• Users Online: 352
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 


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

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


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

Date of Submission24-Oct-2018
Date of Decision07-Nov-2018
Date of Acceptance19-Nov-2018
Date of Web Publication24-Jan-2019

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
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2542-4157.248607

Rights and Permissions
  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).

Keywords: bone non-union; pathohistology; atrophy; hypertrophy; long bone; cross-sectional study


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;3:101-6

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 2019 Aug 22];3:101-6. Available from: http://www.clinicalto.com/text.asp?2018/3/4/101/248607


  Introduction Top


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 Top


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 [Additional file 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 [Additional file 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 [Additional file 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 Top


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: Histomorphological comparison of the types of non-union

Click here to view


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: General histological characteristics (hematoxylin-eosin staining).
Note: (A) Hypercellular fibrous tissue; (B and C) loose connective tissue, vascularized, edematous and congestive; (D and E) densely collagenized, hypocellular connective tissue; (F and G) bone neoformation; (H and I) osteonecrosis. Original magnification, 40× for A, D, H, and I; 100× for B, E, and F; 200× for C and G.


Click here to view


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: Histological characteristics of hypertrophic and oligotrophic non-union (hematoxylin-eosin staining).
Note: (A) Lines of osteosclerosis; (B) osteoclastic or resorptive activity; (C and D) neovascularization; (E) endothelial proliferation; (F and G) chronic inflammation; (H and I) synovial non-union. Original magnification, 100× for A, D, and I; 200× for B, E, F, and G; 40× for C and H.


Click here to view


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: Morphological comparison of aseptic and septic non-union

Click here to view
Figure 3: Histological characteristics of septic and aseptic non-union (hematoxylin-eosin staining).
Note: Aseptic non-union: (A) Fibrous and inflammatory tissue; (B) collagenization and calcium concretions; (C) granulation tissue; (D) osteoid matrix deposit. Septic non-union: (E) extensive osteonecrosis; (F) fibrin deposits. Original magnification, 100× for A–C, F; 200× for D; 40× for E.


Click here to view



  Discussion Top


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: X-ray lateral (A) and anteroposterior (B) views of the left femur.
Note: A mid-third diaphyseal hypertrophic non-union can be observed (white arrows).


Click here to view
Figure 5: X-ray anteroposterior (A) and lateral (B) views of the right humerus.
Note: A distal-third diaphyseal oligotrophic non-union can be observed (white arrows).


Click here to view
Figure 6: X-ray lateral (A) and anteroposterior (B) views of the left knee.
Note: A mid-third diaphyseal and distal metaphyseal atrophic non-union can be observed (white arrows).


Click here to view


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.

 
  References Top

1.
Megas P. Classification of non-union. Injury. 2005;36 Suppl 4:S30-37.  Back to cited text no. 1
    
2.
Rubin C, Bolander M, Ryaby JP, Hadjiargyrou M. The use of low-intensity ultrasound to accelerate the healing of fractures. J Bone Joint Surg Am. 2001;83-A:259-270.  Back to cited text no. 2
    
3.
Escarpanter Buliés JC. Factores de riesgo para la aparición de seudoartrosis en las fracturas diafisarias. Rev Cubana Ortop Traumatol. 1997;11(1-2):50-55.  Back to cited text no. 3
    
4.
Meskens MW, Stuyck JA, Feys H, Mulier JC. Treatment of nonunion using pulsed electromagnetic fields: a retrospective follow-up study. Acta Orthop Belg. 1990;56:483-488.  Back to cited text no. 4
    
5.
Pretell Mazzini JA, Ruiz Semba C, Rodriguez Martín J. Trastornos de la consolidación: Retardo y pseudoartrosis. Rev Med Hered. 2009;20:31-39.  Back to cited text no. 5
    
6.
Liow RY, Montgomery RJ. Treatment of established and anticipated nonunion of the tibia in childhood. J Pediatr Orthop. 2002;22:754-760.  Back to cited text no. 6
    
7.
Smith VA, Wright TW. Nonunion of the distal radius. J Hand Surg Br. 1999;24:601-603.  Back to cited text no. 7
    
8.
Harley BJ, Beaupre LA, Jones CA, Dulai SK, Weber DW. The effect of time to definitive treatment on the rate of nonunion and infection in open fractures. J Orthop Trauma. 2002;16:484-490.  Back to cited text no. 8
    
9.
Weber BG, Brunner C. The treatment of nonunions without electrical stimulation. Clin Orthop Relat Res. 1981;24-32.  Back to cited text no. 9
    
10.
Marsell R, Einhorn TA (2011) The biology of fracture healing. Injury. 42:551-555.  Back to cited text no. 10
    
11.
Tsiridis E, Upadhyay N, Giannoudis P. Molecular aspects of fracture healing: which are the important molecules? Injury. 2007;38 Suppl 1:S11-25.  Back to cited text no. 11
    
12.
Carano RA, Filvaroff EH. Angiogenesis and bone repair. Drug Discov Today. 2003;8:980-989.  Back to cited text no. 12
    
13.
Bajada S, Marshall MJ, Wright KT, Richardson JB, Johnson WE. Decreased osteogenesis, increased cell senescence and elevated Dickkopf-1 secretion in human fracture non union stromal cells. Bone. 2009;45:726-735.  Back to cited text no. 13
    
14.
Reed AA, Joyner CJ, Brownlow HC, Simpson AH. Human atrophic fracture non-unions are not avascular. J Orthop Res. 2002;20:593-599.  Back to cited text no. 14
    
15.
Urist MR, Mazet R Jr, McLean FC. The pathogenesis and treatment of delayed union and non-union; a survey of eighty-five ununited fractures of the shaft of the tibia and one hundred control cases with similar injuries. J Bone Joint Surg Am. 1954;36-A:931-980.  Back to cited text no. 15
    
16.
Miska M, Findeisen S, Tanner M, et al. Treatment of nonunions in fractures of the humeral shaft according to the Diamond Concept. Bone Joint J. 2016;98-B:81-87 .  Back to cited text no. 16
    
17.
Pihlajamaki HK, Salminen ST, Bostman OM. The treatment of nonunions following intramedullary nailing of femoral shaft fractures. J Orthop Trauma. 2002;16:394-402  Back to cited text no. 17
    
18.
Nandra R, Grover L, Porter K. Fracture non-union epidemiology and treatment. Trauma. 2016;18:3-11.  Back to cited text no. 18
    
19.
Zura R, Della Rocca GJ, Mehta S, et al. Treatment of chronic (>1 year) fracture nonunion: heal rate in a cohort of 767 patients treated with low-intensity pulsed ultrasound (LIPUS). Injury. 2015;46:2036-2041.  Back to cited text no. 19
    
20.
Ferreira N, Marais LC, Aldous C. Management of tibial non-unions: Prospective evaluation of a comprehensive treatment algorithm. SA Orthop J. 2016;15:60-66.  Back to cited text no. 20
    

Author contributions
Study design, manuscript drafting and English translation: ACV, LDBF, JGA, and JLV; sample collection: AMP and RGR; sample processing and biostatistics review: JQW and EPH. All authors approved the final version of this manuscript.
Conflicts of interest
None declared.
Financial support
None.
Institutional review board statement
All procedures performed in studies 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 and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts would be made to conceal their identity.
Reporting statement
This study followed the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) statement.
Biostatistics statement
The statistical methods of this study were reviewed by Dr. Pérez-Hernández and Dr. Quiroz-Williams at UMAE “Dr. Victorio de la Fuente Narváez” IMSS, México.
Copyright license agreement
The Copyright License Agreement has been signed by all authors before publication.
Data sharing statement
For data sharing, individual participant data will not be available.
Plagiarism check
Checked twice by iThenticate.
Peer review
Externally peer reviewed.


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Subjects and Methods
Results
Discussion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed424    
    Printed53    
    Emailed0    
    PDF Downloaded74    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]