Abstract
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Long bone non-unions represent a serious clinical and socioeconomical problem due to the prolonged episodes, frequent sequelae, and variable treatment effectiveness.
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Bone grafts, classically involving the autologous iliac crest graft as the ‘gold standard’ bone graft, enhance bone regeneration and fracture healing incorporating osteoconductive and/or osteoinductive/osteogenic capacity to the non-union under treatment.
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Structural alternatives to autologous bone grafts include allografts and bone substitutes, expanding the available stock but loosing biological properties associated with cells in the graft.
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Biological alternatives to autologous bone grafts include bone marrow concentration from iliac crest aspiration, bone marrow aspiration from reaming of the diaphyseal medullary canal in the long bones, and isolated, expanded mesenchymal stem cells under investigation.
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When the combination with natural and synthetic bone substitutes allows for larger volumes of structural grafts, the enhancement of the biological regenerative properties through the incorporation of cells and their secretoma permits to foresee new bone grafting solutions and techniques.
Introduction: non-union definition and epidemiology
Delayed union and non-union fractures are well-defined medical conditions that represent the inability of bone to heal without further intervention, which is usually identified based on radiological, clinical, and temporal criteria. Classically, these were considered ‘pseudarthrosis’ due to the abnormal motion identified in severe non-unions in long bones, developing a flexible fibrotic union or even a true neo-articulation at the fracture site. Patients resent from pain at the fracture site, inability to bear weight, and associated disability, even if significant motion perceived at examination is today rare (1).
Consolidation timing varies from bone to bone and from fracture to fracture. Timing to heal fractures is important not only for the patient and surgeon but also for the definition. Delayed union has been interpreted as the absence of bone bridging and the presence of fracture line after 3–6 months, becoming by definition a non-union 9 months after the original fracture with lack of progressive signs of bone healing over three consecutive months (2, 3, 4). However, long bone fractures with more than 6 months without consolidation are frequently considered non-unions, in part due to patient demands, frequently prompting the indication for surgical intervention (5, 6, 7). A well-accepted pragmatic definition of non-union is a fracture that, in the opinion of the treating physician, has no possibility of healing without additional intervention (8). In case a fracture fails to heal after a first (or several) non-union therapeutic procedure(s), the complications and complexity on the patient and the fracture, the sequelae and the already high socioeconomic burden (9) may even increase, and the non-union can be defined as recalcitrant (10).
Infected or uninfected non-unions
Not all non-unions are equal. A general classification differentiating septic and aseptic, or more generally speaking, infected or uninfected non-unions may drive the treatment, as incorporating hardware and grafts in the infected bone may not heal the infection, and bone healing may not occur. Effective debridement, hardware removal, and pharmacological treatment of infected non-unions is usually required before attempts to enhance bone healing are successful.
Infected non-unions can be estimated in 1–2 out of 10. The fracture healing cascade may be disturbed or disrupted in those cases, ultimately leading to insufficient consolidation of the fracture. However, many complex long bone fractures were originally open fractures, and initial contamination is not rare. While acute treatment may avoid or limit clinically conspicuous, highly inflammatory cases of fracture infection or even osteomyelitis, a low-grade infection with low-pathogenic bacteria in the absence of flogotic clinical signs may be present. Diagnosis can be difficult due to the clinically subtle signs (lack of/discrete hyperthermia and redness), no/small increase in infection parameters, and lack of wound secretion. The following factors (3Ps and 3Ss) predispose to the development of infected non-union:
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Presence of an open fracture with a higher degree of soft tissue damage
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Prolonged interval between trauma event and surgical debridement (>6 h)
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Polytraumatized patient
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Serious comorbidities
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Selection of implants
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Surgical technique
Atrophic or hypertrophic non-unions
A non-union supposes the failure of attempts to complete fracture healing. The amount of bone reaction, from the fractured bone ends to form the callus, defines the potential treatment and requirements of a non-union, differentiating atrophic and hypertrophic. However, this separation is not always that clear. Radiological signs of intramedullary canal obturation, maintained gap between bone endings, and insufficient callus formation can be seen in the common final stage of any non-union, concluding that the differences between atrophic (less bone reaction) or hypertrophic (more bone reaction) are somewhat artificial, and different degrees of bone reaction in a continuum, between more atrophic to more hypertrophic the non-union, may be closer to reality.
Atrophic non-union
The main reason behind an atrophic non-union is related to impaired vascularity. Bone devascularization can be due to injury of adjacent vessels, soft tissue stripping and/or fragment denudation during fracture stabilization, as well as by any ipsilateral additional injury resulting in decreased blood supply. An open fracture with interrupted soft tissues increases the risk of impaired or absent vascularization with potential interference of revascularization, due to the traumatic interruption of the endosteal and periosteal blood supply (6, 11).
But not only deficient blood supply has been related to atrophic non-unions. A decrease of progenitor cells and subsequent decrease in bone reaction at the site of atrophic non-unions has also been detected (12) and also a decrease in proliferation and differentiation of mesenchymal stem cells (MSCs) (13), which may suggest a systemic mesenchymal and osteogenic cell pool defect in patients sustaining this type of lesion. Those are the non-unions that require a biological augmentation as a major part of its treatment.
Hypertrophic non-union
Contrary to atrophic non-unions, the blood supply in hypertrophic non-unions is not impaired. The main reason in the development of a hypertrophic non-union is insufficient primary fracture stabilization due to an unstable and/or insufficient osteosynthesis, or a lack of bone contact due to primary bone loss or soft tissue interposition. Other causes include a too early mobilization increasing the interfragmentary relative motion, with or without secondary implant failure, or inadequate immobilization after conservative treatment (14). Whatever the origin, increased motion in the fracture zone leads to an increased bone healing response developing an external callus as an attempt to increase stability. Ultimately, the increased relative motion may lead to the development of fibrocartilaginous tissue with a lack of mineralization of the hypertrophic callus, thus resulting in a non-union. Fracture stabilization is the major intervention in its treatment (11).
Epidemiology of non-unions
The frequency of non-unions after acute long bone fractures is highly variable, classically reported between 5% and 10% of total long bone fractures. Recent reports confirm that, from 2015 until today, the percentage of non-unions in long bone fractures is not decreasing, with 7–10% non-unions reported in surgically treated fracture cases (15). The non-union occurrence is more likely depending on factors such as the anatomical site (e.g. tibia), fracture type (e.g. open tibia fractures, bone loss, high energy trauma with comminution), or local infection. Patients’ habits (smoking/alcohol abuse), medications (e.g. bisphosphonates, chronic NSAIDs), or the presence of certain conditions (e.g. diabetes, peripheral vascular disease, obesity) can also increase the likelihood of non-union after fracture (16, 17, 18, 19, 20). A large study using the Victorian Orthopaedic Trauma Outcomes Registry of Australia determined the rate of failure of non-unions according to the type of bone fracture, finding 2.3% of failures in the proximal humerus, 7.8% in the humerus shaft, 4.2% in the subtrochanteric fracture, 13.5% in the femur shaft, 8.4% in the distal femur, and 11.7% in the tibia shaft (21). Mills et al. (19) reported an annual prevalence of non-union per acute fracture in the Scottish population of 1.9% with a risk up to 9% for certain fractures in specific age groups (22). A 2–10% percentage of long bone fractures evolving to non-union means a very substantial number of patients due to the incidence of long bone fractures. Results from a Spanish study suggest that the rate of non-union fractures remains mostly stable over time, from 3.7/100 000 inhabitants in 2000 to 4.8/100 000 inhabitants in 2015 (23), supporting the role of a cause not sufficiently addressed with current fracture treatments, despite significant progress and innovation.
Background and rationale of bone grafting
To heal delayed union or non-union fractures, bone regeneration is expected at the fracture site. In addition to an adequate mechanical environment with stabilization of the fracture, a local biological stimulus for bone formation is considered necessary.
Grafting of the bone in non-unions has classically supported and facilitated bone healing, through enhancement of osteoconduction, osteoinduction, and even osteogenesis. Estimations of bone graft usage are imprecise, but the amount of procedures involving bone grafts or substitutes is probably very high. It seems that more than 1 million bone grafts are required each year in Europe, which would make bone the most transplanted tissue in humans. Over 2 million bone grafting procedures were also estimated a decade ago to be performed worldwide annually (24). In any case, bone grafting is a current orthopaedic procedure with the aim of promoting bone regeneration and healing through a sequence of cellular events with influence of molecular and transcription factor pathways in what has been called the ‘fracture healing cascade’ (25). Together with the creation of a mechanically favorable environment at the fracture site, this concept for bone regeneration has been promoted as the ‘diamond concept’ (26, 27).
Many techniques and substances, whether biological or synthetic, have been proposed to facilitate bone healing. The rationale of bone grafting includes structural solutions (with osteoconductive properties, eventually including osteoinductive and/or osteogenic capabilities) and biological solutions (not oriented to osteoconduction, but to osteoinduction and/or osteogenesis) (Table 1).
Overview of the possible bone grafting techniques and their positive and possible negative effects upon usage.
| Bone Grafts | Pros | Cons |
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| Structural bone grafts | ||
| Autograft | ||
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| Allograft | ||
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| Bone substitute | ||
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| Biological bone grafts | ||
| Bone marrow aspirate | ||
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| Bone marrow concentrate | ||
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| Isolated, expanded MSC | ||
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Structural bone grafting techniques
Autologous
Autologous bone graft can be considered the ‘gold standard’ and has been in use for decades, recognized as the best available biological option for everyday surgery (Fig. 1). Its major advantage is to provide osteoconduction (through spongious chips supplying extracellular matrix), osteoinduction (with bone marrow (BM) growth factors, as the extracellular matrix contains a comprehensive reservoir of signaling proteins at physiological dose) (28), and local osteogenesis (with cell precursors in the BM).
Intraoperative image of an atrophic diaphyseal femoral non-union after debridement (A) and after iliac crest autograft (B). The spongious bone is already introduced in the intramedullary canal, whereas the tricortical block is later flipped toward its final position. (C) Callus formation at 3 months post surgery.
Citation: EFORT Open Reviews 9, 5; 10.1530/EOR-24-0032
Autologous grafts enhance bone regeneration and thus heal the bone injury, with accepted advantages such as shorter healing times, better bone quality, lower material costs, no risk of disease transmission or antigenicity due to its autologous nature, and predictability in the repair, with limited resorption except in the case of infection or other complications.
The most established and widely accepted source of bone graft is the iliac crest. Besides a practical and intuitive surgical approach, the iliac crest (both anterior or posterior crests) constitutes a bone reservoir in the human body, offering cortical and trabecular bone. Due to its limited function and without a mechanical role in most daily activities, the harvesting of graft from the iliac crest is expected to be well tolerated.
However, iliac crest autologous bone graft has drawbacks due to its limited stock (about 30 cc), and the absence of regeneration after harvesting (replaced by hypocellular fibrous tissue). Complications after posterior iliac crest harvesting in spine surgery include residual persistent pain, even with functional limitation (29). A general evaluation on the complications of autologous bone graft obtention (30) were classified as minor (in 20% graft harvests), including pain during months (5%); hematoma, seroma, superficial infection (13%); ilio-inguinal neuralgia (eventually requiring neuroma excision) (2%); or major complications (in 5% of graft harvests) including hernias through the bony defect, vascular damage, or even sciatic nerve injury, deep infection, deep hematoma, and iliac bone fracture.
Limited regeneration efficacy from autograft due to limited amount of cells, difficulties to harvest after previous use, decreased number of osteoprogenitors with age and a reported rate of failure up to 26% have been also claimed for iliac crest autograft (31). In the presence of bone defects, the bone regeneration may not be obtained from bone graft unless there is an active biological surrounding, and this relates to the study and development of an induced membrane to facilitate engrafting, in what became the Masquelet technique (32). Despite these advances, the limitations of autologous bone graft became obvious and it is understandable that alternatives have been thoroughly investigated. In conclusion, Hernigou et al. (30) promoted a BM concentration as an alternative technique representing ten times less complications than those from iliac crest graft harvesting.
Allograft
Allogeneic bone grafts have been long time used to fill bone defects as an osteoconductive technique. Hospital bone banks (local, regional or even national) have been designed, from living donors (femoral heads from arthroplasty) or from cadaver (large and small grafts, diaphyseal and metaphyseal, osteochondral, and even soft tissues) to provide fresh-frozen allografts (33). Also, commercially available prepared bone grafts have become increasingly used from highly regulated cadaver donors followed by professional processing (usually freeze-dried or lyophilized). Allografts in all conceivable preparations do not contain living cells and some matrix proteins containing growth factors are destroyed by virus-inactivation treatment and freezing process; thus, they only guarantee osteoconductive properties, with highly variable osteoinductive properties and lack of osteoprogenitor cells (34). On the negative side, allografts associate high non-union rates in the junction with normal bone, and a small risk of disease transmission (35, 36, 37, 38). Furthermore, allografts are associated with a higher risk of fracture when used for larger defects (39) and may require augmentation with vascularized grafts or BM cells (40) to accelerate incorporation and consolidation. Due to these disadvantages and their high associated costs, other strategies have emerged, including the development of a variety of synthetic bone substitutes (41).
Substitutes
The use of synthetic bone substitutes has become widespread in recent decades. The great advantage lies in their rapid and ubiquitous availability and the absence of comorbidities such as those associated with the removal of autologous bone material or the risk of infection with allografts.
Early synthetic bone substitutes were designed based on normal bone mineral composition, biodegradable to undergo resorption and substitution by new bone, differing in the percentage of the two main components beta-tricalcium phosphate and hydroxyapatite. While beta-tricalcium phosphate (TCP) is less stable and has a rapid absorption capacity, hydroxyapatite (HA) is more stable but less rapidly absorbed. The evolution of research has led to new composite biomaterials with intrinsic osteoinductive properties and high strength for load-bearing applications as well as understanding the biological mechanisms of osteogenesis, and many new solutions have reached the market (42). However, a high non-union rate is expected when using synthetic bone substitutes alone, such as HA and/or TCP, and a combination with autologous bone graft at a ratio of 1:3 to 1:1 may be recommended (37, 43, 44).
Furthermore, the synthetic bone replacement materials are also available in different application forms to adapt to the surgical needs: solids not only in the form of granules of different sizes but also as powders, blocks, and wedges or kneadable and moldable material as well as in liquid or pasty form that may harden after implantation.
In the UK, 59 synthetic bone substitutes have been reported (45), used in a third of cases requiring bone graft (44).
Despite different structural bone grafting solutions being widely used, the need for biological augmentation prevails, due to the inherent limitations to heal bone just relying on scarce local cells.
Biological bone graft techniques
The concept of ‘orthobiologics’ has been popularized for the biological solutions in orthopaedics, and bone grafting has benefited from different ways to incorporate cells and their secretome, composed of growth factors and cytokines in a physiological dose, into the non-union (46).
Bone marrow concentrate
Despite attempts to incorporate blood or BM to the allografts or bone substitutes, it is not until the idea to concentrate the BM (BM) that some favourable clinical results have been obtained (47). It has been demonstrated that there is a positive relation between the number of implanted MSCs and the healing of tibia non-union (48). The procedure and surgical technique proved to be simple and feasible (49), while the harvesting technique is considered safe (50).
The initial technique of bone marrow concentration (BMC) was performed in cell therapy units but has evolved to a close system that can be managed inside the operating room under different protocols and commercially available disposable and centrifugation systems, with a concentration factor of 4 to 7 for the nucleated cells of the BM. Percutaneous grafting is thus possible and the results may be satisfactory to bring this concept to the clinical arena (51). Varying regulatory burdens in different countries should be mentioned as a potential limitation for the use of this technique.
Bone marrow aspirate
Other sources of BM to obtain sufficient number of cells were considered. In particular, endosteal reaming performed in many intramedullary nailing procedures for diaphyseal fractures and non-unions was considered as a potential augmentation to obtain bone healing. Therefore, aspirating the BM from the endosteal reaming was seen as an opportunity for harvesting osteoprogenitor cells (52, 53, 54). This bone marrow aspirate (BMA) in toto, which can clot as a result, holds a recognized potential for tissue regeneration (55). A reamer–irrigator–aspirator (RIA) system was designed to provide irrigation and aspiration during a single-stage reaming procedure in which the device can be used to clear the medullary canal of BM and reaming debris, specially at the femur, but has gained popularity when harvesting this material from the diaphyseal canal as a graft, with clinically proven effectiveness (56, 57, 58, 59).
Less favorable is the comparison of RIA and iliac crest harvesting in terms of complications, although the initially detected transfusion rate with RIA harvesting (up to 44%) (60) has been substantially decreased (down to 14%) (61) in other reports. The risk of cortical perforation and the frequently reported relevant intraoperative blood loss are complications that should be anticipated in perioperative management and ultimately considered when using the RIA system, but the system has demonstrated an overall low prevalence rate of complications in a recent meta-analysis (62). Besides the discussion about complications, the effectiveness has been compared to iliac crest grafts, with evidence suggesting an equal healing potential (63). Therefore, autologous graft harvesting through different methods may be feasible and equivalent, but highly variable in the effectiveness to enhance bone healing. Furthermore, this technique may be useful to enhance other forms of structural grafting (Fig. 2).
(A) Intraoperative image of a segmental bone defect of the metaphyseal distal femur after debridement and a vascularized contralateral fibula in the distal intramedullary canal. (B) After positioning the graft in the proximal intramedullary canal, autologous bone aspirate (RIA) was inserted in the fracture site and osteosynthesis with a plate stabilized the bone.
Citation: EFORT Open Reviews 9, 5; 10.1530/EOR-24-0032
Isolated, expanded MSCs
The cell composition of the precedent biological grafts depends on the reservoir at the time and location of harvesting, and on the patient cell population, while the success has been associated to higher numbers of cells (48, 51).
The frequency of MSCs in BM is very low, estimated in about 0.001–0.01% of total nucleated cells. Therefore, the cells harvested from the BM may be insufficient, despite concentration. The rationale of isolating MSCs from the harvest and performing a cell expansion under Good Manufacturing Practice (GMP) criteria will return to the operating room an standardized cell product with a higher cell dose, which is known at the time of release from the GMP facility and may rise up to 200 million cells from a single autologous donor as an advanced therapy medicinal product (ATMP) (64, 65). Many other aspects require standardization, including differentiation toward the osteogenic line (66), transportation to the clinical centers (67), or combination with biomaterials (68). Bringing these proposals into clinical trials is also a significant challenge, due to the considerable regulatory hurdles of these ATMP techniques given that the expanded cells are considered in the UE a medicament (69).
The combination of BM MSCs with osteoconductive scaffolds can be a most effective and suitable therapy for complex non-unions with bone defects (Fig. 3), and this has been tested in early clinical trials (70, 71), where successful clinical and radiological consolidation was obtained in 26 out of 28 patients (92%) with diaphyseal or metaphyseodiaphyseal non-unions of femur, tibia, or humerus at 1-year post surgery. However, these experimental techniques are not yet available to treat patients outside of clinical trials, despite solid proofs of safety and early efficacy.
Intraoperative images of an atrophic tibia non-union. After debridement (A) with visible intramedullary nail and after insertion of a combination of autologous MSCs and a biphasic synthetic bone substitute (B). Postoperative radiograph (C).
Citation: EFORT Open Reviews 9, 5; 10.1530/EOR-24-0032
Remarks and future directions
After so many years and efforts to improve the quality and quantity of bone regeneration to heal fractures and especially non-unions, it has become evident that mechanical approaches alone may still fail, despite the significant advancements in the field. Biological approaches are needed, particularly when bone healing has failed, as is the case of non-unions. The available bone grafting have been tested in many procedures and patients, and therefore provide available products to try to enhance this bone healing, but limitations and downsides need adequate understanding to perform the best choice, in benefit of the patient. In the biological therapies under development, cells are the most promising actor to be incorporated. Whether autologous aspirated, concentrated, or expanded, further research on cell therapy will provide refined solutions of sufficient potency to predict and obtain earlier and better healing. The development of a universal and affordable therapy for fractures with delay or failure to heal is strongly needed. ATMP research, in autologous or allogeneic cell products, may lead to an effective off-the-shelf combined ATMP ready to be used as soon as surgery is indicated. With the invaluable support of structural grafting to provide the new bone of adequate mechanical properties, this or other new pathways could make a real progress in the management of the disease and its consequences.
ICMJE Conflict of Interest Statement
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this instructional lecture.
Funding Statement
EGB and CE report research funds from the European Union's Horizon 2020 Programme (H2020-SC1 2016-2017), with the ORTHOUNION Project (under G.A. 7333288), and European Union’s Horizon Europe Programme (HORIZON-HLTH-2023-TOOL-05), with the ORTHO-ALLO-UNION Project (under G.A. 101137464).
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