1 INTRODUCTION

Bone healing is a complex biological process aiming at restoring the affected area to its pre-injury levels. This is achieved through repair and regeneration of the cellular and extracellular components, regaining its former biochemical and biomechanical properties.1, 2 Successful bone healing requires the orchestrated interaction between the biological (cellular, signalling molecules and extracellular matrix) and mechanical environments.3 Moreover, according to the ‘Diamond Concept’, other parameters that are considered essential for a successful healing include the local vascularity and the patient's biological fitness and comorbidities.4

The definition of non-union has been inconsistent in the literature. The FDA (Food and Drug Administration), however, defines non-union as incomplete fracture healing within 9 months following injury, coupled by the lack of progression in radiological signs of healing over the course of three consecutive months.5 Despite the advancement in both the understanding of fracture healing and some of the pathways that regulate it, the rates of fracture non-union remain largely unchanged over the years. To date, fracture non-union remains common, occurring in 5%–10% of the 850,000 fractures seen yearly in the UK.6 This poses a significant direct and indirect socioeconomic burden through prolonged medical treatments and productivity losses.6 Further understanding of the biological processes and underlying mechanisms, along with their interactions, leading to fracture non-union need to be elucidated in order to reduce this risk.

We have previously published a systematic review outlining the biological and molecular profile of ‘non-union tissue’.1 Nevertheless, one critically relevant and important aspect not previously considered because of the scarce evidence at the time was the relevance of tissues harvested from sites away from the non-union site, such as peripheral blood and bone marrow products. Moreover, the accelerated improvement in laboratory techniques over the last decade also meant the biological and molecular understanding of the multiple pathways involved in bone healing is everchanging. Consequently, the herein study provides an up-to-date review on the knowledge that has been acquired in this important clinical condition. We aim to summarize the current evidence on (i) macroscopic and microscopic characteristics; (ii) cellular characteristics and function (cell surface protein expression, morphology, viability, proliferation, senescence, mineralization and alkaline phosphatase [ALP] activity); (iii) molecular characteristics (protein, mRNA, miRNA and gene expression) of non-union tissue and relevant tissues; (iv) differences between atrophic and hypertrophic non-unions; (v) effect of intervention(s) on non-union tissue and relevant tissues; and (vi) genetic predispositions to fracture non-union.

2 MATERIALS AND METHODS

This systematic review was conducted according to the PRISMA guidelines.7 Our protocol was similar to that of our previous publication, with the only difference being the addition of other types of tissues not harvested from the non-union site (‘relevant tissue’) in our inclusion criteria.1 We define ‘relevant tissue’, as bone marrow or peripheral blood derived products, investigated to identify associations with progression to non-union. The reason for including studies assessing relevant tissue was due to the growing body of evidence demonstrating the correlation of these tissues with the occurrence of non-union, which we feel could be helpful to guide clinicians in their practice.

2.1 Eligibility criteria

The inclusion criteria were as follows: (i) tissue obtained from the non-union site and processed for defining its characteristics and properties, OR studies assessing tissue relevant to non-union as defined above (‘relevant tissue’); (ii) only tissue acquired from human subjects was included; (iii) articles were published in English language; (iv) the full text of each article was available; and (vi) for non-union tissue, articles published between August 2014 (date of our previous publication) and 2 October 2021; for relevant tissue, no publication date restrictions were imposed. Studies that did not fulfil the eligibility criteria were excluded from further analysis.

2.2 Search strategy and information sources

Adhering to our previously published protocol, the following databases were used during literature search: PubMed Medline; Ovid Medline; Embase; Scopus; Google Scholar; and the Cochrane Library. The full search strategy is as detailed in Table 1. Briefly, the search terms included non-union(s), nonunion(s), human, tissue, bone morphogenic protein(s) (BMPs) and MSCs. Bibliographies of all identified articles were collected in Endnote X9, manually reviewed and searched for any potentially eligible studies.

TABLE 1. PubMed search strategy (searched 2 October 2021) 1. (("non-union"[All Fields] OR ("nonunion"[All Fields] OR "nonunions"[All Fields])) 2.

("mesenchymal stem cells"[MeSH Terms]

OR ("mesenchymal"[All Fields] AND "stem"[All Fields] AND "cells"[All Fields])

OR "mesenchymal stem cells"[All Fields]

OR ("mesenchymal"[All Fields] AND "stem"[All Fields] AND "cell"[All Fields])

OR "mesenchymal stem cell"[All Fields]

3 "MSC"[All Fields] 4.

("mesenchymal stem cells"[MeSH Terms]

OR ("mesenchymal"[All Fields] AND "stem"[All Fields] AND "cells"[All Fields])

OR "mesenchymal stem cells"[All Fields]

OR ("mesenchymal"[All Fields] AND "stromal"[All Fields] AND "cell"[All Fields])

OR "mesenchymal stromal cell"[All Fields])

5. "bone morphogenetic proteins"[MeSH Terms] OR ("bone"[All Fields] AND "morphogenetic"[All Fields] AND "proteins"[All Fields]) OR "bone morphogenetic proteins"[All Fields] OR ("bone"[All Fields] AND "morphogenetic"[All Fields] AND "protein"[All Fields]) OR "bone morphogenetic protein"[All Fields] 6. ("tissue s"[All Fields] OR "tissues"[MeSH Terms] OR "tissues"[All Fields] OR "tissue"[All Fields]))) 7. (humans[Filter]) 8. (english[Filter])) 9. 2 OR 3 OR 4 OR 5 OR 6 10. 1 AND 9 11. 10 AND 7 AND 8 2.3 Study selection

Two of the authors (MP and JV) performed the eligibility assessment independently, in an unblinded, standardized manner. Title and abstract sift were conducted first, followed by review of full text by MP and JV. Only studies fulfilling the eligibility criteria were included. Data of each eligible study were independently extracted by MP and JV, with results checked by the third author (IP). Any disagreement between reviewers was resolved by consensus, and if necessary, the senior researcher (PVG) was consulted.

2.4 Extraction of data

Information on author, year of publication, patient demographics, non-union site, the duration and type of non-union, characteristics of non-union tissue (macroscopic/microscopic), cellular characteristics and functions (cell surface protein expression, morphology, viability, proliferation and cellular senescence), molecular characteristics (gene expression, protein expression) and effect of additional interventions were all carefully extracted.

2.5 Data analysis

Outcomes of interest as mentioned in ‘Extraction of data’ section were inserted in an electronic database. Wherever possible, each characteristic of tissue samples was compared across different studies. We also evaluated the effect of any interventions documented in these studies. Qualitative results were summarized and presented in tables, whereas quantitative results are presented with p values if stated by the study. Statistical comparison was not made between studies, due to the heterogeneity in terms of study methodologies observed in each of these in vitro studies.

3 RESULTS 3.1 Literature search

The electronic literature search retrieved 342 citations, of which 24 met the inclusion criteria for the final analysis (Figure 1).8-31 Overall, 10 studies8-17 assessed non-union tissue (Table 2), whereas 14 studies18-31 investigated relevant tissue (Table 3).

PRISMA 2020 flow diagram—study selection

TABLE 2. Non-union tissue: patient demographics Author Year Time frame Number of specimens Site of non-union Patients’ age (mean ± SD) Amount of tissue Cuthbert8 2020 Not mentioned Atrophic non-union: 20 (11 males); critical size defects requiring induced membrane/Masquelet procedure: 15 (10 males); BMA: 8 (3 males) Not mentioned Atrophic non-union: median age 53, range 23–81; critical size defects requiring induced membrane/Masquelet procedure: median age 61, range 19–80; BMA: median age 38, range 19–52 Atrophic non-union: not mentioned; induced periosteum: 1 cm of membrane tissue from centre of bone defect area; BMA: not mentioned Wei9 2020 Not mentioned Atrophic non-union: n = 3; controls (healed fractures): n = 3 Not mentioned Not mentioned Not mentioned Wang10 2018 Not mentioned 8 non-unions compared to 8 with uneventful healing Not mentioned Not mentioned Not mentioned Vallim11 2018 Not mentioned 15 (9 male) Tibia: 3; femur: 4; humerus: 7; ulna: 1 46.4 ± 12.5 Approximately 1 cm3 Takahara12 2016 Not mentioned 4 (2 male) Femur: 1; humerus: 2; clavicle: 1 65.3 ± 5.4 "Small amount" Schira13 2015 Not mentioned 80 (77 male) Scaphoid 24.6 years (range, 18–71 years) Not mentioned Han14 2015 2009 to 2010 11 Not mentioned 40 years (range 27–81 years) Not mentioned Wang15 2014 October 2010 to March 2014 Hypertrophic non-union: 20 (15 male); atrophic non-union: 20 (14 male)

Hypertrophic non-unions: femur 8; femoral neck 1; tibia: 2; humerus: 9.

Atrophic non-unions: femur 5; tibia: 8; humerus: 7.

Hypertrophic non-unions: 39.35 ± 11.67 years

Atrophic non-unions:

33.75 ± 8.37 years

Not mentioned Schwabe16 2014 Not mentioned Atrophic non-union: 44 (22 male) (Histology: 25; GF-quantification: 19); healed fracture: 13 (7 male) (Histology: 5; GF-quantification: 8)

Non-union: Femur: 16; tibia; 12; clavicle: 9; ulna: 4; humerus: 3.

Control group: tibia: 4; ulna: 4; femur: 2; radius: 1; metacarpus: 1

49 years (range 20–74 years) Not mentioned Ismail17 2013 Not mentioned 5 (5 male) Tibia: 1; femur: 3; humerus: 1 27.40 years ± 7.64 (range, 18–17 years) 10 mls of BMA Abbreviation: BMA, bone marrow aspirate. TABLE 3. Relevant tissue: Patient Demographics. Author Year Time frame Number of specimens Site of non-union Patients’ age (mean ± SD) Amount of tissue Burska18 2020 Not mentioned 15 (study group - 10 union; 5 non-union); 18 (healthy controls) Femur, tibia 15 (study group - 10 union; 5 non-union; range 18–70 years); 18 (healthy controls; range 26–64 years) Not mentioned El-Jawhari19 2019 Not mentioned 71 (46 male) Femur, tibia, humerus Non-union group: 49 years (range: 18–76); union group: 44 years (range: 20–75); healthy controls: 42 years (range: 23–60) BMA: 15mls from ASIS; peripheral venous blood: 12mls; serum from healthy controls: not stated Ouyang20 2019 Not mentioned Not mentioned Not mentioned Not mentioned BMA: 2 ml McCoy21 2019 Biobank (Not mentioned) 131 (47 male) compared to 1627 (588 male) with uneventful healing Upper or lower extremity fractures Control group: 64.3 ± 15.0; non-union group: 66.8 ± 12.7 Not applicable Zhang22 2018 May 2012–April 2015 24 (11 male) compared to 24 (11 male) with uneventful healing Fibular head fracture Control group: 41.5 ± 11.6; non-union group: 40.4 ± 11.1 Not mentioned Huang23 2018 2012–2016 1229 (346 non-unions of which 199 males; 883 unions of which 505 males)

Tibial diaphysis: 113/315; femur diaphysis: 98/233; humeral shaft: 82/188; ulnar shaft: 39/117; femur neck: 14/30

(Non-union/Union)

Non-union: 46.1 ± 8.1;

Union: 44.7 ± 8.3

Not applicable Granchi24 2017 Not mentioned 26 (15 male) Tibia: 11; femur: 11; humerus: 3; not reported: 1 39.6 ± 14 Not applicable Sathyendra25 2014 2005–2010 Atrophic non-union: 33 (14 male); normal healing: 29 (18 male)

Non-union: femur: 13; tibia; 18; ulna: 2.

Normal healing: femur: 10; tibia; 15; humerus: 4.

Atrophic non-union: 48.6 years; normal healing: 47.3 years Not applicable Zeckey27 2011 2000–2008 50 compared to 44 patients with uneventful healing Femur: 21; tibia: 29 37.5 ± 2.0 Not applicable Dimitriou28 2011 2005–2007 62 (45 male) compared to 47 (33 male) with uneventful healing Tibia: 41; femur: 18; humerus: 2; ulna: 1 43.9 years (range, 19–65 years) Not applicable Marchelli26 2009 Not mentioned Atrophic non-union: 16 (16 male); healed - 6 months: 18 (18 males); healing - 1 month: 14 (14 males)

Atrophic non-unions: Tibia: 7; radius: 1; radius + ulna: 3; humerus: 2; femur: 3.

Healed: Tibia: 9; radius: 2; radius + ulna: 4; humerus: 1; femur: 2.

Healing: Tibia: 8; radius + ulna: 2; humerus: 2; femur: 2.

Atrophic non-union: 28.1 ± 5.9 years; healed: 32.2 ± 5.7 years; healing: 31.4 ± 7.1 years Not mentioned Xiong29 2009 Not mentioned Not mentioned Not mentioned Not mentioned Not mentioned Seebach30 2007 Not mentioned Not mentioned Male: 41 ± 15; female: 42 ± 13 Not mentioned Not mentioned Henle31 2005 Jan 2002–Jan 2004 15 (12 males) from non-unions and matched group with uncomplicated unions Tibia: 11; femur: 2; humerus: 1; forearm: 1 47 years (range, 20–75 years) Not applicable Abbreviations: ASIS, anterior superior iliac spine; BMA, bone marrow aspirate. 3.2 Studies characteristics

The study characteristics of the non-union tissue and relevant tissue are outlined in Table 4.8-31 Non-union was defined based upon radiographic and clinical examination, with minor variations between studies. Samples of non-union tissue and relevant tissue were mostly obtained during the surgical treatment of non-unions.

TABLE 4. Study characteristics of non-union tissue and relevant tissue Author Duration of non-union (months) Classification Definition of non-union Isolation of tissue Cells/material isolation Cuthbert*8 Not mentioned Atrophic Not mentioned Non-union: Fibrotic tissue lying directly between the fractured bone fragments was excised and collected; induced periosteum from centre of bone defect area; and bone marrow Colony forming unit fibroblast (CFU-F) assay; trilineage differentiation; histological analysis of vessel number, size and area; immunohistochemistry (CD45, SDF1, VEGF, BMP-2); flow cytometry; qPCR; matrigel-based angiotube formation assay Wei*9 Not mentioned Atrophic Not mentioned Tissue samples were collected intra-operatively from (i) non-union tissues of atrophic bone; and (ii) healing callus around internal fixation plates in normal controls. Collected tissues were cut into “small” pieces RNA isolation, miRNA microarray, bioinformatics of target genes, qPCR, Western blot, luciferase reporter assay Burska**18 Not mentioned Not mentioned Failure of the fracture to progress to healing radiographically with the presence of bridging callous on at least 3 cortices by a period of 9 months Peripheral blood ELISA El-Jawhari**19 Not mentioned Atrophic Absence of radiological features of fracture healing (lack of callus formation in at least 3 cortices) either on plane radiographs or computed tomography scans after 9 months from fracture fixation and with ongoing pain at the NU site during ambulation BMA; peripheral venous blood FACS cell sorting; flow cytometry surface cytokine receptor measurement; flow cytometry—immunosuppression assay: levels of IDO, PGE2 and TGF-β transcripts; osteogenic differentiation; RNA extraction; RT-qPCR; proliferation (XTT colorimetric assay); ELISA Ouyang**20 Not mentioned Not mentioned Not mentioned BMA circRNA microarray, RNA FISH, Osteogenic differentiation assay (ALP and Alizarin red staining), cck-8 assay, RNA pull-down assay, double luciferase reporter assay, qPCR, RNA immunoprecipitation, Western Blot McCoy**21 Not mentioned Not mentioned Not mentioned Peripheral blood DNA was extracted from blood samples Zhang**22 Not mentioned Not mentioned Not mentioned Peripheral blood DNA was extracted from blood samples Wang*10 Not mentioned Not mentioned Not mentioned Not applicable Cell viability; mineralization assay; gene expression Vallim*11 34 months (range 9–120 months) Not mentioned Lack of bone healing after 9 months of the fracture Fibrous tissue interposed between the bone ends was excised, along with adjacent osseous fragments Histology; population doubling; cell senescence; flow cytometry; osteogenic / adipogenic differentiation Huang**23 >9 months Not mentioned The cessation of all healing processes and failure to achieve union within 9 months without radiographic signs of progression of the fracture callus Peripheral blood DNA was extracted from blood samples Granchi**24 >3 months Not mentioned Not mentioned BMA, peripheral blood Immunoenzymatic assays Takahara*12 14.8 months (range 4–26 months) Pseudoarthrosis (1) gross motion at the fracture site on physical examination; (2) bridging bone on 0 of 4 cortices on anteroposterior and lateral radiographs; (3) CT showing no purpose- ful cross-sectional area of healing; and (4) evidence showing the existence of pseudocapsule and fluid collection between the fracture gap at the surgery A small amount of pseudoarthrosis tissue (pseudocapsule) was obtained during the surgical treatment

Alizarin Red S staining, ALP activity assay, and RT-PCR after osteogenic induction. Chondrogenic differentiation capacity was assessed via Safranin O staining and RT-PCR after chondrogenic induction.

Histological analysis and cell cultures

Schira*13 18.3 months (range, 3–100 months) Not mentioned Non-unified fractures >3 months with a resorption zone wider than 1 mm (as determined by a mandatory CT scan) with no apparent potential to heal without surgical intervention Non-union tissue (excluding the cortex) and cancellous bone from the ipsilateral radius has been obtained at the time of operative repair Histology, immunohistochemistry, gene expression Han*14 11 months (range, 6–30 months) Not mentioned Failure of the fracture to heal 6 months or more after surgery or non-surgical treatment Fracture and scar tissue during surgery, which was divided into bone stump tissue, marrow cavity contents, and sticking bone scars according to the sites Histology, immunohistochemistry, gene expression Wang