Journal of Biomedical and Biological Sciences

An Update on the Management of Long Bone Non-Unions-A Comprehensive Review

Kulvinder Kochar Kaur1*ORCID ID, Gautam Allahbadia2 and Mandeep Singh3

1Centre for Human Reproduction, Jalandhar, India

2Ex-Rotunda-A Centre for Human reproduction, Mumbai, India

3Swami Satyanand Hospital, Punjab, India

*Correspondence: Kulvinder Kochar Kaur, Scientific Director, Centre for Human Reproduction, 721, G.T.B. Nagar, Jalandhar, Punjab, India. E-mail: kulvinder.dr@gmail.com

Citation: Kaur KK, Allahbadia G and Singh M. An Update on the Management of Long Bone Non-Unions-A Comprehensive Review. Journal of Biomedical and Biological Sciences. 2021;1(1):1-23.

Copyright: © 2021 Kaur KK, et. al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Received On: 28th August, 2021   Accepted On: 12th September, 2021   Published On: 20th September, 2021

Abstract

Earlier a lot of emphasis has been given to the diamond concept for long bone non-unions treatment at the fracture site that is dependent on the biological in addition to mechanical environment. The former requires molecular mediators, progenitor cells, besides, matrix, immuno-regulatory cells as well as others. For mechanical environment both direct primary cortical healing takes place when total stabilization of bone surfaces is ensured for keeping an intricate contact of under 0.15mm, with least interfragmentary strains below 2% that gets attained with the utilization of compression lag screws or compression plating. Moreover, the indirect or secondary repair gets promoted by comparative stability. The diamond concept gives equivalent significance to the mechanical stability in addition to the biological surroundings. Here we detail the diamond concept, besides the update with regards to utilization of different bioreactors that is based only on fluid dynamics, shear strain where osteoblastic lineage gets stimulated by the preconditioning of the bone marrow mesenchymal stem cells (BMSCs) with the appropriate fluid stimuli where need for any chemical stimuli gets obviated just with the use of these fluid dynamics in the linear 3D bioreactor made. Further the role of pulsed electromagnetic fields (PEMFs), other types of 3D scaffolds and their augmentation with nano/microporous technology, as well as besides animal studies where early mobilization is advocated how in human’s immobilization required only during active repair phase as detailed in recent studies. With the use of these 3D scaffolds requirements of recombinant Bone morphogenetic protein (BMP), Stem cells, plus autologous bone grafts might get obviated with further work in future.

Keywords: diamond concept, BMSCs, PEMFs, linear 3D bioreactor, 3D scaffolds, mechanical environment, biological environment.

Introduction

Since its introduction, the diamond concept has demonstrated evidence to a significant framework for getting insight in the least needs for the repair of a fracture. Furthermore, it has demonstrated itself to be specifically of use when trying to plan for surgical treatment of fracture non-union of upper along with lower extremities [1-12].

A lot of controversies have existed over the definition of non-union, with non-consensus among 55% clinicians as regards to timing [13]. As per the US FDA it represents the inability to achieve union by 9 months since the injury as well as for whom no signs of repair are obvious for 3 months. Rest have advocated for long bones a revision is required to a duration of 6 months if no proof of fracture repair is obvious [14]. Existent of unstable fracture site is actual non-union, that is mostly correlated with, as well as clinical signs are as significant in diagnosis like the radiological evaluation [13,15].

Fracture sites that are well vascularized with plenty of fracture haematoma, yet an unstable mechanical environment will mostly then generate hypertrophic non-union, while in case of poor vascular supply in combination with local strain concentration has been pointed to result in atrophic non-union[16].All these definitions are dependent on the radiographic picture of the non-union[16].The existence or its lack of infection is further significant as far as classification is concerned, that can complicate the scenario in addition to treatment strategy [17].

Variable documentation of incidence of non-union has been done in context of literature, based on the study size, patient demographics, injury placement as well as robustness, among 2-30% [18], with a determined 100,000 fracture non-union episodes yearly in USA [15]. An Australian study conducted on 853 patients demonstrated, a total 8% of patients having fractures, requiring admission in hospital yearly for the complications of fracture healing [19]. Nevertheless, a recent much greater population that was studied in Scotland illustrated a lesser total incidence in contrast to the prior documented rate of 1.9% in the adult population, with the incidence of non-union for pelvic as well as femur fractures of 13/1000,humerus of 30/1000, tibia of 55/1000.This incidence was observed to peak in the 25-44 years age group [20].This is correlated with significant financial complications with total documented cost among 21.183₤ as well as ₤33,752/patient [21].

Methods

Here we conducted a systematic review utilizing search engine PubMed, google scholar, web of science, Embase, Cochrane review library utilizing the MeSH terms like DKD, Epigenetics, DNA methylation, Histone post-translational modifications, Histone acetylation, Histone crotonylation, Histone-β-hydroxy butyrylation, Apelin, curcumin analogs, Apabetalone, BET Proteins, BRD’s, TET, BMP’s, sodium butyrate from 1975 to 2021 till date with the utilization of PRISMA Guidelines.

Results

We found a total of 3050 articles out of which we selected 70 articles for this review, it being an update on earlier therapies. No meta-analysis was done see (Figure 1).SNI-JBBS-21-04-Figure 1

Figure 1: Selection Criteria

Chances of Non-union

Chances of non-union by definition can be patient based or independent, in addition to local as well as systemic factors, certain of which get modulated for escalation of fracture repair.

A recent systematic review into the chances of categorization demonstrated that proof with regards to quantification relative risk differs, varying from level 1 to level 5 proof [22].

Efforts have been made for the generation of scoring systems for anticipation of the chances of non-union, inclusive of the Non-union scoring systems (NUSS) [23], along with the Moghaddam risk score [24]. Certain authors have pointed that the proper treatment method of non-union needs to be dependent on the robustness of the NUSS utilized.

Diamond Concept along with fracture Repair

An adequate fracture Repair reaction is based upon the biological microenvironment at the fracture region (availability of the molecular modulators, progenitor cells in addition to matrix, immune-controlling cells, besides others) along with ideal mechanical surrounding which yield the fracture region with proper stabilization, that promotes the physiological event which results in a successful bone repair reaction. Generally, 2 modes are present by which bone repair occurs

  • Direct or primary cortical bone healing takes place in which case there is absolute stabilization of bone surfaces with intimate contact of under0.15mm, as well as least inter fragmentary strains of <2 % which can only get obtained via compression lag screw or compression plating [15, 25-27].
  • Secondly indirect mode, or secondary repair gets promoted by relative stabilization. In toto the phases of fracture repair are stratified into i) fracture haematoma generation ii) inflammation iii) the cellular proliferation iv) differentiation v) remodelling/refashioning [28].

The Diamond Concept is a conceptual frame for getting a favourable bone healing reaction, i) that provides equivalent significance to mechanical stabilization ii) in addition to the bone microenvironment. Furthermore, enough vascularity of bone as well as the physiological situation of the host are believed to be necessary in the framework with regards to the healing of the fracture. A deficiency in the biological microenvironment/mechanical environment or inability of estimating/account the comorbidities of the host, besides absence of vascularity can result in an impaired fracture repair reaction (akin to NU). In toto, the Diamond Concept is the existence of osteo-inductive modulators, osteogenic cells, a matrix that is osteoconductive alias scaffold, ideal mechanical surroundings, enough vascularity, besides taking into account any kind of comorbidity that is associated in the host (Figure 2) [1, 2, 29]. The significant components of the Diamond Concept are detailed here.

SNI-JBBS-21-04-Figure 2

Figure 2: Courtesy reference number -29-Schematic representation of molecular pathways activated by pulsed electromagnetic fields (PEMFs). Abbreviations: A2A (adenosine receptor); alp (alkaline phosphatase gene); BMPs (bone morphogenetic proteins); ibsp (bone sialoprotein gene); col1 (collagen type 1 gene); ERK ( extracellular signal-regulated kinase 1/2); GH (growth hormone); GHR (growth hormone receptor); IGF (insulin-like growth factor); JAK-STAT (Janus kinase- signal transucer activating the transcription); MAPK (mitogen-activated protein kinase); mTOR (mammalian-mechanistic target of rapamycin); NICD (Notch intracellular domain); ocn (osteocalcin gene); opn (ostepontin gene); PTH (parathyroid hormone); runx-2 (runt-related-transcription factor 2 gene); SMAD proteins (small mothers against decapentaplegic); TGF-β (transforming growth factor-β); TGF-β RI/RII (TGF-β receptor I/receptor II); VEGF (vascular endothelial growth factor); VEGFR-2 (vascular endothelial growth factor receptor 2).

Osteoconductive modulators

Immediate bleeding subsequent to a fracture starts the coagulation cascade which results in the generation of a fracture haematoma [29]. This possesses platelets in addition to macrophages, that liberate a series of cytokines of various kinds, that results in activation of a cascade of processes to get the repair started. These are inclusive of proinflammatory interleukin S-1 (IL-1), IL-6, IL-8, as well as IL-12], tumour necrosis factor alpha (TNFα), activated protein C(APC), monocyte chemoattractant protein 1(MCP1), macrophage colony stimulating factor (M-CSF), Receptor Activator of nuclear factor κβ ligand (RANKL), in addition to osteoprotegerin (OPG) [1, 30, 31]. Metalloproteinases along with angiogenic growth factors like Vascular endothelial growth factor (VEGF) play a significant part in the total bone healing event [1, 30, 31]. Nevertheless, the modulators liberated carrying maximum significance that possess a direct action on the progenitor cells to go via the event of mitogenesis as well as osteoblastic differentiation are platelet-derived growth factor (PDGF), Fibroblast growth factor (FGF), insulin –like growth factor (IGF) transforming growth factor beta (TGF-β) proteins, that are inclusive of Bone morphogenetic protein (BMP)-2,4,6,7 (Figure 3) [1, 30, 31].

SNI-JBBS-21-04-Figure 3

 

Figure 3: Courtesy reference number 29-Diagrammatic representation of fracture haematoma composition. Key: IL interleukin, MCP monocyte chemo attractive protein, M-CSF monocyte colony-stimulating factor, BMP bone morphogenic protein, PDGF platelet-derived growth factor, VEGF vascular endothelial growth factor, RANKL receptor activator of nuclear factor kappa-B ligand, OPG osteoprotegerin, SOST sclerostin.

Osteogenic Cells

Osteogenic Cells possess both committed osteogenic progenitor cells from the periosteum in addition to undifferentiated multipotent mesenchymal stem cells(MSC’s) from bone marrow, as well as endothelial progenitor cells get activated as well as per the local fracture surroundings in the haematoma[1].The cytokines liberated result in making sure an inflammatory phase, possessing the properties of escalated blood flow in addition to vascular permeability, chemotaxis, with the complement cascade .Osteoclasts as well as fibroblasts stimulate crosstalk of haematoma with granulation tissue, that lay down fibrin meshwork, that then gets invaded by new capillary network that aids in more MSC’s getting migrated . Subsequent to activation, cytokines further get liberated by the endothelial cells, MSC’s, chondrocytes, osteocytes as well as osteoblast themselves [1, 14, 30, 32]. Proliferation as well as differentiation of 2018 MSC’s results in the generation of concomitant hard as well as soft callus generation, that get markedly implicated via the mechanical microenvironment along with fracture biology [25, 27, 33].

Greater oxygen (O2) tension at the periosteal surfaces distal to the fracture areas in addition to other mineralization factors, stimulate a preference of MSCs differentiation into Osteoblasts [15]. In the cortical alias peripheral zone, osteocalcin stimulates periosteal osteoblasts for generation of type 1 collagen fibres resulting in intramembranous ossification (hard callus). In the central alias medullary zones, MSCs generate into Chondrocytes, to start with they lay down type 2 collagen alias soft callus that is referred to as endochondral ossification (soft callus), by week 3. enhancement of osteocalcin resulting in calcification along with hard callus generation (figure 4). Mineralization of fracture callus into an osteoid type matrix as well as type 1 collagen fibres resulting in closure of the fracture area with the ill organized ‘’woven bone ‘’ generation [27, 34-36]. Key in driving this event is the BMP’s that are implicated in the induction of Ostegenic action in mesenchymal stem cells in addition to maturation of lamellar bone, along with aiding in coordination of osteoclastic action [27, 36, 37]. Inhibitory, along with fibrinolytic molecules further possess a crucial part in controlling the event in the absence of which bone healing has been demonstrated to delay bone healing [27, 31, 38]. Subsequently basic multicellular unit (BMU) of bone remodelling in an event of activation, resorption, reversal in addition to generation, using at least 6 months to finish. This ill organized woven bone that is relatively weak, generates into a more robust, organized laminar bone that is following the general principles of Wolf’s law, who illustrated that the trabecular bone is the one meant for the mechanical stresses exerted on it [40, 41].

SNI-JBBS-21-04-Figure 4

Figure 4: Courtesy ref no-29-Diagrammatic representation of ossification: an Intramembranous ossification. Osteoinductive mediators induce osteogenic MSCs to differentiate into osteoblasts, which lay down osteoid (collagen-1 rich); this mineralises to form an ossification centre, whence mineralisation extends. There is terminal differentiation into osteocytes, becoming entombed in the bone matrix. b Endochondral ossification. Osteoinductive mediators induce osteogenic MSCs to differentiate into chondrocytes; a cartilage matrix is secreted which forms the template for endochondral bone formation. Chondrocytes then undergo hypertrophic differentiation and mineralise the surrounding matrix. They eventually undergo apoptosis—resulting in vascular invasion. Invading blood vessels convey osteoblasts which form bone on the cartilage template.

Extracellular Osteoconductive matrix (scaffolds)

An Osteoconductive extracellular matrix (ECM)working in the form of a scaffold in addition to facilitating migration as well as adhesion of osteoinductive along with osteogenic cells to the fracture area is necessary for fracture repair. In case a good bone apposition exists, necrotic bone at the fracture area does this part. In case there is inadequate natural scaffold, then autograft or allograft demineralized bone matrix (DBM), that possesses inherent Osteoinductive capacity secondary to growth factors (GF) that get retained inclusive of BMP, can get utilized for the treatment of non-union of bone deficits [1, 27, 36].

 Mechanical Surroundings

Proof exists that cells possess the capacity to pick up the surroundings with the context of mechanical environment, via the electrochemical signalling that gets developed by the fluid flow in the canaliculate in the osteocytes in addition to the other cell kinds that possess cell membrane mechanoreceptors along with direct communication among the cell nucleus as well as local cytoskeleton, that get further affected by the chemical environment in addition to molecules associated with cellular signalling [33]. Depending on studies conducted on cells in culture, cellular generation has been illustrated, to get significantly implicated by the local mechanics with the mechanical in addition to physiological conditions that strongly influence the further lineage differentiation of multipotent mesenchymal stem cells. With the existence of proper growth factors, tension results in stimulation of fibroblasts, shear induces chondroblasts, with a combination of compression with distraction stimulating osteoblasts that points to the mechanical surroundings in which cells generally get generated [25].

By definition strain is the extension/unit length in association with the force utilized that suggests that the load in addition to the micromotion at the fracture area is significant for starting the repair event as documented by Perren [16, 26]. Axial micromotion appears to result in activation of fracture repair in the initial stages; by 8 weeks, this association gets reversed, as shown in normal repair event by escalation of the callus stiffness, whose normal goal is to cause reduction of movement at this stage [25, 26]. Lower strain rates facilitate intra membranous ossification, nevertheless, endochondral ossification has greater chances of getting started in case of escalation of the strain rates. On escalation of the strain rates beyond a certain limit, nevertheless escalated differentiation on the soft tissue pathway becomes the dominant one, that results in delayed/non-union [31]. For the ossification to take place, the fracture gap needs to have undergone reduction to a proper amount, influenced further by the comparative stiffness of the tissues which surround it. Ideally these need to be under 2 mm as well as definitely under 6 mm as illustrated at experimental level over which hardly any callus gets generated [27, 34].

Host Factors as well as Vascularity

In case vascular supply or fracture haematoma gets interfered with or deleted, a greater chance of non-union occurs in view of inadequate Osteoinductive along with Ostegenic cells would be existing at the fracture area for the starting of ostegenesis, refashioning along with repair [2, 39]. Its risk significantly escalates in case of high energy in addition to open fractures, or in case of primary surgical repair in which case the fracture biology, periosteum as well as soft tissue envelope are not given adequate respect [24, 42]. Periosteum along with provision of key blood supply, has its individual bone regeneration potential [43]. Akin to that in case of changed systemic physiology of the host or associated comorbidities, further repair would get influenced by these [2, 39, 42].

Biological Chamber

This thought with regards to Biological Chamber is dependent on the requirement of containing, like in case of a long bone non-union has got the treatment keeping in mind the diamond principle, it has to be understood that any biological escalation which got placed at the non-union area, needs to be contained locally to get the maximum action getting impacted. It is the generation of the Chamber that lets in an influx of biological actions for facilitation of the repair in a timely manner. That is what we are discussing is the generation of a bioreactor. Confining the selected treatment method for non-union can get attained via modulation of soft tissues, biological membranes, sealants etc. Particularly this is significant on consideration of the comparative mean retention times of these specific ingredients [2, 44].

Discussion on Diamond Concept

The lookout for the best strategy is continuing. Specifically, the ones that are resistant to treatment are the toughest for management with long-term therapy as well as financial coverage going out of pocket. The diamond concept strategy gives a new prototype with regards to the same. Additionally mechanical microenvironment requires to be taken care of. A robust biological stimulus gets given on supplementation with a scaffold, growth factors, in addition to multipotent mesenchymal stem cells along with simultaneously giving respect to the local blood supply as well as fracture biology. Further more patient comorbidities need to be taken into account for getting over the restrictions imposed by the host’s physiological conditions.

With the context of upper limb Calori et al. [8] conducted a study comprising of 54 patients with upper limb non-unions, where they contrasted polytherapy diamond concept vis a vis mono therapy for non-unions. Patients that received polytherapy constituted by bone morphogenetic protein-7(BMP-7), MSCs, autologous bone graft (ABG) alias re-osteosynthesis) possessed the worst subjects of non-unions to start with. Statistical evaluation documented polytherapy to be significantly superior to mono therapy in case of Clinical, radiological in addition to functional results, as well as greater (89% vs 63%) of union getting attained. Inspite of restriction of fracture variability, sample size as well as retrospective kind of study, these outcomes are quite impressive. Miska et al. [11], evaluated 50 patients presenting with humeral non-unions that received treatment, on the basis of their risk profiles with separate angles of the diamond concept, where just 6 patients possessed the total spectrum of the diamond getting utilized. Such patients received therapy with the utilization of angular stable plating (i.e., mechanical stability) autologous bone graft (MSCs as well as scaffolds) in addition to BMP7 getting applied as an Osteoinductive agent. In toto union rate of 80% was observed. Nevertheless, it is not certain how the results in the diamond concept performed in contrast to others.

 Haubruck et al. [5], managed 156 patients presenting with lower limb non-unions of which 69 were femurs whereas 87 tibias, where they contrasted BMP2 from BMP7 for one and 2 steps revisions with the utilization of ABG to give a scaffold in addition to MSCs along with conducted a re-osteosynthesis for escalation of stability. Total union rate of 91% with BMP2 along with 58% with BMP7(p<0.001), with akin rates of total repair of femurs as well as tibias. In case when combination of BMP groups was utilized it was at 64 as well as 63% respectively, nevertheless in BMP2 group they were 79 as well as 97% respectively that demonstrated a promising potential of the diamond concept.

Giannoudis PV et al. [4], documented, in a retrospective cohort comprising of 14 patients with Subtrochanteric femoral fracture non-unions, of which 4 were open fractures. Impressive results on utilization of all the 4 principles of diamond concept namely debridement, blade plate or revision IM nail (mechanical stability), RIA (reamer, irregular aspirator) from the opposite femur for ABG to get primarily utilized in the form of a scaffold. BMP7 as well as Bone marrow aspirate concentrate (BMAC) for provision of MSCs from the iliac crest, with water tight closure in layers to make sure that containment of the bioactive materials within the biological chamber. In toto union rate of 90% was observed. This study emphasized the existence of varus of fixed acute fractures, stressing that absence of mechanical stability got successfully treated when a part of diamond concept concentrated management. Golf et al. [7], documented, a very good result on application of all the components of the diamond concept when attempting treatment of delayed non-union in the femoral inter trochanteric, by the utilization of two stage modulation of Masquel et al. method that was escalated with BMP7 as well as RIA utilized to provide ABG. Giannoudis et al. [45], conducted another study for evaluation of non-unions from all areas (upper as well as lower limb). Application of diamond concept for the treatment of 64 patients out of which 65% had undergone high energy injuries, in which a non-unions rate was 98% by 12 months, again that illustrated the significance of the diamond concept for the management of these complicated cases.

Moghaddam et al. [9] documented in a study comprising of 88 patients with subtrochanteric femoral fracture non-unions, that was inclusive of 21% with open fractures. Application of all constituents of the diamond concept in case of patients considered to have high risk fracture for repair as part of a single or and 2 steps method. RIA samples were obtained from the femur or ABG for provision of MSCs from the iliac crest in addition to scaffold with BMP7 as well as tricalcium phosphate. 72 Clinical results got a total supplementation of diamond concept, whereas 13 just received ABG, as well as 3 just had a revision of the metal work. No total diamond concept-particular results were given. In toto 69 patients (78%) attained good repair, on application of this strategy, with union in single step method depicted to be greater (95.1%) in contrast to2 steps (63.8%) method in total. Union rates were significantly greater in the femoral diaphysis in contrast to 2 distal femur (84 vs 70% respectively) particularly on utilization of an intramedullary nail. Larger gaps (5-10 cm) in the diaphysis that got treated with a 2 steps method possessed bad repair rates (58%), with cigarette smoking imposing significant influence. Only in subjects in which the total diameter did not cross the whole diameter, with an osseous bridge still persisting did repair take place. This study illustrated that existence of bad vascularity in addition to challenges in attaining mechanical stability resulted in dysfunctional fracture healing, pining their significance in being inclusive in the diamond.

Moghaddam et al. [10], further tried to do evaluation of the treatment of non-unions of tibia with observed results of union in 84% in single stage (group 1) in addition to 80% in two stage(group2) methods, with escalation of elements belonging to the diamond concept, as per the risk profile, in which 76% of patients, in total got a combination of RIA, BMP7 as well as tricalcium phosphate, besides re-osteosynthesis. Akin to first study no total diamond concept-particular results were provided. The outcomes, demonstrated that the application of the ideas with regards to diamond concept that is dependent on the risk profile on attempting treatment of both kinds of fracture resulted in union rates that can get accepted. Inspite of possessing worst initiating position, patients in group2 possessed akin results later. Moghaddam et al. [10], pointed to a greater quantity of earlier surgical procedures in group 2 patients (mean 3.4 vs 2.4) aiding in worst repair secondary to scar tissue interfering with the blood flow, nevertheless, no absolute Figure s or multivariate evaluation with p values for validation the results of this sub group. Group1 as well as group 2 both belonged to medium risk category for non-union on the Moghaddam prediction score in addition to the NUSS, with behaviour as anticipated, further the patients in group 2 who possessed greater scores took longer duration for repair to occur. Microbiology outcomes also pointed that in earlier non-picked up atrophic non-unions, low grade infection might be the etiology as well as in these subjects Masquelet methods can get utilized with greater efficacy. The outcomes further pointed that antibiotic osteitis prophylaxis in the group 1 who received treatment with gentamycin-coated nail was more advantageous.

Douras [6], documented a case study, where all the components of diamond concept had been utilized for the treatment of medial malleolus non-union, where total union got attained by 6 months, with patient possessing normal function, that demonstrated that this strategy is efficacious in ankle fracture non-union as well.

Olivier et al. [12], conducted a study on 20 patients possessing resistant tibial non-union, with the object of application of diamond concept components in a variable way. Besides debridement as well as re-ostosynthesis, a composite graft with the utilization of synthetic re-absorbable calcium phosphate bone substitute in combination with BMP rather than ABG, that might be of utility in patients possessing poor bone stock. What was significant was that no stem cells aid was provided. Union rates were 90%, pointing that it might be safe as well as efficacious in case ABG cannot be attempted.

All the studies detailed here mostly represent practically the whole of literature where in particular utilization of diamond concept has been done for guiding the management in case of long bone non-unions. As is obvious once all the principles with regards to diamond concept got utilized in addition to its elements struck to, as well as escalated at the time of surgery, excellent union rates were observed in upper as well as lower limb non-unions. Biological, in addition to mechanical surroundings, systemic factors in addition to local blood supply along with fracture pattern, position, as well as earlier attempts at management in addition to stability of implants. Nevertheless, just occasional of the earlier studies utilized bone marrow aspirate concentrate (BMAC), in the form of direct ways of concentrations of mesenchymal stem cells to work in the form of osteoprogenitors, with maximum dependent on the MSCs existing in RIA or autologous bone graft (ABG)from the iliac crest. With the experience of Giannoudis group with the RIA maximum of the MSCs out of the bone stock get washed, thus resulted in reduction of the usefulness in the kind of provider of robust osteogenic cells. Earlier their group had documented that following the age of 55, the iliac crest further becomes redundant in the form of supplier of MSCs, possessing a greater yellow look with lesser stem cells to help in the union [46]. Their belief is that in subjects that are compromised utilization of BMAC, as a provider of MSCs that is a dependable source of MSCs, in the form of the diamond that can aid in attainment of good outcomes.

The proof present to demonstrate that utilization of polytherapy with the diamond concept was possessing advantage over mono therapy has given convincing evidence yet the total quantity of subjects treated continues to be small, as detailed by Calori et al. [47] as well. Thus, conclusions of Giannoudis PV group were that despite studies they had quoted just high-lighted all angles of the ’diamond concept for high-risk cases, that might still cover the total picture, besides advocated utilization of prospective randomized studies in future to further highlight this. A single-site retrospective database of older adults with long bone non-unions treated via “diamond concept” were evaluated by Tanner et al. [48]. All medical records of patients receiving surgical treatment of non-unions amongst 01/01/2010 and 31/12/2016 got reviewed. Clinical in addition to radiological out results following non-union therapy were analysed. Overall, 76 patients (37 patients received treatment with one-step and 39 patients with Masquelet therapy) who suffered from a non-union older than 60 years that received treatment as detailed earlier for the duration in their hospital were included into the present study. Bone consolidation was attained in 91.9% following one-step and 76.9% following the Masquelet therapy. Evaluation of age as a risk factor in the outcome of non-union therapy illustrated no significant variation in patients received treatment with one-step procedure as per the “diamond concept”. Conversely, age had a significant negative impact on the results of the Masquelet therapy (p = 0.027). Thus, the conclusions that were drawn by them was that non-union therapy as per the “diamond concept” is an efficacious, besides a strategy on which one can rely on treatment strategy in elderly patients. As per the observation of the present study, older adults that suffered from an infected non-union had advantage with a two-stage procedure, while in patients suffering from a non-infected non-union, a one-step surgical treatment is better [48].

Role of optimization of the mechanical environment in reduction in fracture healing time

The impact of the local mechanical environment in the fracture gap on the bone healing process has been extensively investigated. Whilst it is widely accepted that mechanical stimulation is integral to callus formation and secondary bone healing, treatment strategies that aim to harness that potential are rare. In fact, the current clinical practice with an initially partial or non-weight-bearing approach appears to contradict the findings from animal experiments that early mechanical stimulation is critical. Therefore, we posed the question as to whether optimizing the mechanical environment over the course of healing can deliver a clinically significant reduction in fracture healing time. In reviewing the evidence from pre-clinical studies that investigate the influence of mechanics on bone healing, we formulate a hypothesis for the stimulation protocol which has the potential to shorten healing time. The protocol involves confining stimulation predominantly to the proliferative phase of healing and including adequate rest periods between applications of stimulation. Suffering, beneficial [49].

Role of generation of a laminar flow bioreactor

Bone possesses a restricted ability of spontaneous repair of key deficiencies occurring secondary to injury, inflammation, or therapeutic resection [50]. At present accelerated surgical treatment is needed for the restoration of structure in addition to function, that is usually associated with a lot of postoperative complications [51]. Hence a requirement urgently for tissue engineering approaches dependent on biomaterials in addition to multipotent cells (MC) as well as growth factors (GF) in an alternative approach for the bone regeneration [52]. 3D microporous scaffolds, that simulate the structure of bone matrix, get essential for regulation of spatiotemporal cells getting redistributed as well as bioactive clues. A porous structure facilitates the osteogenic differentiation of multipotent cells. Particularly, significantly porous structure with pore sizes 100-600 µm, seem to promote cell adhesion growth, along with mineral generation in addition to blood vessel generation, in vivo in the construct [53]. A hurdle encountered technically for its clinical utilization for cell treatment with scaffold generation is its size. In a cell dependent tissue engineering strategy, 3D scaffolds along with MC as well as the core of the engineered construct [54]. With the escalation of volume of 3D scaffolds, the cells become prone to a deficiency of nutrient in addition to gas provision, that results in the worsening of cell viability along with multipotency [55].

Perfusion of the culture medium via scaffolds ensures homogenisation of nutritional provision in addition to deletion of waste generated [56]. Different kinds of bioreactors have got generated for this, inclusive of spinner flasks bioreactors, rotating wall vessel bioreactors as well as laminar flow bioreactors [57]. Furthermore, fluid flow implicates cell behaviours, hence these bioreactors systems utilization can be done for regulation of growth as well as differentiation of progenitors mechanically [58]. Utilization of flow bioreactors has received specific highlighting in bone tissue engineering 3D in view of bone remodelling being intricately associated with the fluid surroundings secondary to little bone deformation following a little bit of physical deformation [59].Conversely it has been documented that mammalian cells have a predisposition to fluid shear, with a shear stress that is not proper, results in cell demise [60].The shear stimulated injury gets further exaggerated by turbulent blood flow, that possesses greater chances of generation in the spinner flask in addition to rotating wall vessel bioreactors [60, 61]. Laminar flow bioreactors promote appropriate regulation of fluid pattern via engineered constructs that is believed to result in less injury as well as greater anticipation event [62]. Furthermore, laminar flow bioreactors give uniform nutrient along with gas provision within the constructs that possesses relatively lesser shear rate [55]. Hence the utilization of laminar flow bioreactors appears favourable for the regulation of the fate of progenitors by the fluidic stimuli.

In case of bone tissue engineering, various studies in 2dimensional (D) flow systems documented the mechanical stimulation of Osteogenesis in the absence of Osteogenic supplements, that is inclusive of dexamethasone, β–glycerophosphate in addition to ascorbic acid or bone morphogenetic protein 2(BMP2). Like as early as 1h following perfusion Osteoblast precursors, MCT3-E1, responded to shear stress at 2 Pa by up regulation of a critical transcription factor, Runt –related transcription factor (RUNX2), for Ostegenesis [63]. Moreover, the up regulation of other Osteogenic markers like type 1 collagen (Col1), osteocalcin (OCN), alkaline phosphatase (ALP), was seen following 3d of persistent perfusion [64]. Akin to that mesenchymal stem/stromal cell (MSCs) were. documented to go via Osteogenesis is simply secondary to mechanical stimuli. In case of studies where human bone marrow obtained MSCs documented that shear stress at 0.4 -2 Pa 2.2 escalated by the expression of BMP2, bone sialoprotein (BSP), osteopontin (OPN), as well as ALP was seen along with escalated calcium getting deposited in 7days [65, 66]. Similar outcomes were documented in MSCs obtained from rodents at 1.09 Pa-1.03 Pa [67]. In contrast to 2Dmodels, nevertheless restricted proof on fluid flow – stimulated Osteogenesis in the 3D surroundings. Human fetal Osteoblasts (hFOB) 1.19 that received cyclic fluid shear stress at 3.03mpa x 28days on functionalized polycaprolactone/ hydroxyapatite scaffolds, illustrated escalated ALP action, extracellular matrix (ECM) generation along with mineralisation [68]. Occasional studies of MSCs in a 3D surrounding have documented, facilitation in addition to hampering of Osteogenesis in fluid flow. Nevertheless, maximum of these studies was carried out in the existence of either chemical supplementation or Osteoinductive biomaterials like decellularized matrix constructs. ECM-coated or hydroxyapatite laden scaffolds [62, 69, 70]. The lack of proof validating fluid flow stimulated Osteogenesis in 3D scaffolds might further get accounted by the complicated nature of the 3D culture systems. A relatively complicated bioreactor setup is needed for the generation of stable culture systems like systems for monitoring the surrounding along with regulation. Furthermore, the evaluation of flow pattern in culture chambers existing in 3D constructs has its own challenges. Different from the 2 D surroundings, fluid actions in culture chambers get applied in the form of a lot of directional shear force (implying a sliding force exerted parallel to the surface) as well as pressure (meaning a compression force exerted perpendicularly onto the surface). Hence computationally costly for evaluation of fluid in addition to a full factor checking of the experimental configurations is a must for attaining greater anticipation power [71].

Hence it is not certain if MSC osteogenesis can get stimulated just by 3D constructs, i.e., without addition of osteogenic supplements. Thus Yamada et al., aimed to precondition the MSCs for bone regeneration by generation along with trying to optimizing a laminar flow bioreactor. It was posited that proper fluid flow would guide MSCs towards the Osteoblastic lineage when there was lack of Osteoinductive biomaterials/supplements. More objective was to estimate the ideal flow for aiding the cell growth, where severe osteogenesis got stimulated. Bone marrow obtained MSCs (BMSCs) from Lewis rats (r BMSCs) got seeded onto the 3D microporous scaffolds made of synthetic copolymer, poly-L- (lactide-co-trimethylene carbonate) lactide (LTMC). This was made to fluid flow at various rates for21 days in a custom-fashioned laminar flow bioreactor. They managed to stimulate osteogenesis in the bioreactor without the existence of osteogenic supplements, that got corroborated by the expression patterns of osteogenic in addition to multipotent markers, cell proliferation, morphology, ECM generation along with calcium deposition. This strategy will open the probability of clinical translation of a laminar flow bioreactor in the stimulation of osteogenesis without utilization of chemicals that could result in reduction of unknown adverse actions of the drugs along with is anticipated to escalate bone regeneration following their transplantation of preconditioned constructs into injured areas (Figure 5, 6, 7) [72].

SNI-JBBS-21-04-Figure 5

Figure 5: Courtesy reference number 72-(a) The geometry of the culture chamber and the inlet/outlet was computationally reproduced. In the culture chambers, the scaffolds and rectifiers were placed as those in the actual experimental setting, (b) from the inlet, fully developed laminar flow of 0.8 ml/min (FL-L) or 1.6 ml/min (FL-H) was applied, and (c–f) the mean values and the range of velocity, Reynolds number, share stress, hydrodynamic pressure and their distribution within the scaffold construct were depicted.

SNI-JBBS-21-04-Figure 6

Figure 6: Courtesy reference number 72-Characterisation of rBMSC used in the study: (a) rBMSC used in the study possessed a plastic adherent property and showed spindle morphology, (b) rBMSC were capable of differentiating into osteoblasts, adipocytes and chondrocytes under inductive culture conditions, (c) the gating strategy for the flow cytometry analysis for live cells and (d) fixed cells. Cells were distinguished from debris in the FSC-A VS SSC-A plots and then singlets were distinguished in the FSC-A VS FCS-H plot, and (e) rBMSC exclusively expressed putative rat MSC markers including CD44H, CD73, Sca-1/Ly6 and CD90 while they did not express haematopoietic markers including CD34, CD45 and CD79. Stro1 expression was only found in approximately 4% of the population.

SNI-JBBS-21-04-Figure 7

Figure 7: Courtesy reference number 72-Morphological assessment of rBMSC subjected to differential flow rates for 7 days: (a and b) immunofluorescent images of αTubulin, F-actin and ROCK 1, (c) mRNA expression of ROCK1, and (d) image quantification of F-actin and ROCK1 intensity. Under the effect of fluid flow, rBMSC altered their morphology and alignment accompanied with the upregulation of ROCK1.

FL-L: 0.8 ml/min, FL-H: 1.6 ml/min. Scale bar = 100 μm.

*p < 0.05. **p < 0.01. ***p < 0.001.

Role of Low intensity pulsed ultrasound (LIPUS)

Low intensity pulsed ultrasound (LIPUS) is a non-invasive therapy for non-union treatment that can improve the long-term outcome. The purpose of this study is to summarize the available literature assessing LIPUS potential to improve the union rate in instrumented, infected, and fragility non-unions.

They did a utilization of review of the literature from the 3search engines namely PubMed, Embase as well CINAHL as from A literature search was conducted in the MEDLINE, EMBASE, and for all relevant literature on the healing rates of LIPUS utilized in instrumented, infected, and fragility non-unions. Study properties were summarized for every one of the studies that got included. Hence the percentage of healing rate as decided from the healed patients, for instrumented, infected, in addition to fragility fracture non-union patients which got pooled from every study that had been included.

Identification of an overall number that was inclusive of 326 articles, while searching for the reference lists, besides grey literature that got identified along with 3 articles. There was a total of 29 articles included in this review, with 20 articles included within the quantitative synthesis of healing rates. The most common design of included studies was case series (17 articles), followed by case reports (9 articles). Studies were primarily retrospective (18 studies), with an additional 10 prospective studies. Non-union healing rates were 82% (95% CI: 76 to 87%) in instrumented, 82% (95% CI: 70 to 95%) in infected, and 91% (95% CI: 87 to 95%) in fragility fracture patients with non-unions.

This study has provided a thorough overview of the current literature on LIPUS treatment for instrumented, infected, and fragility fracture non-unions. The healing rates for non-unions in these subgroups were comparable to healing rates observed with LIPUS use in general non-union literature. LIPUS treatment should be considered as a conservative non-surgical treatment option to potentially reduce the socioeconomic impact and improve the quality of life of these unfortunate patients [73].

Role of Pulsed Electromagnetic Fields (PEMFs)

Subsequent to reviewing 2-3 articles with regards to Pulsed Electromagnetic Fields (PEMFs) that is being carried out by certain Israeli workers recently Caliogna et al., exhaustively reviewed the recent available documentation with regards to the signalling pathways that get modulated by Pulsed Electromagnetic Fields (PEMFs) in addition to the clinical application of PEMFs. They did a utilization of review of the literature from the 2 search engines namely PubMed as well as Embase from 3 to 5 March 2021. The evaluation was carried out by 3 of the authors using studies and the data extraction from them. All studies for this review were based on these inclusion criteria: i) studies written in English, ii) studies where full text was traceable iv) besides, studies that had got published in peer-reviewed journal. Identification of cell membrane receptors. besides pathways implicated in bone healing, in addition to the molecular biology, along with evaluation of the PEMFs target of action have provided a robust basis for clinical applications of PEMFs (see Figure 8). Nevertheless, greater biology studies in addition to clinical trials with clarification of the standardisation of factors that were inclusive of intensity, frequency, dose, duration, type of coil are all needed for getting the exact dose-response association, besides getting an insight with regards to the actual applications in clinical scenario of PEMFs [74].

SNI-JBBS-21-04-Figure 8

Figure 8: Courtesy reference number 74-Schematic representation of molecular pathways activated by pulsed electromagnetic fields (PEMFs). Abbreviations: A2A (adenosine receptor); alp (alkaline phosphatase gene); BMPs (bone morphogenetic proteins); ibsp (bone sialoprotein gene); col1 (collagen type 1 gene); ERK ( extracellular signal-regulated kinase 1/2); GH (growth hormone); GHR (growth hormone receptor); IGF (insulin-like growth factor); JAK-STAT (Janus kinase- signal transducer activating the transcription); MAPK (mitogen-activated protein kinase); mTOR (mammalian-mechanistic target of rapamycin); NICD (Notch intracellular domain); ocn (osteocalcin gene); opn (ostepontin gene); PTH (parathyroid hormone); runx-2 (runt-related-transcription factor 2 gene); SMAD proteins (small mothers against decapentaplegic); TGF-β (transforming growth factor-β); TGF-β RI/RII (TGF-β receptor I/receptor II); VEGF (vascular endothelial growth factor); VEGFR-2 (vascular endothelial growth factor receptor 2).

Anticipation Scoring System for Tibial Fracture Non-union

To evaluate the available tibial fracture non-union anticipation scores in addition to evaluate their strengths, weaknesses, and restrictions, Chloros et al., attempted this evaluation. The first part comprised a systematic strategy of finding the presently attainable clinico-radiological non-union anticipation scores. The second part of the evaluation comprised of contrasting the corroboration of the non-union anticipation scores in 15 patients with tibial shaft fractures that had been recruited at random from a Level I trauma centre with prospectively collected database who were treated with intramedullary nailing. Four scoring systems were found: The Leeds-Genoa Non-Union Index (LEG-NUI), the Non-Union Determination Score (NURD), the FRACTING score, and the Tibial Fracture Healing Score (TFHS). The demographics of the patients were as follow: i) Non-union group: five male patients, mean age 36.4 years (18–50); ii) Union group: ten patients (8 males) with mean age 39.8 years (20–66). The following score thresholds were used for estimation of positive in addition to negative anticipative values for non-union: FRACTING score ≥ 7 at the immediate post-operative period, LEG-NUI score ≥ 5 within 12 weeks, NURD score ≥ 9 at the immediate post-operative period, and TFHS < 3 at 12 weeks. For the FRACTING, LEG-NUI and NURD scores, the positive anticipative values for the generation of non-union were 80, 100, 40% respectively, whereas the negative anticipative values were 60, 90 and 90%. The TFHS could not be retrospectively calculated for strong precisions. Thus, their Conclusions drawn were that the LEG-NUI had the best combination of positive and negative anticipative values for early detection of non-union. Dependent on this study, all presently available scores possess inherent strengths as well as limitations. Thus, different recommendations were provided by the group of Giannoudis PV for the escalation of future score designs are detailed by them for more advantageous handling of this devastating, nevertheless, unresolved issue [75].

Role of nano- and micro-topographical cues for escalated healing

Basis of achievement of success for fracture healing is the need for an ideal mechanical in addition to biological surroundings at the fracture site. Impairment in fracture healing (non-union) besides key size bone defects, where void volume is greater in contrast to the self-healing ability of bone tissue, remain great challenges for the orthopedic surgeons. With this context to handle these challenges, new surgical implant concepts have been recently generated to attain the ideal mechanical conditions. Whanfert et al., first reviewed the mechanical surroundings on bone in addition to fracture healing. In this regard, a new implant concept, variable fixation technology, is introduced. This implant has the special ability to alter its mechanical characteristic from “rigid” to “dynamic” over the time of fracture healing. This results in escalated callus formation, that is accompanied by greater homogeneous callus distribution and hence escalated fracture healing. Second, recent advances in the nano- along with micro-topography of bone scaffolds for guiding osteoinduction were detailed, specifically highlighting the simulation of natural bone. Thus, they summarized that an ideal scaffold should needs micropores of 50–150 µm diameter, that allows vascularization in addition to migration of stem cells along with nano-topographical osteoinductive cues, preferably pores of 30 nm diameter. Next to osteoinduction, such nano- and micro-topographical cues may further cause reduction in inflammation, besides possessing an antibacterial activity to further facilitate bone regeneration [76].

Conclusions

Thus, the long bone non-unions keep on remaining a significant challenge world over. Earlier to overcome that the group of Giannoudis PV provided the ’diamond concept’’, that represents a framework that is significant for getting insight into the least needs for fracture repair. As per the USFDA, council, the definition of non-unions is the inability of attaining union within 9 months from the injury, besides that displays no evidence of healing for3 months Here we have detailed the, epidemiology of non-unions besides the molecular science behind this ’diamond concept’’. Further the fate of the bone marrow mesenchymal stem/stromal cells (BMSC) possesses an intricate association with the mechanical environment, besides biochemical hints surroundings. Thus Yamada et al. provided the answer in how in the absence of these biochemical cues when a custom designed laminar flow is present. By seeding BMSC over the synthetic microporous scaffolds in addition to exposed through the sub physiological concentration of the fluid flow for 21 days. Significant hampering of the cell proliferation was observed. Besides that, morphological alteration in addition to F-actin polymerization was observed along with upregulation of ROCK1. Noticeably, the BMSC that were put through this flow, a significant upregulation of the mRNA of the osteogenic markers in addition to RUNX2 were localized to the nuclei. Moreover, a higher concentration of the type 1 collagen fibres awa calcium deposition occurred in the scaffolds. Thus, their study validated that with the utilization of proper amounts of fluid stimuli preconditioning of BMSC occurred by towards ideal osteoblasticlinege in 3dimensional (3D) scaffolds when there was no existence of chemical stimulation, that emphasizes the significance of utilization of fluid bioreactors in tissue engineering. Furthermore, we detailed the potential part of PEMF In future therapy along with role of pulsed ultrasound for escalation of fracture repair.

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