascularity, embryological origin of bone) and load-sharing pattern of the fixation method; and, therefore, the results and the potential clinical use should be interpreted with caution [2,13,33,76,123,130].
According to the preliminary clinical reports, the time period for complete regeneration of bone in the mandible is three months, whereas long bones require more than two times the same period (seven months) [33,57]. This is most likely to be attributed to the greater vascularity of the mandible and the surrounding soft tissues as well as to the different mechanical environment and less stress-shielding of the fixation method used. Furthermore, it may also be explained by the different pathways of bone formation during the regeneration process due to the different embryological origin of the mandible (intramembranous ossification) compared to long bones (endochondral ossification) . Considering these differences, the 'ideal' barrier membrane may be different for maxillofacial and orthopedic applications. For example, in the case of long bone defects, the 'ideal' membrane may require improved mechanical properties, a prolonged degradation period in the case of an absorbable membrane, and even different membrane porosity to allow vascular ingrowth from the surrounding soft tissues to optimize bone formation within the defect.
Despite the fact that experimental evidence is well established and preliminary results from clinical studies are encouraging, there are still several points which prevent the safe and wide use of bioabsorbable membranes in humans. Healing potential in humans is different from that of animals and it occurs with various speeds in different bones (for example, mandible versus tibia), mainly due to the difference in vascularity and/or embryological origin. Therefore, the size of the segmental defect, able to be bridged using membranes, is not yet defined in humans . Additionally, the load-bearing of different bones varies widely. Even if the bone gap may be successfully bridged by the regenerated bone, more evidence is required regarding the time it will be structurally mature to cover the functional requirements. Since load-bearing is vital for the formation and progression of bone formation, the load sharing capacity of the fixation method is of utmost importance. There is no information yet on how the new bone will develop and mature in various types of fixation methods, that is, which may be considered the optimal fixation for bone regeneration in humans.
Other major parameters affecting the efficacy of bone regeneration are the characteristics of the membranes, such as composition, thickness, porosity, and perforation size [13,132]. These variables are yet to be defined in humans, because they may act in conjunction with the healing potential of each bone and may be used to optimize bone regeneration in bones with low healing potential or with a deficient local environment.
Bioresorbable membranes are currently being used mainly for bone regeneration in oral and maxillofacial surgery in humans. However, their use in various orthopedic conditions also represents a field of interest, especially since the number of revision surgeries [136,137] and limb salvage procedures is increasing [138,139]. For example, such membranes can be shaped as tubular chambers, thus preserving the continuity of the diaphysis for the repair of large diaphyseal bone defects . By forming a 'tube-in-tube implant' using two concentric perforated membranes in combination with cancellous bone-graft, the reconstitution of the 'neocortex' with well-defined thickness was possible for the treatment of segmental diaphyseal defects in sheep tibiae . Barrier membranes can also help to prevent significant absorption of the bone graft which is estimated to be up to 40% to 50% at four weeks  and seems to be due to absorption of bone that is not mechanically functioning . As these membranes are radiolucent, they allow assessment of bone formation with conventional radiographs, CT or MRI , which is important for monitoring the regeneration process.
The evidence on the efficacy for cortical perforation (decortication) during GBR procedures in an effort to enhance bone formation remains controversial . Studies have shown that cortical perforations increase the blood supply, facilitate angiogenesis, and allow access for progenitor cells from the bone marrow into the 'chamber'  whereas other studies showed that bone formation occurred from a non-injured cortical bone surface and that perforations were not required as they did not increase bone formation [59,81]. However, since there are no relevant human clinical studies and the relevant animal studies refer to mandibular defects, where local vascularity is superior to long bones, recommendations for additional bone decortication cannot be made for orthopedic GBR applications .
Finally, barrier membranes can be used in combination with bone grafting to augment osseointegration of orthopedic implants in the case of bone defects . 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 [144,145], as the preliminary results are encouraging.
The interest in accelerating bone formation has led researchers to combine the membrane technique with osteoinductive or growth factors. Although the concept of additional biological enhancement of bone formation using growth factors that enhance proliferation, chemotaxis, and differentiation of osteogenic cells seems promising, results are often controversial. In a study evaluating the long-term outcome of oral implants 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), no statistically significant differences were observed regarding the clinical and radiological outcomes . On the contrary, numerous in vivo and in vitro studies have demonstrated improved bone formation when barrier membranes are loaded with platelet-derived growth factor (PDGF-BB) , basic fibroblast growth factor (FGF2) , and rhBMP-2 [99,146,149].
Controversial evidence may be secondary to insufficiency in maintaining therapeutic concentrations of growth factors within bone defects due to rapid clearance and use of different delivery methods with supraphysiological non-standarized doses to obtain therapeutic efficacy . Furthermore, current research usually evaluates one or a combination of two growth factors, which does not reflect the complex physiological process of bone formation. Research is ongoing to develop novel membranes and scaffolds with improved growth factor delivery systems to accelerate bone regeneration of critically-sized segmental bone defects with promising preliminary results . Moreover, with a controlled spatiotemporal delivery of growth factors, adequate local protein concentrations can be improved and maintained for optimal regenerative efficacy, avoiding the currently used supraphysiologic doses and the concomitant adverse effects . Finally, the optimal 'combination' of growth factors to be delivered has also to be established.
Aiming to maximize or accelerate bone formation, supplementary strategies have been investigated in combination with barrier membranes and grafting. The potential use of low-level laser therapy (LLLT) has been evaluated as an adjunct for the regeneration of long bone defects in animal studies with positive results [114,152]. Supplementary treatment with hyperbaric oxygen has also shown synergistic regenerative effects in the past . Additionally, preliminary results have shown that systemic administration of synthetic salmon calcitonin accelerated bone regeneration of the defects .
Research is ongoing to evaluate other methods to enhance bone regeneration, such as local administration of parathyroid hormone (PTH(1-34))  and other growth factors  with promising preliminary results. Moreover, methods to optimize surface microtopography of the membranes have also been investigated to enhance bone formation at the cellular and molecular level . Finally, in the future, improved barrier membranes can be used as part of the bone-tissue engineering approach combined with osteoprogenitor cells and/or osteopromotive factors or even gene therapy, aiming to produce improved composite grafts . Preliminary research is promising. For example, a novel three-dimensional porous polymer poly(ε-caprolactone) (PCL) scaffold coated with adeno-associated virus encoding BMP2 using both ex vivo or in vivo gene therapy, led to increased bone ingrowth with increased mechanical properties in a rat femoral defect model .
The concept of barrier membranes for restoration of large bone defects has been developed in an effort to simplify their treatment by offering a sinlge-staged procedure and to overcome the limitations of current bone regeneration strategies. Research in this field is ongoing, with evidence being mainly gained from preclinical studies. Preliminary human studies have also shown promising results in maxillofacial, oral and orthopedic surgery. Nevertheless, before clinical applications can be recommended, future research should aim to generate and establish the 'ideal' barrier membrane. The additional use of bone-grafting materials within the membrane to fill the defect should also be evaluated, aiming to 'mimic' or even accelerate the normal process of bone formation. Finally, reproducible results and long-term observations with certified barrier membranes in animal models are required, and especially in large animal long bone defect models, as well as well-designed clinical studies to evaluate their safety, efficacy and cost-effectiveness.
ABG: autologous bone graft; ADM: acellular dermal matrix; BAMs: bioabsorbable membranes; BBM: bovine bone marrow; BCM: bovine collagen membrane; BG: bone graft; BMP: bone morphogenetic protein; BP: bovine pericardium; CAF: calcium alginate film; CaP: calcium phosphate; CM: collagen membrane; CCM: cross-linked collagen membrane; CSD: critical size defect; CT: computed tomography; DPPA: diphenylphosphorylazide; e-PTFE: expanded polytetrafluoroethylene; FGF: fibroblast growth factor; GBR: guided bone regeneration; GDF: growth differentiation factor; GTR: guided tissue regeneration; HA: hydroxyapatite; HFL: human fascia lata; HFT: human fascia temporalis; HP: human pericardium; ICBG: iliac crest bone graft; IMN: intramedullary nailing; MRI: magnetic resonance imaging; muCT: micro-computer tomography; nHA: nano-HA; PCL: poly(ε-caprolactone); PDLLCL: poly(dl-lactide-epsilon-caprolactone); PDGF: platelet-derived growth factor; PDTE: poly desaminotyrosyl-tyrosine-ethyl ester; PES: polyethersulfone; PLCL: poly lactide-co-ε-caprolactone; PLGC: poly L-lactide-co-glycolide-coepsilon- caprolactone; PLLA: poly(L-lactide) acid; PLGA: poly(L-lactide)-co-glycolide acid; PRP: platelet-rich plasma; rh-BMP: recombinant human BMP; RIA: Ria/Irrigator/Aspirator; SIS: small intestine submucosa; TCP: tricalcium phosphate.
The authors declare that they have no competing interests.
RD and GIM contributed in the preparation of this manuscript in terms of literature review and writing-up. GMC and PVG contributed in the writing of specific sections of the manuscript and in revising it critically for important intellectual content. All authors read and have given final approval of the final manuscript.
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