Clinical Trials in Orthopedic Disorders

RESEARCH ARTICLE
Year
: 2018  |  Volume : 3  |  Issue : 4  |  Page : 101--106

Clinicopathological characterization of long bone non-union: a prospective cross-sectional study


Avelino Colin-Vazquez1, Luis Dario Bernal-Fortich1, Joel Galindo-Avalos2, Juan López-Valencia1, Rafael Grajales-Ruiz3, Adrián Miguel-Pérez3, Jorge Quiroz-Williams4, Elizabeth Pérez-Hernández5,  
1 Department of Orthopedics, UMAE “Dr. Victorio de la Fuente Narváez” IMSS-UNAM, Ciudad de México, México
2 Department of Orthopedics, Arthroscopic Surgery, Centro de Ortopedia y Medicina del Deporte, Centro Médico Puerta de Hierro, Zapopan, Jalisco, México
3 Osteoarticular Rescue Department, UMAE “Dr. Victorio de la Fuente Narváez” IMSS, México
4 Division Head of the Health Research Division, UMAE “Dr. Victorio de la Fuente Narváez” IMSS, México
5 Division Head of the Health Education and Research Division, UMAE “Dr. Victorio de la Fuente Narváez” IMSS, México

Correspondence Address:
Joel Galindo-Avalos
Department of Orthopedics, Arthroscopic Surgery, Centro de Ortopedia y Medicina del Deporte, Centro Médico Puerta de Hierro, Zapopan, Jalisco
México

Abstract

Background and objectives: The exact biological process that leads to a non-union remains obscure and it is well accepted that any intervention to reverse this process must be timely and well directed to re-establish both biological and mechanical deficiencies. The purpose of the present work was to identify the possible morphological patterns of bone tissue in a state of non-union and in the presence or absence of infection. Methods: A prospective, observational, cross-sectional study was performed. Bone tissue samples obtained from patients with radiologically and clinically diagnosed long bone non-union undergoing revision surgery were included, without distinction of age and sex. Histopathological analysis and semiquantitative evaluation of tissue samples were performed using conventional optical microscopy. Clinical data related to the study variables were obtained, such as type of consolidation, time of evolution, and corresponding bacteriological analysis. Results: Several morphological pattern variables such as bone quality, osteosclerosis, areas of bone devitalization, osteoclastic activity, vascularization, and in particular cell density were related to the subtype of long bone non-union. They were also associated with septic and aseptic variants of long bone non-union. Conclusion: The biological bone profiles, in particular the histomorphological characteristics, are related to the subtype of non-union and reflect the physiopathological environment that involves an anabolic and catabolic imbalance. These can be incorporated into the classification system and favor the stratification of non-union. Ethics and trial registration: This study was approved by Comité Local de Investigación y Ética en Investigación en Salud at UMAE “Dr. Victorio de la Fuente Narváez” (R-2017-3401-8) on July 19, 2017 and registered with SIRELCIS (identifier: R-2017-3401-8).



How to cite this article:
Colin-Vazquez A, Bernal-Fortich LD, Galindo-Avalos J, López-Valencia J, Grajales-Ruiz R, Miguel-Pérez A, Quiroz-Williams J, Pérez-Hernández E. Clinicopathological characterization of long bone non-union: a prospective cross-sectional study.Clin Trials Orthop Disord 2018;3:101-106


How to cite this URL:
Colin-Vazquez A, Bernal-Fortich LD, Galindo-Avalos J, López-Valencia J, Grajales-Ruiz R, Miguel-Pérez A, Quiroz-Williams J, Pérez-Hernández E. Clinicopathological characterization of long bone non-union: a prospective cross-sectional study. Clin Trials Orthop Disord [serial online] 2018 [cited 2024 Mar 29 ];3:101-106
Available from: https://www.clinicalto.com/text.asp?2018/3/4/101/248607


Full Text

 Introduction



Epidemiology

Non-union (pseudarthrosis) is defined as the formation of a false joint where a fibrocartilaginous cavity is lined with synovium producing synovial fluid. Non-union of the fracture is defined as the cessation of all reparative processes of healing without bone union. Unless there is bone loss, a non-union is usually declared between 6 and 8 months following the fracture.[1]

The incidence of non-union ranges from 5–10% of all fractures.[2] Approximately 53% of non-union occurs in the lower limbs, the tibia being the most affected bone, followed by the femur,[3],[4] then the humerus, the bones of the forearm and the clavicle.[5] Risk factors of non-union include high energy trauma, open fractures, multifragmentation, bone loss, post-reduction instability, diabetes, obesity, alcoholism, peripheral vascular disease and scleroderma.[6],[7]

Revision surgery with bone grafts, usually from the iliac crest,[8] is currently the best way to stimulate bone regeneration. However, several approaches have been described to promote and improve bone tissue regeneration, such as extracorporeal shockwave therapy (ESWT), ultrasound, bone morphogenic proteins (BMPs) and platelet-rich plasma (PRP).[9]

Cellular repair process in the bone tissue

Whenever there is a fracture, direct bone healing occurs only with absolute stability and is a biological process of osteonal bone remodeling.[10],[11] Indirect bone healing depends on the formation of fibrocartilaginous calluses and occurs in most of the cases.[10] This type of bone healing follows a specific biological pathway. It involves an acute inflammatory response, the recruitment of mesenchymal stem cells to generate a primary cartilaginous callus, which later undergoes revascularization and calcification, and is finally remodeled to fully restore a normal bone structure.[10],[11],[12]

Macroscopic and microscopic structure

In a histological study on non-union tissue, it was found that tissue vessels often appear occluded by thrombotic material, concluding that the cells that populate non-union tissue can induce mineralization of the cell matrix, but have an insufficient blood supply to provide them with a normal amount of calcium, which is the real cause for non-union development.[13] Fibrocartilaginous tissue that contains occasional bony islands has been found in atrophic non-union; in hypertrophic non-union, areas of new bone formation by both endochondral and intramembranous ossification have been observed.[13],[14] The exact biological process that leads to a non-union remains obscure and it is well accepted that any intervention to reverse this process must be timely and well directed to re-establish both biological and mechanical deficiencies.

Objective

The purpose of the present work was to identify the possible morphological patterns of bone tissue in a state of non-union and in the presence or absence of infection.

 Subjects and Methods



Study design

This prospective, observational, cross-sectional study was conducted at Hospital de Ortopedia UMAE “Dr. Victorio de la Fuente Narváez” from May 2017 to February 2018. All procedures involving human participants were in accordance with the ethical standards of Comité Local de Investigación y Ética en Investigación en Salud at UMAE “Dr. Victorio de la Fuente Narváez” (R-2017-3401-8) on July 19, 2017 (Additional file 1 [SUPPORTING:1]) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement (Additional file 2 [SUPPORTING:2]).

Bone tissue sample harvesting

Pathological bone tissue (in a state of non-union diagnosed radiographically) was obtained from patients who underwent revision surgery in our hospital after they had provided written informed consent (Additional file 3 [SUPPORTING:3]). The conditions of aseptic and septic non-union were determined by clinical and laboratory means (biopsy culture). A total of 34 samples of bone tissue were collected under such conditions. To avoid any disruption in tissue morphology, the samples were collected using an osteotome instead of a bone saw.

Fixation methods

The samples were fixed in 10% neutral buffered formalin, then decalcified with 7% nitric acid and neutralized. Afterwards, the excess chemical was washed off, and samples were dehydrated by immersing tissue in a series of ethanol solutions of increasing concentrations until 100% water-free alcohol was reached. Then ethanol was replaced with xylol and the samples were immersed in liquid paraffin to form blocks. The blocks were then cut into 3 μm-thick sections. The tissue sections were then mounted on a glass microscope slide and treated with a conventional hematoxylin-eosin stain. Subsequently, they were covered with epoxy resin for visualization using conventional optical microscopy (Nikon Instruements Inc., Melville, NY, USA).

Histopathological observation and semiquantitative evaluation

Histopathological changes of bone tissue sample were observed and then descriptive and semiquantitative evaluation was performed.

 Results



Baseline data

Thirty-four bone tissue samples were obtained from 34 patients aged 40 (range 23–57 years) years. The male to female ratio was 2.4:1. The mean time from clinical diagnosis to surgery was 17 (range 9–36 months) months. Among these patients, 26 were classified as aseptic (76.5%) and 8 as septic (23.5%). In the septic non-union, men were more affected than women with a 7:1 ratio.

Bone segments

The most affected bone segments were the tibia and femur (32.3%), followed by the humerus (20.5%), the radius and the ulna (11.7%), and finally the clavicle (3.2%). The femur was the most commonly affected in aseptic non-union and the tibia in septic non-union.

Bacteria

The most common infectious agents associated with septic non-union were Staphylococcus aureus in 75% of the cases, and Escherichia coli in 25% of the cases.

Clinicopathological classification

Regarding the clinicopathological evolution, we found 24 patients with oligotrophic non-union (70.6%), 6 patients atrophic non-union (17.6%), and 4 patients with hypertrophic non-union (11.8%). A higher incidence of infection was found in oligotrophic non-union (6 patients), followed by atrophic non-union (2 patients). Hypertrophic non-union was not found in any patient.

Disease severity

Regarding the severity of the disease, 11 patients were classified as Paley A (32.4%), that is, with a bone defect of less than 1 cm; and 23 patients as Paley B (67.6%), that is, with a bone defect bigger than 1 cm. From the cases of septic non-union, 7 of them were classified as Paley B (87.5%), and only 1 as Paley A (12.5%).

Microscopic description

The histological sections evaluated showed heterogeneity of the morphological patterns, with variability in the proportion, however, no specific statistical significance was observed for the types of non-union. These characteristics are summarized in [Table 1].{Table 1}

Cell population was characterized by fibroblasts of normal morphology, arranged in short beams, with the formation of nuclear palisades and with variable proportions of extracellular matrix (collagen), including densely collagenized, hyalinized areas [Figure 1]A.{Figure 1}

Two types of cartilage, reparative fibrocartilage, and hyaline cartilage were identified [Figure 1]B, [Figure 1]C, [Figure 1]D, [Figure 1]E). The fibrous cartilage was mostly associated with the hypertrophic variety of non-union, as a part of the exuberant bone callus. Likewise, cystic degenerative changes were identified, especially in the oligotrophic type. The presence of hyaline cartilage arranged in islands or lobes between the fibrous tissues was observed in the atrophic as well as in the hypertrophic forms, however, in the latter, the arrangement of the chondrocytes and their morphological characteristics simulate the hypertrophic cartilage of the growth plate.

New bone formation was a common feature in all types of non-union, either from endochondral ossification (predominantly in the atrophic and oligotrophic varieties) and/or from areas of intramembranous ossification (more frequent in the hypertrophic form) [Figure 1]F, [Figure 1]G.

Areas of bone necrosis were more prevalent in the atrophic and oligotrophic forms. These changes were also observed in addition to fragmentation of bone spicules and identification of osteosclerosis lines [Figure 1]H and [Figure 1]I.

Osteoblastic activity was observed in some samples in both the oligotrophic and hypertrophic forms, whereas the osteoclastic activity was predominantly identified in the atrophic form [Figure 2]A and [Figure 2]B.{Figure 2}

Vascularization was observed in different types of non-union, with minimal variations in the vascular proportion [Figure 2]C and [Figure 2]D, and in some samples, it was associated with lymphoplasmacytic inflammatory cell aggregates, with diffuse and perivascular disposition, as well as with areas of edema and proliferation of connective tissue fibers [Figure 2]E, [Figure 2]F, [Figure 2]G.

Another change associated with the hypertrophic form was synovial non-union characterized by vascularized fibrotic tissue with cell proliferation similar to synoviocytes and pseudomembrane formation [Figure 2]H and [Figure 2]I.

The septic and aseptic forms of non-union also showed morphological heterogeneity and variable proportions of morphological patterns. These characteristics are summarized in [Table 2] and [Figure 3]A, [Figure 3]B, [Figure 3]C, [Figure 3]D, [Figure 3]E, [Figure 3]F.{Table 2}{Figure 3}

 Discussion



Non-union or pseudoarthrosis represents a public health problem with adverse consequences in a patient’s quality of life. The mechanisms that lead to non-union are multifactorial and therefore the treatment has evolved since the prolonged immobilizations in the 1950’s.[15],[16],[17],[18],[19],[20]

This study has some limitations, the most important of them being that, because of the small sample size, a histopathological classification could not be reached, which is the primary objective of this study.

Few articles in the scientific literature examine the global characterization of non-union that includes radiographic, clinical and histological aspects, likewise, the current classification systems do not include histopathological findings, which could be determinant for treatment of the disease, together with the biomolecular aspects.

As described in the literature, the Weber-Cech classification includes both radiographic observations and fixation stability. Based on this characterization, different types of non-union are recognized: hypertrophic [Figure 4], where there is adequate curative potential due to abundant callus formation and it is associated with hypervascularity; oligotrophic [Figure 5], which is vascularized but with minimal callus formation; and atrophic [Figure 6], in which there is absence of callus formation and no bone vascularity.[1]{Figure 4}{Figure 5}{Figure 6}

In this study, the histopathological characteristics of different forms of non-union were described, from the atrophic, oligotrophic to hypertrophic variants. As observed, there is a broad, heterogeneous morphological spectrum, which correlated proportionally with the formation of scarce to exuberant bone callus, however, histological patterns characteristic of a specific subtype were not identified, which leads to difficulty in the integration of scales or associated morphological patterns.

However, variable proportions of cellularity, as well as the components of the extracellular matrix, participate in the phenomenon of non-union. In this regard, as mentioned in some publications, the elements of the connective tissue can be targeted by growth factors to stimulate bone consolidation effectively and in a controlled manner. The use of in vitro biological models, such as cultures and cell cocultures, construction of monolayers, use of stimulating and inhibiting factors during the healing process, etc., are still in research.

Likewise, the influence of concomitant factors and demographic variables, such as comorbidities, especially in populations like ours, are also the research areas of this study. However, the association with infectious processes, the type of etiological agents and their mechanisms of antimicrobial resistance, etc., are also important aspects to be considered in bone consolidation. They are emphasized in the education of the personnel that intervenes in the healthcare process and in the education to the community to prevent associated complications.

To conclude, long bone non-union is currently one of the major orthopedic challenges. This is due not only to the complexity of the disease but also to its devastating effects when it is not treated effectively and timely.

There is heterogeneity in the histopathological characterization of non-union, which correlates proportionally with the formation of bone callus. However, it is not possible to integrate specific morphological patterns for classification purposes.

The association of non-union with an infectious process (septic non-union) showed a higher degree of tissue lysis, which clinically delays and interferes with the process of bone consolidation.

Additional files

Additional file 1: Ethics committee approval.

Additional file 2: STROBE checklist.

Additional file 3: Model consent form.

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