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Tissue engineering approaches for bone repair: Concepts and evidence
Injury, Int. J. Care Injured 42 (2011) 609–613
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Injury journal homepage: www.elsevier.com/locate/injury
Tissue engineering approaches for bone repair: Concepts and evidence Josh E. Schroeder, Rami Mosheiff * Orthopedic Surgery Department, Hadassah Hebrew University Medical Center, Jerusalem, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 17 March 2011
Over the last decades, the medical world has advanced dramatically in the understanding of fracture repair. The three components needed for fracture healing are osteoconduction, osteoinduction and osteogenesis. With newly designed scaffolds, ex vivo produced growth factors and isolated stem cells, most of the challenges of critical size bone defects have been resolved in vitro, and in some cases in animal models as well. However, there are still challenges needed to be overcome before these technologies can be fully converted from the bench to the bedside. These technological and biological advancements need to be converted to mass production of affordable products that can be used in every part of the world. Vascularity, full substation of scaffolds by native bone, and bio-safety are the three most critical steps to be challenged before reaching the clinical setting. ß 2011 Elsevier Ltd. All rights reserved.
Keywords: Scaffold Stem cells BMPs Osteoconduction Osteoinduction Bone regeneration PRP Bone defect
The clinical problem with critical size bone defects Bone is a very forgiving tissue. It withstands multiple insults and can regenerate itself into a healthy bone. One of the strengths of bone is its ability to build new osteones when the native structure of the bone is injured. The entire process of bone healing is beyond the scope of this paper; however, a basic understanding of the concepts of bone healing is essential for understanding the pathopysiology of critical bone defects. When bone is injured, a gap is created. This gap is filled with necrotic bone debris; blood from broken blood vessels and inflammatory cells, chemo mediated to the damage site by a set of signals, not yet fully understood.13,20 The healing process is margined by a combination of osteoconduction – a material acting as a scaffold for the new bone to grow into, and osteoprogenitor cells allowing osteoinduction – a combination of signals and cells getting rid of the necrotic bone and putting down the scaffolds for the newly generated bone.23 However, a gap beyond two and a half times the bone radius (termed as a critical size bone defect) remains a significant clinical problem.12,36,43 Such defects can be caused by blunt or penetrating trauma,38 surgical treatment of tumors12,17 or necrosis caused by radiation,74 or various chemical substances.96 Traditional therapeutic approaches in treating large bone defects include bone grafts16 and transplants90 (autologous – from the iliac bone or fibular grafts, allograft – fresh or frozen after
* Corresponding author. Tel.: +972 2 6776342; fax: +972 26778052. E-mail address: [email protected] (R. Mosheiff). 0020–1383/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.injury.2011.03.029
cleaning, or xeno-grafts). These grafts are supported by different fixtures, in hope that native bone will bridge the gaps and a boney fusion will occur. Other options include specialised implants that can serve as internal prosthesis (for example, tumour prosthesis after large bone resection, due to bone tumors91), shortening of the limb,44 with or without secondary distraction osteogenesis,5 bone transport methods (i.e. Ilizarov technique45), or in the unfortunate result, an amputation of the involved limb.42 The best classic solution for a large bone defect is the use of autologous bone graft.16 These grafts do not cause immunoreaction and contain the osteoconductive scaffolds, osteogenic cells and, if preserved, a viable blood supply (via connected arteries88). However, the use of bone grafts in clinical practice is limited due to high percentage of donor and recipient site complications.8 Vascularised grafts need a more extensive surgical team, with an over all, relatively low, artery patency.81 The use of allograft or xenografts prevents the problems involved with donor site morbidity, and allows larger substitutes. However, since they undergo sterilisation and purification, allografts and xenografts do not provide osteoinduction signals, and do not have living cells. In addition, they also present the potential risk of viral or bacterial infections and of an immune response of the host tissue after implantation.59 In addition, full integration of the graft is rare, ending at most cases with only bone substitution at the ends of the grafts, leading to late graft fracture, reported as high as 60% at 10 years.94 The use of large prosthesis for bone grafts is a well-known solution. They provide a medial to long term solution, and new coatings provide better osseous coating; however, there is no
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biological interface – once the material gives in (as a result of chronic ware) or loosening occurs, the patient is in need of further surgery. The rate of revision is high, with over 30% of the patients needing revision after 7 years.63 Distraction osteogenesis is a well-established technique, by means of which an acute shortening is preformed at the bone defect site, and a slow distraction process is started elsewhere in the bone. This technique takes advantage of the bone’s regeneration potential, avoiding the troubles associated with graft’s integration.45 However, it requires extreme patients’ compliance, since it takes a long period of time and it is often complicated with infections.4 In addition, this process calls for a bone that can regenerate; thus if the patient’s regeneration capacity is compromised, this technique cannot work. However, in animal models, the chemotherapy, commonly given for cancer, did not effect the cells contraindicating distraction osteogenesis.87 In summary, the current situation is suboptimal at best, and a new, better and wider substitution for large bone defects is needed. As stated above, in order to create new bone (osteogensis) a combination of osteoinduction and osteoconduction is required. One cannot function without the other. In the classic approaches, each part of this equation was separately tackled, however, in the advanced biochemical methods there is a combination of the two, with sophisticated materials integrating osteoconduction and induction allowing a surplus of osteogenesis. In the next sections, we will discuss the concepts and evidence in each of these elements. Osteoconduction – mind the gap The major problem with critical size bone defects is that cells cannot skip from one edge of the bone defect to the other; they need a solid platform on which to build bone and unite the fracture. Fracture ends that are distracted or in motion will not heal, and heterotrophic nonunion will develop.14 When a critical size bone defect develops, the body cannot heal itself and the gap needs to be surpassed, all the whilst keeping the limb in the proper length and angulations. Navarro et al.68 wisely divides materials used in the orthopaedic clinical usage into three generations according to their biofunction: in the first generation, there are bio-inert materials – these materials are used in most cases as fillers for the gap and in the modern scenario they are surrounded by materials that encourage the in-growth of bone. As a result, as in dental implants, a wide variety of surface treatments are applied to most metallic implants before their implantation.54 The second-generation group of materials include composites that are bioactive and biodegradable. These materials interact with the biological environment around the fracture to enhance the biological response and the tissue/surface bonding. In addition, these materials are bio absorbable and have the ability to undergo a progressive degradation whilst new tissue regenerates and heals. These materials can be metal,55 ceramics22 or polymers.19 The aim is to obtain a material with mechanical properties similar to those of bone, strong enough to allow operating room manipulation that can bond with bone. The material needs a degradation process that matches the healing period of the fracture or lesion. Too fast or too slow degradation pace is not acceptable in the clinical setting.58 One of the ongoing challenges is the bonding process,32 in which the scaffold unites with the adjacent bone. This is achieved by polymers binding at the interface between the organic matrix and the inorganic supplements.62 This surface modification is achieved by phosphorilation of proteins and peptides on the surface of the implant, as well as by modification of the insert to induce the mineralisation of the endplates and unite with the adjacent bone, via HA layers placed on the outer surface of the implant.56
Third-generation materials are designed to stimulate specific cellular responses at the molecular level.67 The implants are threedimensional structures that are biodegradable and biocompatible with degraded by-products that are non-cytotoxic. The degradation must happen at the same rate the tissue is repaired. These scaffolds hold a highly interconnected porous network, allowing integration of osteoblasts and osteoclasts. It has been shown that pores sized 100–350 mm are optimal for bone progenitor cells.79 The mechanical properties of the scaffold must be appropriate to regenerate bone tissue in load-bearing sites.68 Many materials have been tried for this group. One of the most commonly used is demineralised bone matrix.39,86 This provides a solid scaffold and includes bone morphogenetic proteins (BMPs). Other materials are nanocrystalline structures,84 organic–inorganic composites,69 nanofibres,60 biodegradable glass,26 microspheres,71 three-dimensional cross-linked hyaluran sponges (ACP) scaffolds,25 hydroxyapatite,40 glass micro-beads,95 hierarchically organised structures and hydrogels containing calcium and phosphate.92 These materials can be customised to any threedimensional scaffold needed,83 or press fitted to the defect to allow maximal surgeon ease.85 Despite these advances several main challenges remain. The introduction of blood vessels to the grafts is problematic, thus impairing the integration and migration of the cells to the scaffold site.33 In addition, the durability of these new composites has yet to be tested and designed in such a way that surgeons will be able to use them freely in a clinical setting. It is important to remember that most of these materials are still in the in vitro/animal model phase, and it will take 10–20 years before they are cleared for everyday use. However, despite these limitations the inclusion of engineers, chemists, physicists and biologists and the constant influence of surgeons can bring great advances, resulting in new and improved materials to our present day operating room. Osteoinduction – or signals and cells When a fracture occurs, a set of signals is triggered. These are both local signals and systemic ones; some of these signals are mediated by neuronal impulses,70 by the haematoma at the site of the fracture35 and by the trauma caused to the tissues surrounding the fracture.24 These signals can be divided into two interactive and interchangeable categories: inflammatory signals (i.e. IL-1, IL6, and TNF-a), and bone building signals (BMPs and WNTs).24 These factors mitigate the migration of phagocytotic cells to the area of the fracture, removing the necrotic tissue and propagating the in-growth of new blood vessels to the site of the fracture, thus providing nutrients and cells to the fracture site and starting the healing cascade.20 As stated above, if at the end of the healing process osteo-integration (of the new bone together with the native bone) is not achieved, even with the best type of scaffolds, the chances of long-term success are dismal.7 The addition of growth factors such as bone morphogenetic proteins and growth factors to scaffolds or to the area of the bone defects has proven to increase bone formation both in vitro and in animal models.11 However, when converting these studies to humans, the concentrations needed are higher and, at times, supraphysiologic, with possible related side effects and high costs (with a strong industry drive).3,28 Furthermore, most current clinical techniques for the use of bone growth factors result in fast release of the growth factor shortly after position, with only few clinical studies investigating the long-term release of these factors.46,48 An attractive approach for the addition of growth factors to increase bone regeneration is the addition of platelet rich plasma (PRP) to the fracture site.2 PRP has been shown to enhance osteoid
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formation both in vitro and in vivo (not just in animals but in humans as well).17,37 Since PRP is easy to create, and since it does not cause immune reaction, it can be considered a natural augmenter to bone formation.76 There are several limiting factors in the use of PRP. First of all, there is no precise knowledge of the component in PRP that enhances bone formation.6 Secondly, there are no case controlled studies regarding the effect of PRP on osteoinduction in humans, and the data are consisted mainly of case reports.27,89 Lastly, most of the work is done in the maxiofacial area, with little data regarding its effect in critical bone defects in the axial skeleton.29 Osteoprogenitor cells are necessary to replace the inserted scaffold and to create new bone tissue. These cells can come from the periostum at the fracture site, or from chemotaxis and blood vessels entering the haematoma.13,23 The cells that migrate are mesenchymal stem cells (MSC); these cells are the body’s reservoir for muscle, fat, cartilage and bone.73 Specific mechanical and biological stimulants cause the cells to differentiate into osteoblasts, which are the bone forming cells. However, in critical size bone defects the natural migration of osteoprogenitor cells does not suffice for fracture healing. In normal conditions MSCs are rare (one in 10 million cells).80 However, when a bone is broken, these cells, using special probing signals, roam in the blood and settle in the fracture site, differentiate into bone cells and start to construct the callus.15 The number of stem cells differs from person to person and is affected by age, sex and environmental factors (for example, it was found in our institution that smoking reduces the number of stem cells).10,21,64 These cells are found in higher concentration in the bone marrow, therefore the addition of bone marrow aspirates, mainly from the iliac crest, to problematic fracture sites (with or without a scaffold) has been practiced for the last 20 years.72 In the recent decades methods for the isolation of these cells have been developed. In a sterile, minimally invasive way, tens of millions of mesenchymal stem cells can be isolated and purified. These cells are aspirated from the bone marrow, purified, proliferated ex vivo, and added to the scaffolds or to the fracture site.9,77 Trials driving stem cells from the adipose tissue and directing them to the regeneration of bone are in progress. Comparative studies of these cells indicate both similarities and differences when compared to bone-marrow derived stem cells.57 More work is needed to evaluate the comparative differentiation potential of the two cell types and the optimal conditioning for clinical use.41 The ex vivo proliferation of cells poses many questions as methylation patterns of cells expanded ex vivo may change when they are removed from their native atmosphere, thus changing gene expression and the behaviour of the cells.82 In addition, levels of cleanliness matching that of an industrial ‘‘clean room’’ must be kept in order to prevent contamination of the cells. Tests to prevent malignant transformation of the cells must be conducted as well. Once all the materials are placed in the fracture site, they can easily be depleted by the blood circulation and a method is required to keep them in place. In addition, after debridement of a fracture site, the migration of adjacent soft tissues that might hamper the fracture healing must be prevented.1 To this end, a method of protected bone regeneration was developed using membranes that close off the fracture site and create a semipermeable ‘‘compartment’’ around the bone defect site.66 Membranes must have micropores that will allow vascular in-growth. The membranes can be composited from many materials – polytetrafliroethlen (ePTFE), polyglacin, polylactic acid, calcium sulphate and collagen.31 They can be biodegradable or not.47 The membranes can be rigid or flexible. Rigid membranes provide a stronger scaffold with increased bone in-growth in animal
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models.30 The use of these membranes is common in oral bone defects. In a meta-analysis, Parrish et al.75 demonstrates that the use of these membranes is better than open bone grafts, and that there is no significant difference if the membrane is biodegradable or not. Another advantage related to the use of membranes is the possibility to incorporate medications in high doses, without their leaking into the systemic circulation.18 This approach has lead to different coatings of membranes incorporating positive osteogenic signals. Vascularity of the scaffolds is critical. If blood vessels do not develop and the scaffold remains ischemic, the cells will die and no integration will take place.78 The use of growth factors, such as VEGF, PDGF and FGF is used to enhance vascularity and angiogenesis in the grafts.49,49,93 There have been studies looking at implanting grafts into environments rich in vascular supply (subcutaneous,52 intramuscular,53 or intraperitoneal sites51). However, in such implementation, formation of new vessels within the implanted bone happens at random patterns and the graft is not suitable for revascularisation in the clinical setting. Alternative approaches include the utilisation of a blood vessel located centrally in the graft or the application of an arteriovenous shunt that goes through the graft.34 A two-stage approach takes a different route61: in the first step, the scaffold is implanted and a haematoma is created around it (most likely resulting from the medical procedure of the scaffold’s insertion). On the third or fourth day, during the chronic inflammation phase, blood vessels and fibroblasts proliferate in the fibrin clot, thus forming a granulation tissue. Osteoprogenitor cells are injected into the scaffold at that time. This approach increases the chances that as a result of the injection, new blood vessels will invade into the haematoma, thereby guaranteeing a sufficient supply of oxygen and nutrients, and thus securing the survival of the implanted cells. However, the downside of such an approach is the increased risk of infection involved with the double procedure. The use of adipose derived stem cells might be advantageous with regards to the regeneration of blood vessels, as they were found to regenerate blood vessels at a higher rate when compared to bone marrow derived stem cells.50 The cost of the new science When all of these new technologies are put together in the clinical setting, one must take into account the price. With BMPs costing more than their weight in gold (or diamonds), and scaffolds that cost thousands of dollars per cubic centimetre, the solution to this dire problem must be made more accessible. There is no use to develop a technology that will not be of use where it is most needed – i.e. the developing world countries.65 A major challenge the medical and the research world must encounter is finding an accessible, simple and reasonably priced solution to the healing of critical size bone defects; otherwise surgeons might revert to the old solution presented by Dr. Illizarov, namely, using used bicycles’ pins for fixation and distraction osteogenesis.45 Summary Over the last decades we have advanced in many aspects of bone defects treatment. We have good understanding of the components involved in the healing of bone. We have strived forward in defining different components of bone regeneration and have achieved a good combination of biology and technology leading to solid and reproducible answers to the in vitro and animal in vivo problem of bone defects. However, there is still one more step to take – the human in vivo step. There are scant data with respect to this part of the question, and in the next few years this field must undergo a transition, giving clinicians tools to deal with
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these critical everyday problems. The solution will come from a collaborative work of engineers, chemists, biologist and surgeons who possess the social understanding that there has to be a limit to the cost that the patient (and the society) can bear for healing a fracture.
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Contents lists available at ScienceDirect
Injury journal homepage: www.elsevier.com/locate/injury
Tissue engineering approaches for bone repair: Concepts and evidence Josh E. Schroeder, Rami Mosheiff * Orthopedic Surgery Department, Hadassah Hebrew University Medical Center, Jerusalem, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 17 March 2011
Over the last decades, the medical world has advanced dramatically in the understanding of fracture repair. The three components needed for fracture healing are osteoconduction, osteoinduction and osteogenesis. With newly designed scaffolds, ex vivo produced growth factors and isolated stem cells, most of the challenges of critical size bone defects have been resolved in vitro, and in some cases in animal models as well. However, there are still challenges needed to be overcome before these technologies can be fully converted from the bench to the bedside. These technological and biological advancements need to be converted to mass production of affordable products that can be used in every part of the world. Vascularity, full substation of scaffolds by native bone, and bio-safety are the three most critical steps to be challenged before reaching the clinical setting. ß 2011 Elsevier Ltd. All rights reserved.
Keywords: Scaffold Stem cells BMPs Osteoconduction Osteoinduction Bone regeneration PRP Bone defect
The clinical problem with critical size bone defects Bone is a very forgiving tissue. It withstands multiple insults and can regenerate itself into a healthy bone. One of the strengths of bone is its ability to build new osteones when the native structure of the bone is injured. The entire process of bone healing is beyond the scope of this paper; however, a basic understanding of the concepts of bone healing is essential for understanding the pathopysiology of critical bone defects. When bone is injured, a gap is created. This gap is filled with necrotic bone debris; blood from broken blood vessels and inflammatory cells, chemo mediated to the damage site by a set of signals, not yet fully understood.13,20 The healing process is margined by a combination of osteoconduction – a material acting as a scaffold for the new bone to grow into, and osteoprogenitor cells allowing osteoinduction – a combination of signals and cells getting rid of the necrotic bone and putting down the scaffolds for the newly generated bone.23 However, a gap beyond two and a half times the bone radius (termed as a critical size bone defect) remains a significant clinical problem.12,36,43 Such defects can be caused by blunt or penetrating trauma,38 surgical treatment of tumors12,17 or necrosis caused by radiation,74 or various chemical substances.96 Traditional therapeutic approaches in treating large bone defects include bone grafts16 and transplants90 (autologous – from the iliac bone or fibular grafts, allograft – fresh or frozen after
* Corresponding author. Tel.: +972 2 6776342; fax: +972 26778052. E-mail address: [email protected] (R. Mosheiff). 0020–1383/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.injury.2011.03.029
cleaning, or xeno-grafts). These grafts are supported by different fixtures, in hope that native bone will bridge the gaps and a boney fusion will occur. Other options include specialised implants that can serve as internal prosthesis (for example, tumour prosthesis after large bone resection, due to bone tumors91), shortening of the limb,44 with or without secondary distraction osteogenesis,5 bone transport methods (i.e. Ilizarov technique45), or in the unfortunate result, an amputation of the involved limb.42 The best classic solution for a large bone defect is the use of autologous bone graft.16 These grafts do not cause immunoreaction and contain the osteoconductive scaffolds, osteogenic cells and, if preserved, a viable blood supply (via connected arteries88). However, the use of bone grafts in clinical practice is limited due to high percentage of donor and recipient site complications.8 Vascularised grafts need a more extensive surgical team, with an over all, relatively low, artery patency.81 The use of allograft or xenografts prevents the problems involved with donor site morbidity, and allows larger substitutes. However, since they undergo sterilisation and purification, allografts and xenografts do not provide osteoinduction signals, and do not have living cells. In addition, they also present the potential risk of viral or bacterial infections and of an immune response of the host tissue after implantation.59 In addition, full integration of the graft is rare, ending at most cases with only bone substitution at the ends of the grafts, leading to late graft fracture, reported as high as 60% at 10 years.94 The use of large prosthesis for bone grafts is a well-known solution. They provide a medial to long term solution, and new coatings provide better osseous coating; however, there is no
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biological interface – once the material gives in (as a result of chronic ware) or loosening occurs, the patient is in need of further surgery. The rate of revision is high, with over 30% of the patients needing revision after 7 years.63 Distraction osteogenesis is a well-established technique, by means of which an acute shortening is preformed at the bone defect site, and a slow distraction process is started elsewhere in the bone. This technique takes advantage of the bone’s regeneration potential, avoiding the troubles associated with graft’s integration.45 However, it requires extreme patients’ compliance, since it takes a long period of time and it is often complicated with infections.4 In addition, this process calls for a bone that can regenerate; thus if the patient’s regeneration capacity is compromised, this technique cannot work. However, in animal models, the chemotherapy, commonly given for cancer, did not effect the cells contraindicating distraction osteogenesis.87 In summary, the current situation is suboptimal at best, and a new, better and wider substitution for large bone defects is needed. As stated above, in order to create new bone (osteogensis) a combination of osteoinduction and osteoconduction is required. One cannot function without the other. In the classic approaches, each part of this equation was separately tackled, however, in the advanced biochemical methods there is a combination of the two, with sophisticated materials integrating osteoconduction and induction allowing a surplus of osteogenesis. In the next sections, we will discuss the concepts and evidence in each of these elements. Osteoconduction – mind the gap The major problem with critical size bone defects is that cells cannot skip from one edge of the bone defect to the other; they need a solid platform on which to build bone and unite the fracture. Fracture ends that are distracted or in motion will not heal, and heterotrophic nonunion will develop.14 When a critical size bone defect develops, the body cannot heal itself and the gap needs to be surpassed, all the whilst keeping the limb in the proper length and angulations. Navarro et al.68 wisely divides materials used in the orthopaedic clinical usage into three generations according to their biofunction: in the first generation, there are bio-inert materials – these materials are used in most cases as fillers for the gap and in the modern scenario they are surrounded by materials that encourage the in-growth of bone. As a result, as in dental implants, a wide variety of surface treatments are applied to most metallic implants before their implantation.54 The second-generation group of materials include composites that are bioactive and biodegradable. These materials interact with the biological environment around the fracture to enhance the biological response and the tissue/surface bonding. In addition, these materials are bio absorbable and have the ability to undergo a progressive degradation whilst new tissue regenerates and heals. These materials can be metal,55 ceramics22 or polymers.19 The aim is to obtain a material with mechanical properties similar to those of bone, strong enough to allow operating room manipulation that can bond with bone. The material needs a degradation process that matches the healing period of the fracture or lesion. Too fast or too slow degradation pace is not acceptable in the clinical setting.58 One of the ongoing challenges is the bonding process,32 in which the scaffold unites with the adjacent bone. This is achieved by polymers binding at the interface between the organic matrix and the inorganic supplements.62 This surface modification is achieved by phosphorilation of proteins and peptides on the surface of the implant, as well as by modification of the insert to induce the mineralisation of the endplates and unite with the adjacent bone, via HA layers placed on the outer surface of the implant.56
Third-generation materials are designed to stimulate specific cellular responses at the molecular level.67 The implants are threedimensional structures that are biodegradable and biocompatible with degraded by-products that are non-cytotoxic. The degradation must happen at the same rate the tissue is repaired. These scaffolds hold a highly interconnected porous network, allowing integration of osteoblasts and osteoclasts. It has been shown that pores sized 100–350 mm are optimal for bone progenitor cells.79 The mechanical properties of the scaffold must be appropriate to regenerate bone tissue in load-bearing sites.68 Many materials have been tried for this group. One of the most commonly used is demineralised bone matrix.39,86 This provides a solid scaffold and includes bone morphogenetic proteins (BMPs). Other materials are nanocrystalline structures,84 organic–inorganic composites,69 nanofibres,60 biodegradable glass,26 microspheres,71 three-dimensional cross-linked hyaluran sponges (ACP) scaffolds,25 hydroxyapatite,40 glass micro-beads,95 hierarchically organised structures and hydrogels containing calcium and phosphate.92 These materials can be customised to any threedimensional scaffold needed,83 or press fitted to the defect to allow maximal surgeon ease.85 Despite these advances several main challenges remain. The introduction of blood vessels to the grafts is problematic, thus impairing the integration and migration of the cells to the scaffold site.33 In addition, the durability of these new composites has yet to be tested and designed in such a way that surgeons will be able to use them freely in a clinical setting. It is important to remember that most of these materials are still in the in vitro/animal model phase, and it will take 10–20 years before they are cleared for everyday use. However, despite these limitations the inclusion of engineers, chemists, physicists and biologists and the constant influence of surgeons can bring great advances, resulting in new and improved materials to our present day operating room. Osteoinduction – or signals and cells When a fracture occurs, a set of signals is triggered. These are both local signals and systemic ones; some of these signals are mediated by neuronal impulses,70 by the haematoma at the site of the fracture35 and by the trauma caused to the tissues surrounding the fracture.24 These signals can be divided into two interactive and interchangeable categories: inflammatory signals (i.e. IL-1, IL6, and TNF-a), and bone building signals (BMPs and WNTs).24 These factors mitigate the migration of phagocytotic cells to the area of the fracture, removing the necrotic tissue and propagating the in-growth of new blood vessels to the site of the fracture, thus providing nutrients and cells to the fracture site and starting the healing cascade.20 As stated above, if at the end of the healing process osteo-integration (of the new bone together with the native bone) is not achieved, even with the best type of scaffolds, the chances of long-term success are dismal.7 The addition of growth factors such as bone morphogenetic proteins and growth factors to scaffolds or to the area of the bone defects has proven to increase bone formation both in vitro and in animal models.11 However, when converting these studies to humans, the concentrations needed are higher and, at times, supraphysiologic, with possible related side effects and high costs (with a strong industry drive).3,28 Furthermore, most current clinical techniques for the use of bone growth factors result in fast release of the growth factor shortly after position, with only few clinical studies investigating the long-term release of these factors.46,48 An attractive approach for the addition of growth factors to increase bone regeneration is the addition of platelet rich plasma (PRP) to the fracture site.2 PRP has been shown to enhance osteoid
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formation both in vitro and in vivo (not just in animals but in humans as well).17,37 Since PRP is easy to create, and since it does not cause immune reaction, it can be considered a natural augmenter to bone formation.76 There are several limiting factors in the use of PRP. First of all, there is no precise knowledge of the component in PRP that enhances bone formation.6 Secondly, there are no case controlled studies regarding the effect of PRP on osteoinduction in humans, and the data are consisted mainly of case reports.27,89 Lastly, most of the work is done in the maxiofacial area, with little data regarding its effect in critical bone defects in the axial skeleton.29 Osteoprogenitor cells are necessary to replace the inserted scaffold and to create new bone tissue. These cells can come from the periostum at the fracture site, or from chemotaxis and blood vessels entering the haematoma.13,23 The cells that migrate are mesenchymal stem cells (MSC); these cells are the body’s reservoir for muscle, fat, cartilage and bone.73 Specific mechanical and biological stimulants cause the cells to differentiate into osteoblasts, which are the bone forming cells. However, in critical size bone defects the natural migration of osteoprogenitor cells does not suffice for fracture healing. In normal conditions MSCs are rare (one in 10 million cells).80 However, when a bone is broken, these cells, using special probing signals, roam in the blood and settle in the fracture site, differentiate into bone cells and start to construct the callus.15 The number of stem cells differs from person to person and is affected by age, sex and environmental factors (for example, it was found in our institution that smoking reduces the number of stem cells).10,21,64 These cells are found in higher concentration in the bone marrow, therefore the addition of bone marrow aspirates, mainly from the iliac crest, to problematic fracture sites (with or without a scaffold) has been practiced for the last 20 years.72 In the recent decades methods for the isolation of these cells have been developed. In a sterile, minimally invasive way, tens of millions of mesenchymal stem cells can be isolated and purified. These cells are aspirated from the bone marrow, purified, proliferated ex vivo, and added to the scaffolds or to the fracture site.9,77 Trials driving stem cells from the adipose tissue and directing them to the regeneration of bone are in progress. Comparative studies of these cells indicate both similarities and differences when compared to bone-marrow derived stem cells.57 More work is needed to evaluate the comparative differentiation potential of the two cell types and the optimal conditioning for clinical use.41 The ex vivo proliferation of cells poses many questions as methylation patterns of cells expanded ex vivo may change when they are removed from their native atmosphere, thus changing gene expression and the behaviour of the cells.82 In addition, levels of cleanliness matching that of an industrial ‘‘clean room’’ must be kept in order to prevent contamination of the cells. Tests to prevent malignant transformation of the cells must be conducted as well. Once all the materials are placed in the fracture site, they can easily be depleted by the blood circulation and a method is required to keep them in place. In addition, after debridement of a fracture site, the migration of adjacent soft tissues that might hamper the fracture healing must be prevented.1 To this end, a method of protected bone regeneration was developed using membranes that close off the fracture site and create a semipermeable ‘‘compartment’’ around the bone defect site.66 Membranes must have micropores that will allow vascular in-growth. The membranes can be composited from many materials – polytetrafliroethlen (ePTFE), polyglacin, polylactic acid, calcium sulphate and collagen.31 They can be biodegradable or not.47 The membranes can be rigid or flexible. Rigid membranes provide a stronger scaffold with increased bone in-growth in animal
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models.30 The use of these membranes is common in oral bone defects. In a meta-analysis, Parrish et al.75 demonstrates that the use of these membranes is better than open bone grafts, and that there is no significant difference if the membrane is biodegradable or not. Another advantage related to the use of membranes is the possibility to incorporate medications in high doses, without their leaking into the systemic circulation.18 This approach has lead to different coatings of membranes incorporating positive osteogenic signals. Vascularity of the scaffolds is critical. If blood vessels do not develop and the scaffold remains ischemic, the cells will die and no integration will take place.78 The use of growth factors, such as VEGF, PDGF and FGF is used to enhance vascularity and angiogenesis in the grafts.49,49,93 There have been studies looking at implanting grafts into environments rich in vascular supply (subcutaneous,52 intramuscular,53 or intraperitoneal sites51). However, in such implementation, formation of new vessels within the implanted bone happens at random patterns and the graft is not suitable for revascularisation in the clinical setting. Alternative approaches include the utilisation of a blood vessel located centrally in the graft or the application of an arteriovenous shunt that goes through the graft.34 A two-stage approach takes a different route61: in the first step, the scaffold is implanted and a haematoma is created around it (most likely resulting from the medical procedure of the scaffold’s insertion). On the third or fourth day, during the chronic inflammation phase, blood vessels and fibroblasts proliferate in the fibrin clot, thus forming a granulation tissue. Osteoprogenitor cells are injected into the scaffold at that time. This approach increases the chances that as a result of the injection, new blood vessels will invade into the haematoma, thereby guaranteeing a sufficient supply of oxygen and nutrients, and thus securing the survival of the implanted cells. However, the downside of such an approach is the increased risk of infection involved with the double procedure. The use of adipose derived stem cells might be advantageous with regards to the regeneration of blood vessels, as they were found to regenerate blood vessels at a higher rate when compared to bone marrow derived stem cells.50 The cost of the new science When all of these new technologies are put together in the clinical setting, one must take into account the price. With BMPs costing more than their weight in gold (or diamonds), and scaffolds that cost thousands of dollars per cubic centimetre, the solution to this dire problem must be made more accessible. There is no use to develop a technology that will not be of use where it is most needed – i.e. the developing world countries.65 A major challenge the medical and the research world must encounter is finding an accessible, simple and reasonably priced solution to the healing of critical size bone defects; otherwise surgeons might revert to the old solution presented by Dr. Illizarov, namely, using used bicycles’ pins for fixation and distraction osteogenesis.45 Summary Over the last decades we have advanced in many aspects of bone defects treatment. We have good understanding of the components involved in the healing of bone. We have strived forward in defining different components of bone regeneration and have achieved a good combination of biology and technology leading to solid and reproducible answers to the in vitro and animal in vivo problem of bone defects. However, there is still one more step to take – the human in vivo step. There are scant data with respect to this part of the question, and in the next few years this field must undergo a transition, giving clinicians tools to deal with
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these critical everyday problems. The solution will come from a collaborative work of engineers, chemists, biologist and surgeons who possess the social understanding that there has to be a limit to the cost that the patient (and the society) can bear for healing a fracture.
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