*Proceedings* **Peculiarities Regarding the Reconstruction of a Broken Grooved Shaft** †

## **Bogdan Bucur \*, Constantin Bucur and Gabriela Andronic**

"George Emil Palade" University of Medicine, Pharmacy, Science and Technology of Târgu Mures, Gh. Marinescu 38, 540142 Targu Mures, Romania<EMAIL_ADDRESS>(C.B<EMAIL_ADDRESS>(G.A.)

Published: 22 January 2021

**Abstract:** The work remarks the peculiarities, which appeared in the reconstruction of a broken grooved shaft. The originality of the subject consists of the realization of a slotting tool, in material changing, which increased the reliability of the piece, and the total time of measuring the allocation to the work. The dimensional aspects specified include the adjustment, the tool making for profiling the groove, the choice of the material, and the required time to design and build the new spare part.

**Keywords:** splintering processing; grooved; CAD; slotting tool; semi-coupling

## **1. Introduction**

Argument: for the machinery park to work with maximum, economic efficiency of mechanization and works automation, in addition to rational exploatation and the proper maintenance, the organization must be ensured and performing current and capitals repairs at a high technical level as well as of assemblies, [1].

The studied grooved shaft can be seen in Figure 1. The first findings on the damaged piece are as follows:

**Figure 1.** Broken grooved shaft–plastic material/part.

## **2. Working Method**

The construction stages of the new grooved shaft are as follows:

## *2.1. Dimensions of the Piece and Execution Drawing*

The dimensions determining the defective piece are shown in Figure 2. It was achieved with some difficulty due to the advanced state of deterioration caused by impact. For dimensional detection it were used two micrometers of the exterior with an aperture of 0–25 and 25–50 mm were used for the measurements, having an accuracy of 0.01 mm and a caliper with a rod having an accuracy of 0.02 mm.

The CAD design media—Inventor Autodesk was used to elaboration on the drawings present in this paper. Groove assemblies are shaft-hub assemblies designed to transmit torque and rotational motion, [2].

**Figure 2.** The execution sketch of the grooved shaft [3].

The shape of the groove protrusion is identified with the rectangular profile, but the piece which is made of polyamide, does not insist on the aspects related to the precision of execution (with external centering, with internal centering or with centering on the flanks). Argument: regardless of the category in which they fall, plastics have a high specific coefficient of expansion due to the Van der Walls bonds (week bonds) between the chains and their conformation, [4,5].

The affected accuracy of the part in question is shown—Table 1.

**Table 1.** The coefficient of linear expansion [4].

## *2.2. Choosing of the Material for the Grooved Shaft*

The main framings of the polyamide are: Polyamide 6 (PA 6) and Polyamide 6.6 (PA 66). However by modifying the chemical structures (chain length and chemical organization), several other families of polyamides are obtained: PA 4.6; PA 6.10; PA 10.10; PA 11 and PA 12, [6]

Determining the type of polyamide that made up the defective piece was not insisted on, since another material will be chosen. The argument for this option as follows:

In order to improve the strength and hardness or of some technological properties (splintering, material castability, etc.), aluminum alloys with Mg, Cu, Mn, Ni, Zn, Ti, Li, Bi, Sb, etc. [7].

For the manufacturing process of the part was used the material: EN AW-6061/AlMg1SiCu (EN 573-3-2007) [5].

## *2.3. Machining of the New Grooved Piece*

Of all technological processes necessary for execution of equipment, machinery, machines, the one of mechanical processing is most complex [8].

The notion of manufacture derives from two latin words *manus* (hand) and *factus* (to do), resulting combination meaning "hand made". The formal term of "hand made" accurately described the manual methods used until the word "manufacture" was coined around 1567 [9], and it used as a verb, it first appears around 1683 [10]. Next, the piece will not be "hand made" totally, so we have the elements of technological process of mechanical processing are: operation, placement/position, phase, passage, handling and movement, [8]. The unique manufacturing system of the piece will use classic machine tools, a universal lathe SNB 400 × 750 [11] and a milling cutter machine FUS 22, [12].

## *2.4. Initial Turning of the Grooved Shaft*

The processings performed on SNB 400 × 750, inside the machine tool laboratory of "George Emil Palade" UMFST of Târgu Mure¸s, are classic cutting operations, so this paragraph will not be developed, except for Figure 3, in order to argue the shape evolution of the semi-couple during its fabrication.

**Figure 3.** Cilindrical lathe of the semi-couple

## *2.5. Mortising the Grooved Profile*

The manufacturing preparation of the FUS 22 machine (equiped with a slotter ram) takes into account the adjustment of MDPT system (machine, device, part and tool) as follows:

**Figure 4.** Measurement of deviations of fixing the part in the universal of the index head of FUS 22 machine tool: measuring the radial beat (**a**); axial deviation out of the X direction measurement (**b**); measuring frontal beat (**c**); axial deviation out of the Y direction measurement (**d**); Three-dimensional (3D) model of the slotting machine process: main cutting movement (*I*), indexing movement, executed by the part (*II*); mortising device (**1**) mortising knife-tool (**2**), part (**3**), screw for fixing the knife-tool (**4**).

When mortising, the main movement (alternating rectilinear) is performed vertically by the tool, and the feed movement (intermittent) is performed, at the end of each inactive stroke of tool [13,14], in the horizontal plane, also by the tool, however, in the transverse direction (particular case on—FUS 22 machine). By slotting, flat vertical surfaces can be generated with curvilinear profiles, channels of wedge, inner or outer grooves and teeth, etc. [13,14]. In the Figure 4, you can see the 3D model of the theoretical sintesis of the mortise process and the measurement of the deviations of fixing the piece in the universal slotter ram on the machine tool FUS 22:

After the mounting index head and slotter ram, both the semi-finished and the slotting tool, Kinematic adjustment parameters are established on the FUS 22 machine and after the positioning adjustments [15]: the main movement (alternative rectilinear) of the slotting tool ncd = 50 dr/min, the cutting advance s = manual, and an empirical calculation—s = 0.0359 mm/dr, is being determined

by knowing the timing (3 5"), to displacement at the cutting depth t = 1.95 mm. About for the splintering knife-tool, the name is generally adopted for a wide range of single-tooth tools used in cutting processes, on universal lathes, revolver lathes, automatic, and semi-automatic, carousel lathes, on mortising, grinders, on boring machine, a.o. [16].

In Figure 5 you can see the scheme of the robot part that defines all the machined surfaces of the finished part.

**Figure 5.** Letter of the surface on the piece [3,13].

Depending on temperature at they work, tools steels for cold processing, non-alloy and alloyed, with working temperature below 200 ◦C and tool steels for hot machining, alloyed, at which working temperature is over 200 ◦C, [7].

In Figure 6 you can see the details of the geometry of the active part of the mortising cutting tool, as well as the fixing in the mortising device.

**Figure 6.** Shape evolution at the mortise knife manufacture.

Only one piece (aluminum) is fabricated, thus the chosen material for the mortise knife is C105U 1.1545 (SR EN ISO 4957:2002), [7].

According to the classification [15,17], the full name of the tool for cutting the groove is: Radial slotting tool of high-speed steel with a profile. In Figure 7 you can see all the geometric and constructive elements of the radial slotting cutting tool.

**Figure 7.** Geometric and constructive elements of the radial slotting tool and holder bar for cutting tool [3,15].

Following is the calculation of the simple indirect division [12,13] for the rotation of the index head axis at each executed channel (out of the 12 required channels): *GD* is the number of holes on a circumference of disk *D*; *k* is the number of traveled holes; *ZP* is the number of divisions on the surface of the piece, relation (1). Therefore each channel will be divided, rotating the crank of the index head to three complete rotations, on a circle of *GD* = 36 holes plus *k* = 12 additional holes displaced on GD.

$$Zp = \frac{k}{G\_D} = \frac{40}{12} = 3 + \frac{40}{12} = 3 + \frac{12}{36} \tag{1}$$

In Figure 8 you can see practical aspects about manufacturing process of tne grooved shaft.

**Figure 8.** Slotting the groove (**a**), cutting the piece (**b**) and milling the square profile (**c**).

## **3. Results and Discussion**

The total time includes: 1 h measurements + 1 h parametric modeling of three drawings + 1 h 55 slotting tool construction + 2 h 40 turning + 2 h 54 mortising + 1 h 19 turning again + 1 h 30 square profile milling, with a total of 11 h 38'. It is not possible to assess whether the realized time (11 h 38 ) is more or less, achived. It would be desirable to conduct a theoretical calculation of time, for edification. Figure 9 shows the final result of the technological method approached for the manufacture of the grooved shaft, as well as the assembly whit conjugate part.

**Figure 9.** The piece after mortising (**a**), in four hypostases—the finished piece (**b**), the part-mounted piece on the worm (**c**).

For the working method presented, all the photo present in this paper are original and were taken in the laboratory M.U.—A014 of the "George Emil Palade" University of Medicine, Pharmacy, Science and Technologie of Targu Mures, Roamania.

## **4. Conclusions**

The dimensions of the defective piece have been determined, in the Inventor-Autodesk enviroment. The necessary 2D and 3D drawings were designed, all the necessary technological steps, necessary tools, and adjustment of the machine tools were completed.

The slotting tool was conceived and built by the auto-endowment, and saving acquisition costs.

It was possible to piece the fabricate in required parameters, according to the endowment of the laboratory. Therefore, no other faster or cheaper fabricatione ways are disputed (3D printing or CAD/CAM). In this sense, it would be interesting to have a comparison.

#### **References**

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


*Concept Paper* **Sustainable Agrifood Value Chain—Transformation in Developing Countries**

**Dwi Ratna Hidayati 1,2,\*, Elena Garnevska 1 and Paul Childerhouse 3**

**Abstract:** To service high-value international markets, many agrifood value chains in developing countries are required to transform to meet the strict quality and safety standards. This transformation process has become further complicated by increased sustainability expectations. The key players in these countries, typically smallholders, are struggling to meet this new sustainability value focus. Economic drivers pervade in this context, whilst the lack of integration often decouples producers from the end market. To address these challenges, this paper develops a framework to enable sustainable agrifood value chain transformation in developing countries. A narrative review was used to analyse the major enablers and barriers in sustainable agrifood value chain transformation specifically in developing countries. The framework novelty lies in the synthesis and prioritisation of transformations actions, by integrating three central dimensions: sustainability, governance, and value addition. The incorporation of sustainability drivers into value chain governance provides a holistic approach that balances profit maximization with social and environmental impacts, thus enabling smallholders in developing countries to access higher value markets. The framework can assist these value chain actors in identifying their transformation trajectory and guide policymakers, along with the public sector, in prioritising their intervention to overcome barriers.

**Keywords:** value chain transformation; sustainability; smallholders; agrifood; developing countries

### **1. Introduction**

To increase income, many agrifood actors in developing countries are attempting to transform their value chains to access higher value markets [1,2]. Many of these actors are smallholder farmers, who are required to interact with multiple actors when transforming their practices to join complex high-value markets, including the global market [3,4]. These smallholders have been compelled to increase their income by shifting their focus towards the value drivers of the final market. Downstream global players are progressively targeting supply sources from developing countries in the high value food industry [5,6]. However, regardless of the profit opportunity, advancing smallholder practices into a high-value market is an area that requires further exploration.

High-value markets place increased expectations on food quality and safety, which contribute to the growing relevance of sustainability as a new component of 'value' [7,8]. To service higher value markets, smallholder's goals are expanded from a singular profit agenda to include socially acceptable practices that also have minimal environmental impact. Transformation requires all value chain players to work towards an acceptable mutual outcome from production to consumption [9]. Global consumers' preference have shifted into higher value, increasingly processed foods [10] that include additional attributes to price [11,12]. Wider stakeholder pressures from society, including the World Health Organization require value chain actors to embrace sustainable production and

**Citation:** Hidayati, D.R.; Garnevska, E.; Childerhouse, P. Sustainable Agrifood Value Chain— Transformation in Developing Countries. *Sustainability* **2021**, *13*, 12358. https://doi.org/10.3390/ su132212358

Academic Editors: Riccardo Testa, Giuseppina Migliore, Giorgio Schifani and József Tóth

Received: 1 October 2021 Accepted: 2 November 2021 Published: 9 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

consumption as a vital component of food security [13]. Therefore, the growing demand for sustainability practice further affects the transformation process to improve not only product quality and efficiency but also social–environmental considerations that enable the business environment [14].

Smallholders face several challenges when attempting to develop sustainable value chain practices. Prior studies have concluded that smallholder farmers in developing countries are often the weakest link during transformation, as they are typically trapped in a traditional system [1–15]. They have limited resources that constrain them from achieving high-value market requirements such as low productivity, inconsistent quality, limited education, and restricted access to market information [16–18]. Many of these barriers impede value chain participation in terms of governance and value addition [1]. For these reasons, smallholder farmers have limited power, dependent relationships, and are marginalized from more profitable markets [15].

Developing countries often undertake value chain transformation as part of a poverty alleviation strategy for smallholders [5–20]. Asia (i.e., Southeast Asia and South Asia) and Africa contain the highest concentration of developing countries with a significant proportion of smallholders in the agrifood value chains [6–21]. A poverty alleviation strategy often prioritises economic growth at the expense of social and environmental concerns [22]. In fact, smallholders, who generally have traditional practices, often lack the capacity to be fully engaged with the ideals of sustainability in high-value markets [8]. Enhanced value-adding activities by smallholders carry the potential to damage the environment and degrade social life. The agro–industrial revolution (through the development of tools, fertilizers, and planting technology) resulted in a substantial increase in land use and productivity [23]. Many of these activities have negative consequences such as overexploitation of natural resources, deforestation, and harmful waste [13].

Many studies have advanced the discussion on smallholder sustainability practice improvement in the high-value markets regarding global value chains [24,25]. Governance enhancement [15–26], higher value market linkage [27,28], and certifications [29] have been proposed as approaches to advance sustainable practice in developing countries in Asia and Africa. Most of these approaches list enablers without a clear structure, and often, they use a top–down lens to enable transformation, where lead firms design and dictate practices throughout the chain. As a consequence, many global players source from developing countries producers by controlling the value-adding activities [1–30]. This prevalent practice clearly demonstrates a marginal discrepancy in sustainable value chains, which minimizes smallholders' participation in enhancement initiatives. Moreover, most of these approaches view wider stakeholders (such as the government) as external, additional functions and overlook them as critical components. Conversely, it is widely acknowledged that stakeholders strongly influence the business environment and frequently enable smallholders' practice improvement [5–31]. While sustainable agrifood value chain transformation approaches have been insufficiently researched, the underlying enabling mechanisms remain unclear, and transformation trajectories have only been partially explored.

To address the aforementioned research gap, this paper aims to develop a framework for enabling sustainable agrifood value chain transformation in developing countries. The framework will assist actors to assess sustainability initiatives quantitatively and qualitatively [32]. The conceptual framework development in this paper uses a narrative review method. By using this method, a broad body of literature can be synthesised under an umbrella idea [33] and thus be able to support assumptions, identify research gaps, and establish integrated frameworks. A literature review, according to Snyder [34], provides the foundation for developing a new conceptual model/theory, and it can be useful to map the evolution of a particular research subject over time. Despite the fact that the narrative method heavily relies on the researcher's interpretation, the narrative structuring generally generates a perceptible pattern [35]. This type of review can be conducted through integrative review by discussing and summarizing the current state of knowledge, noting areas of agreement and disagreement [36]. The literature discussion starts with the fundamentals of agrifood value chain transformation in developing countries. The following section investigates how to incorporate sustainability drivers in value chain thinking. Thereafter, the key elements that enable the transformation process (to balance the profit maximization and social environmental aspects) are synthesized into a holistic framework to operationalize the change process.

Sustainable value chain transformation in developing countries has sparked great interest in the agrifood sector recently. This paper contributes to a deeper understanding of the enabling mechanism in several ways. While previous studies focused on postulating various enablers for sustainable value chain transformation, this paper will advance literature via the prioritisation of actions depend on value chain maturity. This study provides a structured process to assess and advance the sustainability of agrifood value chains. Further, the framework provides practitioners with information on how to enable sustainability, manage the risks of transformation, and therefore gain access to high-value markets. Finally, this study will assist policymakers to provide tailored support by prioritizing interventions to address context specific barriers.

#### **2. Agrifood Value Chain Transformation in Developing Countries**

While agrifood value chain transformation has various definitions, a classical definition by Reardon [37] describes it as the process of reforming the agrifood sector through the procurement of modernized systems. The transformation of the agrifood sector has been triggered by various modernization factors such as globalization industrial structures, technology, and consumerism [12–38]. Miller and Jones [39] elaborated further, stating that the agrifood value chain progresses towards a modern system that delivers higher market value via increased processing and stringent quality and safety standards.

Previous studies have identified a range of value chain characteristics to evaluate agrifood transformation. Boehlje [40] proposed six codependent dimensions: process flow, product flow, financial flow, information flow, incentive, and governance. Subsequently, many scholars have focused on the central role of governance, as it drives the rest of chain's activity and determines a firm's interactions throughout the chain [17–41]. Governance describes market dynamics in arranging and organizing the chain's operational rules. It generally involves vertical and horizontal integration [19–37] and information exchange [19–27]. Governance may also include incentives and assistance such as loans, warranty, recognition programs, and financial assistance through contracts and agreements [42,43]. Going further, Hidayati et al. [1] argue governance activities also have a significant impact on the actual value added activities. Value addition underscores the sequential product transformation, including physical form, space, and time, with each stage potentially contributing value to the market offering [12–45].

Value chain transformation in developing countries generally starts from a traditional value chain state and progresses towards a modern domestic or modern global value chain [2–10]. Defining transformation states and vectors provides each value chain stage with clear boundaries and future orientations. Simultaneously, it indicates how agrifood value chains can progressively become market-oriented [46]. However, market orientation may not adequately describe many immature value chain transformation processes. This is because a significant gap remains in many developing countries' value chain practices, regardless of the market requirements. According to Gereffi [47], there have been variations of governance practice used by value chain actors, despite the development of global markets. Thus, to facilitate a better understanding of agrifood value chain transformation, Hidayati et al. [1] proposed three practice maturity levels by integrating governance and value addition attributes (shown in Table 1).

Source: adapted from Hidayati et al. [1].

Maturity level evaluation facilitates an evolutionary assessment in terms of experience and practice quality [48]. Table 1 provides a means to assess the maturity of practice regarding governance and value addition. Once current status is determined, transformation routes can be identified to advance value chains to service high value global markets. Practice in developing countries necessitates the adoption of an integrative structure as the bases for directing the transformation process, as integration is a fundamental factor to determine the success of value chain operations [49]. The classification of integration structure to detect transformation direction aligns with Collins [12], who highlighted that value chain managerial takes progress through three key stages: traditional chain, managed chain, and best practice management.

Transforming value chains from traditional systems in developing countries is not a straightforward task that will undoubtably face numerous challenges. To addresses this, barriers need to be identified prior to transformation and potentially be exploited to create opportunities [24]. While the discussion in this area is continuously evolving, most of the studies highlight the major barriers of value chain transformation in developing countries relate to smallholders' practice. Smallholders typically operate in a traditional mode, disjointed from advanced value chain systems [1–15]. The main barriers to advancing smallholders' practice are associated with their characteristics, which include low productivity, inconsistent quality, high transaction costs, limited skills, and limited access to market, best practice, and financial information [5–50]. These factors hinder the value chain integration via the disconnection of practices in terms of goal setting, planning, working cultures, and synchronization [51]. In addition to these barriers, several enablers have also been identified in the developing countries context. Table 2 synthesises the most pertinent barriers and enablers for agrifood value chain transformation in developing countries.

**Table 2.** Agrifood Value Chain Transformation Enablers and Barriers in Developing Countries.

**Table 2.** *Cont.*

In transformational actions, setting the boundary is fundamental to clarify the enabling tasks. The key enablers and barriers of value chain transformation in developing countries are categorised into niche, meso, and macro levels in Table 2. By knowing which part drives the value chain transformation, the process can be managed appropriately based on the governance and facilitation requirements [5]. Therefore, transformation studies in the agrifood context increasingly require a Multilevel Perspective (MLP) to analyse transitions [60–62]. Within the MLP approach, value chain transformation in developing countries focuses on smallholder's perspective as the niche level, a value chain perspective at the meso level, and stakeholder's perspective as the macro level. Despite the differences in perspectives, these levels are not opposed to each other. Rather, these perspectives complement one another in terms of providing a consistent focus to enable transformation.

As stated earlier, the first critical investigation regarding value chain transformation is the smallholders' perspectives. The information pertains to smallholders' characteristics along with their intention to participate in the transformation process [28] and their capacity to scale up operations through horizontal coordination [52–63]. The attention then turns to the value chain stage perspective. Through a vertical coordination lens, the value chain perspective explores the relationship between smallholders and buyers. Due to the need to obtain consistent supply, buyers frequently combine buying processes with facilitation approaches to motivate smallholders to participate in the chain [58]. Finally, the last stage is to consider stakeholders' views in order to enable agrifood value chain transformation in a broader context.

Many stakeholders (such as government and NGOs) perceive agrifood value chain transformation as a strategy for reducing poverty in developing countries, which benefits global supply [5]. For this reason, transformation is often seen as the agenda of stakeholders, which often involves capacity enhancement and incentives [21–63]. While most assistances from stakeholders are advantageous, many of these have been associated as transient interventions and project-based operations [27–58]. Hence, despite stakeholders' interventions aimed at improving smallholders' practices [24–31], they are often considered as an additional, somewhat external player.

#### **3. Agrifood Value Chain Sustainability**

The most pressing challenge in the high-value food industry is sustainable practice. Value chain actors are required to refocus on 'value' from the multifunctionality elements of sustainability [7]. In general, Choudhury [64] introduced the sustainability concept as a global system that focuses on environmental, social, and economic elements, which fulfils the needs of current generations' whilst considering future generations' ability to

meets their needs. To respond to the urgency of sustainability in the agrifood sector, many scholars stress sustainability as a foundation for long term food security [55,65]. The World Health Organization (WHO) defines food security as economic and physical access of agrifood activities that adhere to sustainable production and consumption principles [13]. Aligned to this food security definition, sustainable value chains are defined in accordance with FAO [19] (p. 6):

*'The full range of farms and firms and their successive coordinated value-adding activities that produce particular raw agricultural materials and transform them into particular food products that are sold to final consumers and disposed of after use, in a manner that is profitable throughout, has broad-based benefits for society, and does not permanently deplete natural resources'.*

Within the scope of food security, sustainability is not simply a set of indicators. Rather, sustainability is an integrated system of dimensions [54]. A value chain is perceived as an economic-based activity that accesses both social and environmental dimensions. The value chain combines resources such as natural capital, knowledge, and skills within the social structures to deliver products or services [66] in which the products also end up in the environment [67]. Based on this, the dimensions of sustainable agrifood value chains are seen as a layered system. For instance, according to Gidding et al. [67], the economic dimension exploits society and environment dimensions, and Raworth [68], who identified the social foundation and ecological ceiling as an embedded dimension, expressed as much through a doughnut economy approach. A layered or nested system, on the other hand, has a tendency to prioritise certain dimensions above others. Meanwhile, strong sustainability practice necessitates a more balanced interaction of practice. In other words, economy, ecology, and social dimensions are to be accounted for at each value chain stage [69].

A value chain is deemed economically sustainable if each stage's activities generate value that leads to profit [19–54]. Being sustainable in the social dimension refers to a value chain that is both culturally and socially acceptable. However, assessing this social dimension continues to be a daunting task [70]. Higher levels of comprehensiveness and stringency in the social dimension can only be achieved by addressing foundations on standards within scope [71]; therefore, the social dimension direction should not be limited to social acceptability. Being socially engaged would strengthen connectedness and shared meanings with the community [72]. The third dimension, illustrated in Figure 1, is the environment that refers to the actor's ability to minimize any negative environmental impacts from the value-adding activities and, if possible, have a positive impact [19–54]. To represent this practice, some scholars recommend the term 'environmentally friendly' [73,74]. However, an environmentally respectful practice better depicts the act of practicing in a responsible way by respecting the environment [54–75].

A fully sustainable value chain is only possible if all three dimensions are aligned. In a developing country's context, this will be the compelling goal, yet the most difficult task. Enabling synergic incorporation of sustainability into a value chain is an area under intense research but has been incompletely explored in developing countries' literature. In contrast, many sustainability studies have been broadly explored in developed countries [32–77]. The approach for sustainable agrifood value chain transformation has been extensively viewed from various perspectives such as the individual (farm or household), local, global (sector-specific), and plot (ex-post and ex-ante). Despite this, some fundamental principles from developed countries may serve as the foundation for this context. For instance, incorporating sustainability into agrifood value chains begins with emphasizing farm practices [74–78]. This stage plays a significant role that determines the subsequent stages' performance. Farm practices are highly reliant on environmental sources [9], the production of perishable goods [45], and supplying the basic attributes of consumer's value [12].

**Figure 1.** Sustainable Value Chain Dimensions.

The economy dimension is prioritised in developing countries [54–73]. Although the Economic for Common Good (ECG) perspective has also seen the rationale of using economic gain to tailor the other aspects [68], economic growth that jeopardizes nature and human life is no longer deemed acceptable. Nature is an asset priced beyond market value, and human life ultimately depends on the natural environment [79]. The interactions between the three dimensions can be considered as synergies, complementary, competitive, or in conflict [54–80]. To assess transformation directions, the two most contradictory routes can be consolidated as positive and negative. While the competition and conflict relationships can lead to a negative transformation, synergy and complementary relationships can help to achieve a positive transformation.

A positive economic transformation represents an improvement of profit, which can be achieved by enhanced activities such as new processes, products, or functions [75]; elimination of inefficient activities [81]; an increase of productivity [82]; and an expansion of market opportunities [69]. A positive and meaningful social transformation benefits both value chain actors and the wider society [81,83]. Value chains in the agrifood sector in developing countries are characterized by the presence of a multitude of individual smallholder farmers. Individual (or within-group) levels are determined by factors such as education, working conditions [84], farming skills, and experience [54]. However, more accurate social dimension indicators are obtained by observing social components of specific farming systems [71]. Meanwhile, a wider society level is often determined by employment, acceptable cultural practices, and the safety and quality of products and processes [84–86]. A positive environmental transformation results when natural resources are utilized in line with domestic and international regulations [83], waste handling [69], and ecosystem protection and restoration [64].

Negative transformation is the opposite of positive transformation, and value can be added or lost at each stage [19]. Value-adding often puts pressure on natural resources, resulting in environmental degradation and the eroding of social traditional norms [9]. In the same way, social conditions through the interaction of people and nature also influence ecological sustainability [87]. As a consequence, conflict can arise due to natural deterioration caused by a chain's activities [13–55]. Long-term consequences affect not only

the environment but also the economic foundations, as the food industry is highly reliant on nature for the supply of raw materials [50]. Table 3 incorporates sustainability into the agrifood value chain by identifying the major enablers and barriers.

**Table 3.** Key Enablers and Barriers to Incorporate Sustainability into Agrifood Value Chains.

Amidst the variation and complexities of enablers/barriers for a sustainable practice transformation shown in Table 3, more exploration of enabling mechanisms is urgently needed. The underlying method to transform value chain practice in line with sustainability is still far from clear. Many of the enablers may work in tandem or different ways and be applied by various actors without a clear structure. A discussion on a systematic structure to leverage the sustainability enablers has been overlooked to date. Structuring the activities will help provide a clearer view of the mechanisms and synergize between players [1]. It is critical to shed light on prioritizing each stage activity in order to portray the precedence of goals while simultaneously eliminating irrelevant activities to avoid negative transformations.

Preventing detrimental transformation is the most challenging task; numerous studies have suggested the employment of sustainable value sharing as a key governance activity. Value sharing enables sustainable value inclusion into a value chain [100,102], which ultimately contributes to sustainable production and consumption [103]. The merit of sustainable value results from synergizing value chain actors (i.e., farmers and firms) in sharing their sustainability vision and willingness through a common sustainable strategy in order to avoid conflicts [70–103]. Value sharing requires further exploration to accurately capture the needs of developing countries' practice. Value sharing that exclusively focuses on value chain actors may limit the sustainability scope and overlook the critical role of wider stakeholders. Sharing activities in developing countries should address not only internal value chain actors but also external actors [69]. In the meantime, the use of the term 'external actor' for the government tends to disconnect its important function in affecting the business environments [104]. Government and/or NGOs play critical roles in determining value chain guidelines as part of the governance dimension [53]. Sustainable value creation is an ideal target, where all three sustainability dimensions are considered concurrently resulting in a commitment to delivering ecological, societal, and economical value addition [69].

### **4. Sustainability and Agrifood Value Chain Transformation in Developing Countries**

Enabling sustainable agrifood value chain transformation is a burgeoning research area that is relatively underexplored in a developing countries context. Various approaches are often used to address the increased focus on sustainability in agrifood value chain transformation via a combination of variables as enablers. However, the persistent challenge in this area primarily lies in the enabling mechanism. In particular, how to manifest in a myriad of specific practices for smallholder actors of developing countries and convert their orientation towards sustainability requires addressing. Therefore, to advance the current state of knowledge, this paper synthesizes approaches of agrifood value chain transformation and sustainability. An organised and aligned structure of actions is indispensable to transform a value chain in developing countries [1]. The solution offered herein synthesizes three key constructs: sustainability, governance, and value addition, as shown in Figure 2. Incorporating sustainability orientation into value chain governance leads to an enhancement of value addition activities, resulting in a sustainable value chain.

**Figure 2.** Sustainable Agrifood Value Chain Transformation Drivers.

The sustainability element draws attention to prior literature that highlights economic priorities in developing countries. This issue raises concern to balance the economic profit orientation with more social and environmental aspects to achieve sustainable practice. Being economically profitable is the first and primary orientation of smallholders in developing countries. Literature highlights that in order to create a profit, the enhancement activities contain a productivity increase, cost efficiency, premium pricing, and/or market opportunity [69–81].

Being socially engaged is the next important orientation to be embedded in developing countries. This dimension first considers the link between work and life quality [71]. After making a profit from farm work, smallholders would generally enhance their individual well-being via education/knowledge, skill, lifestyle, and working conditions. Meanwhile, simultaneously, literature also suggests smallholders consider activities that have an impact on a wider society [84–86]. Smallholders' consideration for broader societal requirements is primarily concerned with product safety and quality, employment issues, and acceptable cultural practices.

Operating environmentally respectful practices is the last critical and most important orientation that completes the overall sustainability in developing countries. The most fundamental aspect of environmental orientation relates to natural resource management, waste handling, and preservation. It is also important to mention that the key actors in the production of raw commodity are farmers, who thus ultimately determine environmental sustainability [80]. Hence, transformation must focus on the needs of developing technologies and practices that have minimal adverse environmental effects, which are accessible and effective for farmers while also improving productivity [90]. Galdeano-Gómez et al. [80] further state that reducing pressures on natural resources link positively to economic and social elements. Another way to see this is that a long-term economic condition can be achieved at the cost of not only social considerations but also environmental pressures [54].

The governance dimension refers to the degree of multilevel value sharing in order to capture a comprehensive sustainable perspective that suits the developing countries' context. Value sharing starts at the smallholder stage (as a niche level) to establish the scope of the practices and motivation to transform. This level represents smallholders' typology in producing the basic value at the farm stage. Smallholders generally have a heterogeneous typology [29]. Therefore, farmers' demography (within farm characteristics) frequently influences their decision to join higher value markets [28]. Next, smallholders also need to scale up operations in order to transform into a higher value market. To do so, they can develop horizontal coordination by collectively acting as producer organizations (PO) [20–52]. Collective action not only strengthens the members' positions as smallholders but also opens up new opportunities to capture more of the value from high-value markets and improves access to both markets and services [27–63].

In order to further advance activities, value sharing progresses to the wider value chain domain (as meso level). This level highlights the activities between smallholders with key buyers in the chain who play a significant role in sourcing from smallholders [1]. The relationships between farmers and buyers generally comprise transaction terms, negotiation, collaboration, and standard arrangements [1–12]. This type of vertical coordination also often includes a sourcing strategy applied by buyers to enable smallholder farmers to produce commodities that are compatible with high-value-adding chains [27].

In line with the preceding literature review, sustainable value chains in the developing countries' context need to advance the value sharing activities by incorporating stakeholders as key governance actors. Value sharing is complete once stakeholders are included (as macro level). Stakeholders enable value sharing expansion to broader actors, who can become business influencers. For ease of interpretation, stakeholders are commonly classified according to their motivations. Most governmental actions are identified as being relevant to policy setting within program implementation [27–58]. Meanwhile, the public sector is often viewed to be concerned with nonprofit activities conducted by NGOs and aid organisations [5]. Despite the difference in motivation, many of these institutions undertake similar actions to facilitate the advancement of smallholders' activities. Typically capacity enhancement programs are most relevant to harvesting techniques, storage facilities, and financial skills [50]. Meanwhile, incentives are commonly interpreted as input-, price-, and risk-related elements [37].

The value addition dimension in developing countries has generally denoted orientation to create potential value that includes: commodity-based orientation, which indicates smallholder's focus to produce and sell raw material products with minimal treatment; processed-based orientation that indicates an expansion in value-adding by

smallholders via post-harvesting treatments; and branded/certified orientation, which indicates smallholders' orientation in optimizing value creation through branded and certified products.

In addition to ensuring the clarity of enabling mechanism, the direction of the dimensions should be aligned. The alignment of direction will be necessarily constructed progressiveness towards each dimension's goals. A more progressive direction in each dimension increases the possibility to achieve a sustainable value chain status [92] and vice versa. Thus, progression and regression can represent the alignment of sustainable value chain transformation.

The last alignment includes enablers and barriers, which are synthesized in Table 4. The integration of both approaches has generated four key enablers/barriers for sustainable value chain transformation. The literature contains many similarities between approaches, such as the demography within the typology of practices, information sharing, access of market and service, and facilitation. Meantime, there are horizontal coordination and certification elements, which symbolize the uniqueness of each approach. Agrifood value chain transformation approach emphasises the fundamental role of collective action, as smallholder actors are the major stumbling block [28,52], whereas certification is the ultimate goal that verifies sustainability practices [29,101].

**Table 4.** Sustainable Agrifood Value Chain Transformation Enablers/Barriers in Developing Countries.

Once the enablers/barriers are identified, the next stage is to identify who is best placed to drive the transformation. To do so, connecting enablers/barriers with the governance dimension clarifies the enabling mechanism in the sustainable value chain transformation. As illustrated in Figure 3, this starts with the smallholders, progresses into the value chain level, and concludes with stakeholder facilitation. The smallholder level covers the enabling tasks in regards to demography within the typology of farm stage practices. As smallholders' conditions are generally heterogeneous, they may necessitate group-specific support [29]. Meanwhile, the typology of practice encompasses their behaviour and initiatives to better participate and effectively distribute the value to subsequent stages. In addition to this, their initiative in connecting and obtaining resources with other smallholders is vital to scale up operations. Next, the value chain level focuses on vertical integration, which orchestrates information sharing along the chain and provides access and services to end markets. At this level, buyers' involvement is critical to enhancing smallholders' capacity for meeting the sourcing requirements. Finally, stakeholders facilitate the smallholders' transformation via incentives, practice advancement support, and certification.

**Figure 3.** Sustainable Value Chain Transformation Governance.

### **5. Sustainable Agrifood Value Chain Transformation Operationalisation**

Agrifood value chains in developing countries are aspiring to higher value markets and urgently need assistance to transform value chain practices sustainably. Smallholders are the 'transformation agent' in developing countries because they hold the majority role as produce suppliers and are responsible for the base value for any subsequent value chain stages. However, smallholders are the weakest actor in the value chain and are primary focuses on economic gains. Consequently, value chain transformation carries a high risk, as smallholder practices may conflict with social and environmental sustainability. A narrow short-term economic focus degrades the basic value produced at the farm stage, which further hinders full participation in the higher value markets [22–99]. Many social and environmental issues are under the care of government and NGOs as key agrifood value chain stakeholders [5–31]. Therefore, sustainability value has pressured the expansion of the transformational approach from 'the business as usual' in the value chain operation towards a holistic agenda.

The central contribution of this paper is the development of a framework to enable sustainable value chain transformation in the developing countries context. While previous research have focused on unearthing various enablers and barriers, e.g., [77–93], this paper focuses on the enabling mechanism of these factors in order to gain greater clarity on how to find effective transformation trajectories. Building on the preceding synthesis of literature, the development of the mechanism constitutes the structuring of the transformation process based around three major dimensions (sustainability, governance, and value addition) and transformation direction (progression or regression). Figure 4 integrates the three dimensions transformation states.

**Figure 4.** Sustainable Value Chain Transformation Framework for Developing Countries.

The trajectories reflect the sequential practice (in terms of sustainability, governance, and value addition) to transform into a sustainable value chain in the developing countries context. Generally, the current state of sustainable value chain practice in developing countries is assumed as smallholder-centric, with a restricted focus on pursuing their individual profitability resulting in being trapped as a commodity seller, as illustrated in the central red zone in Figure 4. Red is used to indicate this inner zone as a warning, with the lowest level of value chain sustainability. This traditional value chain state should be first transitioned into the yellow zone. Thereafter, to develop a sustainable value chain, the activities need to be shifted gradually into the green zone of Figure 4.

There is a high probability that transformation will not follow a linear stepwise path. Smallholders, for example, may have shifted their practice orientation towards a value chain perspective due to the influence of buyers, by performing more processing (shown in Figure 4 as possible scenario I). However, it is more than likely that they may continue to be driven by economic profit, putting social and environmental aspects at risk. In possible scenario II, smallholders, supported from buyers at the value chain stage and the government/NGO at the stakeholders' stage, expand their consideration towards social and environmental aspects. They might also move into more processing activities; if they are not interested in pursuing the branded and certified products, they will lose out on potential higher value markets.

To help pave the transformation path, sustainability orientation is incorporating into governance, which leads to value-adding activities required in a high-value market. Incorporating sustainability dimension into the value chain practice is fundamental by advancing the economic profit orientation towards a more socially engaged and environmentally respectful practice. To accomplish this, the sustainability dimension should be embedded via the governance dimension, with the smallholder level serving as the key initial stage in the value chain. Smallholders should progress their orientation from the farm stage towards the value chain stage and ultimately level up their orientation towards wider stakeholders. The use of a multiperspective approach is critical to appreciate the dynamics of the agrifood chain at the different scales in regards to power and the interplay of

relationships [105]. By doing so, smallholders are expected to progress their value-adding activities from purely commodity-based to processed-based and to eventually achieve the branded high-value certification. In short, progressive advancement of smallholders' practice in each dimension acts as a gateway in transforming the value chain into enhanced sustainability. As a practical guide, we can position the axis of any future transformation direction by using the detail indicators in Table 5.

**Table 5.** Operationalisation of Sustainable Agrifood Value Chain Transformation in Developing Countries. Bold is required to emphasis and distinguish between the key elements and the derivations aspects.

Providing a practical assessment will benefit both individual players and industries to independently evaluate their position and prioritise their transformation. Using the indicators in Table 5, each player can perform a detailed evaluation of their enablers and barriers. Once value chain players are aware of their enablers and barriers [31], they can develop their unique plan and find the most effective transformation route. This will assist value chain actors to respond and engage with the high-value market requirements. On a larger scale, industries could examine the common issues in their value chains and collectively rectify unsatisfactory and substandard practices. Overall, the development of enabling mechanisms constitutes a powerful framework to guide developing countries' players in attaining sustainability practice, managing the transformation risks, and building strong connection with the high-value market.

Policymakers can also use the framework to evaluate specific agrifood sectors and prioritise tailored assistance activities. Facilitation to smallholders is generally offered in various forms, such as capacity enhancement (in the form of training) and incentives (i.e., input subsidy, tool, financial support) and could be further prioritised based on the urgency of each transformation case. Stakeholders need to stringently 'hit the right button' to intervene in smallholders' actions in order to provide efficacious assistance. For this reason, facilitation should ultimately lead to certification. Although certification might seem to be a long-term goal in many developing countries [29–52], certification can create a tremendous difference on practice. Certification is very empowering for smallholders to securely participate in higher value markets. Subsequently, stakeholders can take control through policies or regulations to make positive changes [19].

The framework in this paper complements and advances the existing value chains frameworks. For instance, the DFID framework [9] aims to integrate the poor (including smallholders and traditional practices) into value chains using three tools: a general tool (value chain analysis and mapping value chain), a qualitative tool (governance, linkages– relationship–trusts, and upgrading demand), and a quantitative tool (margin, income, and employment distribution). While each tool provides detailed, practical, and informative analysis, it falls short in delivering a holistic and interconnected value chain view. Furthermore, the sustainability agenda is not explicitly expressed in these three tools.

In 2014, FAO [19] developed a sustainable value chain framework using vertical coordination (governance), broad commodities scope importance, and value added along with sustainability. In 2016, FAO [50] places more emphasis on developing countries and focuses on three main strategies: equity aspects, smallholders' linkage, and policy along with public investment foundation. The framework developed herein advances the FAO approach by operationalising the actions required to advance and via the provision of transformation pathways.

To sum up, the framework represents transformational trajectories that involve a complex interaction between three primary sustainable value chain dimensions. Prioritization on one dimension at the neglect of the other two will detract from the overall achievement of sustainable value chain transformation. This framework represents the enabling mechanism, where the value given to the society takes wider environmental impacts into account [19].

#### **6. Conclusions**

This paper highlights the adversity developing countries' value chains face when transforming to service higher value markets, given additional sustainability imperatives. The sustainable value chain transformation framework developed in this paper goes beyond previous works by synthesising governance, value addition and sustainability. The framework goes one step further by stressing the need for a distinctive approach to overcoming the major problems in developing countries' transformations: the dominance of powerless actors (smallholders) and their economic orientation.

Theoretical and technical contributions are provided by the synthesised framework. Theoretically, the enabling mechanism for a sustainable value chain transformation approach is structured regarding three dimensions along with transformation trajectories. A systematic transformation approach allows developing countries' value chains to optimally arrange actions and create effective routes for a positive transformation. Technically, the development of a practical guide in this paper assists both practitioners and policymakers to investigate transformation status and improvement trajectories. The guidelines enable practitioners to assess and self-determine their transformation path to fully align with higher value market requirements. Correspondingly, the guidelines assist policymakers in terms of delivering efficacious support for the transformation process by prioritizing and placing their interventions to address specific barriers.

To verify the framework and progress the investigation, empirical tests are proposed in the agrifood sector of developing countries. The empirical testing set for this framework will necessarily focus on high-value food produced mainly by smallholders and traded on the global market. This setting would help to depict the inevitable sustainable value chain transformation, which is currently the concern of the global agrifood industry. This paper focuses on the transformation mechanism that emphasizes the positive and negative directions. These vectors and orientations require broader investigation, as trade-offs between activities on each dimension are empirically explored. Trade-offs sometimes are needed between the degree and rate for the sustainability achievement through vis-a-vis objectives [87]. For instance, it would be crucial to specifically distinguish the relationship between complementary, synergy, competition, and conflicts [54,55]. Thus, future exploration on each element's impacts between dimensions would be beneficial to verify sustainable value chain transformation mechanisms.

**Author Contributions:** Conceptualisation, D.R.H., E.G. and P.C. Writing—Original Draft Preparation, D.R.H. Supervision, E.G., P.C. Writing—Review and Editing, D.R.H., E.G. and P.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by NZAID Scholarship (PhD Scholarship program).

**Institutional Review Board Statement:** Not Applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


*Article* **e-Cigarette Vapour Condensate Reduces Viability and Impairs Function of Human Osteoblasts, in Part, via a Nicotine Dependent Mechanism**

**Thomas Nicholson 1, Lauren Davis 2, Edward T. Davis 3, Matthew Newton Ede 3, Aaron Scott 2,† and Simon W. Jones 1,\*,†**

**Abstract:** Cigarette consumption negatively impacts bone quality and is a risk-factor for the development of multiple bone associated disorders, due to the highly vascularised structure of bone being exposed to systemic factors. However, the impact on bone to electronic cigarette (e-cigarette) use, which contains high doses of nicotine and other compounds including flavouring chemicals, metal particulates and carbonyls, is poorly understood. Here, we present the first evidence demonstrating the impact of e-cigarette vapour condensate (replicating changes in e-cigarette liquid chemical structure that occur upon device usage), on human primary osteoblast viability and function. 24 h exposure of osteoblasts to e-cigarette vapour condensate, generated from either second or third generation devices, significantly reduced osteoblast viability in a dose dependent manner, with condensate generated from the more powerful third generation device having greater toxicity. This effect was mediated in-part by nicotine, since exposure to nicotine-free condensate of an equal concentration had a less toxic effect. The detrimental effect of e-cigarette vapour condensate on osteoblast viability was rescued by co-treatment with the antioxidant N-Acetyl-L-cysteine (NAC), indicating toxicity may also be driven by reactive species generated upon device usage. Finally, non-toxic doses of either second or third generation condensate significantly blunted osteoblast osteoprotegerin secretion after 24 h, which was sustained for up to 7 days. In summary we demonstrate that e-cigarette vapour condensate, generated from commonly used second and third generation devices, can significantly reduce osteoblast viability and impair osteoblast function, at physiologically relevant doses. These data highlight the need for further investigation to inform users of the potential risks of e-cigarette use on bone health, including, accelerating bone associated disease progression, impacting skeletal development in younger users and to advise patients following orthopaedic surgery, dental surgery, or injury to maximise bone healing.

**Keywords:** electronic cigarettes; osteoblast; e-cigarette; vaping; viability; bone; osteoprotegerin; human primary cells

#### **1. Introduction**

Multiple meta-analyses have reported that a history of cigarette smoking is associated with significantly reduced bone mineral density (BMD), increased risk of fracture and reduced fracture healing, in comparison to age, sex and BMI-matched non-smokers [1]. It is also apparent that such smoking-associated effects are cumulative, demonstrating a positive correlation with pack year history [2–4]. Furthermore, fracture risk in smoking cohorts is greater than in non-smokers when corrected for BMD, indicating that smoking may directly

**Citation:** Nicholson, T.; Davis, L.; Davis, E.T.; Newton Ede, M.; Scott, A.; Jones, S.W. e-Cigarette Vapour Condensate Reduces Viability and Impairs Function of Human Osteoblasts, in Part, via a Nicotine Dependent Mechanism. *Toxics* **2022**, *10*, 506. https://doi.org/10.3390/ toxics10090506

Academic Editors: Andrzej Sobczak and Leon Ko´smider

Received: 4 August 2022 Accepted: 25 August 2022 Published: 28 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

impact bone architecture and quality. Indeed, a decrease in trabecular bone mass and increased trabecular separation has been reported in older smokers [5], while in younger individuals smoking is associated with a reduction in trabecular bone volume, independent of age, BMI, activity level and calcium intake [6]. Recent studies have also demonstrated that smoking is independently associated with increased post-surgery complications such as infection and aseptic loosening following arthroplasty [7–10].

While cigarette consumption has declined over the past decade, the use of electronic cigarettes (e-cigarettes) or vaping has risen dramatically, partly due to being regarded as a safer alternative to smoking, although, 8% of current EC users in the UK have never smoked a cigarette [11–13]. Increased use of e-cigarettes will undoubtedly make a significant contribution towards harm reduction in comparison to cigarettes. However, e-cigarette usage still results in systemic exposure to numerous and potentially harmful vapour constituents, particularly to highly vascularised tissues such as the bone. In support of this, Agoons et al. recently reported that e-cigarette users have a 46% higher prevalence of fractures, in comparison to those who have never used e-cigarettes based on a cohort of 4519 individuals [14].

E-cigarette vapour is much less complex than cigarette smoke, yet many harmful constituents of cigarette smoke are found in e-cigarette vapour. Upon thermal decomposition, e-liquid humectants propylene glycol (PG) and vegetable glycerine (VG) form products such as acrolein and formaldehyde, commonly termed reactive carbonyl species (RCS), which are causatively linked to systemic harm [15,16]. Furthermore, since their invention in 2003, e-cigarette device technology has developed rapidly with current 3rd generation devices capable of delivering vapour at a much higher temperature than earlier models due to larger battery sizes. Consequently, this enables greater delivery of nicotine [17–19], increasing user satisfaction but also delivering much greater amounts of harmful RCS [20–22].

As to be expected, the majority of research on e-cigarettes to date has been carried out in models relevant to the lungs. Importantly, we and others have investigated the effect of vaping constituents on lung immune cells, reporting cytotoxic, proinflammatory and anti-phagocytic effects in alveolar macrophages [15,23]. Similar reductions in neutrophil function have also been reported, including reduced neutrophil migration and phagocytosis, suppression of NETosis and increased ROS production [24]. Additionally, dysfunctional cilia beat frequency and motility has been reported in human airway epithelial cells and normal human bronchial epithelial (NHBE) cells following cigarette vapour exposure [25–28].

However, there has been limited investigation into the impact of e-cigarette usage on bone physiology, particularly following long-term use [29,30]. There are also limited in vitro data, particularly utilising human osteoblasts. Typically exhibiting a large, cuboidal morphology, osteoblasts are the primary cell type responsible for bone formation, through secretion of collagenous and non-collagenous proteins and proteoglycans that in turn become mineralised.

Utilising a novel system previously described by our group [23], we have performed the first investigation into the effect of e-cigarette vapour condensate on human primary osteoblast viability and function. Importantly, we report the comparative effects of vaping constituents generated by 2nd generation and 3rd generation devices, which together account for 77% of devices used in the UK [31]. Finally, we have utilised both nicotine containing and nicotine-free vapour condensate, in addition to the antioxidant, N-acetyl cysteine (NAC) to investigate the contribution of the vapour constituents nicotine and RCS, respectively, on osteoblast viability.

#### **2. Materials and Methods**

*2.1. Ethical Approval and Subject Recruitment*

Femoral heads were collected from hip osteoarthritis (OA) patients undergoing orthopaedic joint replacement surgery at the Royal Orthopaedic Hospital (Birmingham, UK). All patient participants were recruited on a volunteer basis, after being fully informed of the study requirements by the clinical research staff, and providing written consent (NRES 16/SS/0172).

#### *2.2. Primary Human Osteoblast Cell Culture*

Trabecular bone chips (<100 mm3) were obtained from the OA patient femoral heads using a Friedman Rongeur, washed three times in phosphate-buffered saline (PBS) [Life Technologies Ltd., Renfrew, UK] and once with high-glucose Dulbecco's Modified Eagle Medium (DMEM) to remove excess fat, blood, marrow, and connective tissue. Bone chips were then cut into small pieces (<5 mm3) and transferred to a 25 cm2 vented flask containing primary human osteoblast media (DMEM, 10% FBS, 100 Units/mL Penicillin Streptomycin, 2 mM L-Glutamine, 1% NEAA, 2 mM β-glycerophosphate disodium salt hydrate, 50 ug/mL L-Ascorbic Acid, 10 nM Dexamethasone). Bone chips were incubated at 37 ◦C and 5% CO2 for 5 days before the initial media change. Following 5 days, differentiation media was changed every 3 days, and bone chips were removed once primary osteoblast cell coverage reached approximately 50% confluency. Upon reaching confluency, cells were passaged into a 75 cm2 flask. For all experiments primary human osteoblasts were limited to passage 5.

#### *2.3. e-Cigarette Devices*

Two popular devices in the UK were chosen for condensate generation, a 2nd generation device and 3rd generation device from Kanger tech Ltd., (Shenzhen, China). The 2nd generation device was fitted with a standard 650 mAh battery with a fresh 1.8 Ohm atomiser for each preparation, generating 7.6 W. The 3rd generation device, the most powerful of the devices, was fitted with a 3000 mAh battery with a fresh 0.15 Ohm atomizer fitted for each preparation, generating 75 W. The same devices were used for each condensate preparation.

#### *2.4. e-Cigarette Vapour Condensate Collection*

e-cigarette vapour condensate (ECVC) or nicotine-free e-cigarette vapour condensate (nfECVC) was collected from 2nd and 3rd generation e-cigarette devices, as previously described by Scott et al. [23]. Prior to use, e-Cigarette devices were cleaned and prepared with either 36 mg/mL nicotine flavourless liquid (Durasmoke® Unflavored e-Liquid (50% PG/50% VG Base), American e-liquid Store, (Wauwatosa, WI, USA) or nicotine-free flavourless liquid (Durasmoke® Unflavored eLiquid (50% PG/50% VG Base), American e-liquid Store, (Wauwatosa, WI, USA). Next, six tracheal suction taps (Unomedical, UK) were arranged in sequence and sealed with parafilm. EC devices were attached to the open end of tap 1, while tap 6 was connected to a vacuum tap by plastic tubing. Taps 2–6 were sealed inside 30 mL universal tubes with parafilm, to provide insulation and prevent cracking upon cooling. Next insulated taps were suspended in a dry ice/methanol bath and allowed to cool, tap 1 was kept outside the bath for observation of vapour production. The optimum puff duration of 3 s (previously determined by Scott et al.) was performed every 30 s until EC liquid was exhausted. Taps were then allowed to warm to room temperature, before centrifugation (2755× *g*, 5 min) to collect condensate. Condensate was pooled into a single 1.5 mL Eppendorf and stored at −40 ◦C for a maximum of 24 h before use.

#### *2.5. Osteoblast Challenge and Intervention*

Challenge with ECVC, nfECVC, PG, VG and N-Acetyl-Cysteine (Sigma-Aldrich, St. Louis, MO, USA) were diluted in osteoblast differentiation media to concentrations detailed in individual Figure legends. Incubation periods are described per experiment as appropriate.

#### *2.6. Primary Human Osteoblast Viability and Cellular Morphology*

Osteoblast viability was determined using CellTiter 96® AQueous One Solution Cell Proliferation Assay (CTA) (Promega, UK) following the manufacturers protocol. Following addition of CTA reagent, cells were incubated in the dark for 3 h at 37 ◦C and 5% CO2. Absorbance at 490 nm was then immediately measured using a Synergy HT (BioTek, Santa Clara, CA, USA) plate reader. Additionally, in order to assess cell morphology, osteoblasts were imaged at 20× magnification using an SP8 Lightning confocal microscope (Leica Microsystems, UK).

#### *2.7. Quantification of OPG and RANK-L Secretion from Primary Human Osteoblasts*

OPG and RANK-L protein in primary human osteoblast supernatants were quantified in duplicate using commercially available ELISAs (Osteoprotegerin/TNFRSF11B (R&D Systems, Minneapolis, MN, USA), Human TRANCE/RANK L/TNFSF11 (R&D Systems, 29, Minneapolis, MN, USA) following the manufacturer's instructions. Absorbance was measured at 450 nm and 570 nm on a Synergy HT (BioTek, USA) plate reader.

#### *2.8. Statistical Analysis*

Data analysis was carried out using GraphPad Prism v8 statistical package. For data sets with 2 variables, significance was determined by 2-way ANOVA followed by Tukey's multiple comparisons test where appropriate. For data sets with one variable, data was analysed using a non-parametric Kruskal–Wallis test, followed by post hoc Dunn's multiple comparison tests. Data is presented as mean ± S.E.M with a *p* value < 0.05 considered statistically significant.

#### **3. Results**

#### *3.1. ECVC from either 2nd or 3rd Generation e-Cigarette Devices Reduces Human Osteoblast Viability and Alters Cellular Morphology*

Osteoblast viability was quantified and cellular morphology observed following 24 h exposure to increasing doses of ECVC (0.25% to 10%), generated from either 2nd or 3rd generation devices. ECVC at 0.25% or 0.5%, generated from either 2nd or 3rd generation devices, had no significant effect on osteoblast viability (Figure 1A) or cellular morphology (Figure 1C,G). However, in contrast to the 2nd generation device, 1% ECVC generated by the 3rd generation device resulted in a significant reduction in osteoblast viability (46.6% ± 10.0% *p* = 0.002), compared to untreated control cells (Figure 1A). At ECVC concentrations of 2.5% and greater, condensate generated by either 2nd or 3rd generation devices significantly reduced osteoblast viability (*p* < 0.0001, Figure 1A). Furthermore, osteoblasts exposed to 2nd or 3rd generation ECVC at concentrations of 2.5% or greater, showed clear signs of altered cellular morphology, with loss of spindle cell shape (Figure 1C,G).

**Figure 1.** *Cont*.

**Figure 1.** The effect of 2nd and 3rd generation ECVC and contribution of nicotine on human Primary osteoblast viability. (**A**) Osteoblast viability following 24 h exposure to 2nd or 3rd generation nicotinecontaining ECVC (0.25–10%). (**B**) Osteoblast viability following 24 h exposure to either nicotinecontaining or nicotine-free 2nd generation ECVC (0.25–10%). (**C**,**D**) Representative images of primary human osteoblasts treated with 2nd gen ECVC or nfECVF (0–10%). (**E**) Osteoblast viability following 24 h exposure to either nicotine-containing or nicotine-free 3rd generation ECVC (0.25–10%). (**F**) Osteoblast viability following 24 h exposure nicotine-free ECVC from either 2nd or 3rd generation devices (0.25–10%). (**G**,**H**) Representative images of primary human osteoblasts treated with 3rd generation ECVC or nfECVC (0–10%). Viability was inferred by 4 h incubation with cell titre aqueous assay. Images captured at 20× magnification. *n* = 3 patient replicates, with 5 biological replicates performed per patient. \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 denoting a significant denoting a significant difference to relevant untreated control. ## *p* < 0.01, ### *p* < 0.001, #### *p* < 0.0001, denoting a significant difference between treatment groups.

#### *3.2. ECVC from Nicotine-Free 3rd Generation Devices Has a Greater Effect on Reducing Osteoblast Viability than Nicotine-Free 2nd Generation e-Cigarette Devices*

To determine the extent that the observed reduction in osteoblast viability was attributable to nicotine content in the condensate, we compared the effects of nicotinecontaining and nicotine-free ECVC (Figure 1B,E,F). As expected, from the 2nd generation devices, nicotine-containing ECVC at 2.5%, 5% and 10% induced a significant reduction in osteoblast viability (*p* > 0.0001) (Figure 1B,D). However, osteoblasts exposed to the nicotinefree 2nd generation condensate experienced a significantly lower loss in viability after; 10% nfECVC (37.2% ± 1.27% loss in viability), vs. just 2.5% ECVC *p* =< 0.0001) (Figure 1B,D). Exposure to 3rd generation condensate did not follow this pattern. 1% nfECVC challenge caused significantly less osteoblast toxicity than 1% nicotine containing ECVC (*p* = 0.001, Figure 1E,H). However, at higher concentrations, both nicotine-containing and nicotine-free condensate elicited a significant reduction in osteoblast viability, compared to untreated controls (Figure 1E,H). Whilst this effect was more pronounced with nicotine-containing condensate there was no significant difference between nicotine containing and nicotine free challenge at these doses (Figure 1E). Directly comparing nfECVC generated by 2nd generation and 3rd generation devices confirmed a greater toxic effect on osteoblasts when exposed to 3rd generation nfECVC at concentrations of 2.5% and above (*p* > 0.0001, Figure 1F).

#### *3.3. Sub-Cytotoxic Doses of e-Cigarette Condensate Alters Human Osteoblast Function*

Next, we investigated the potential for e-cigarette condensate to impact primary human osteoblast function, by assessing the secretion of the pro-osteogenic protein Osteoprotegerin (OPG) after low dose condensate exposure. In untreated cells, OPG secretion increased with each timepoint (Figure 2A). Following incubation with either 2nd generation or 3rd generation ECVC for 24 h, OPG secretion declined in a dose dependent manner (Figure 2A,B). Notably, OPG secretion was significantly reduced after exposure to 0.5% ECVC from either 2nd generation (33% ± 1.43% reduction, *p* = 0.015) or 3rd generation (52% ± 0.08% reduction, *p* =< 0.0001) devices (Figure 2A,B), despite this dosage having no effect on osteoblast viability, or morphology (Figure 1).

Furthermore, over 7 days, OPG secretion continued to increase significantly in untreated osteoblasts in a time dependent manner (Figure 2C). However, continuous exposure to either 0.25% or 0.5% ECVC for 7 days, significantly blunted OPG secretion at each timepoint for both 2nd and 3rd generation devices. By day 7, OPG secretion was approximately 2-fold or 4-fold less than that of untreated control cells for 2nd and 3rd generation devices, respectively, (*p* < 0.0001) (Figure 2C,D), whilst cellular viability and morphology remained unaffected (Figure 2E,F and Figure A1 in Appendix A).

#### *3.4. The Antioxidant N-Acetyl Cysteine Rescues the ECVC-Induced Reduction in Osteoblast Viability*

To elucidate the mechanism by which ECVC may interact with and affect osteoblast function, we challenged osteoblasts with a toxic dose (2.5%) of ECVC, previously identified to negatively impact osteoblast viability for both 2nd and 3rd generation devices (Figure 1A). These experiments assessed the efficacy of the antioxidant and antialdehyde, NAC to rescue osteoblast viability when given concurrently with condensate challenge. NAC alone had no significant effect on osteoblast viability (Figure 3A,B). However, NAC treatment was able to partially mitigate the toxic effects of ECVC challenge, offering a significant protective effect after the 3rd generation condensate challenge (48.5% restoration, *p* < 0.0001). NAC intervention also mitigated effects on osteoblast morphology following treatment with 2.5% ECVC (Figure 3C).

Having observed this NAC mediated rescue effect, we next performed a series of experiments in which we treated osteoblasts with the humectants propylene glycol and vegetable glycerine in isolation, to further validate whether e-cigarette vapour components other than nicotine may reduce osteoblast viability (Figure 4). A significant treatment effect of PG on osteoblast viability was observed (*p* < 0.05, Kruskal–Wallace test), with 10% PG decreasing viability up to 80% (Figure 4A) and clearly altered cellular morphology observed with 5–10% dosages (Figure 4C). VG had no significant effect on osteoblast viability or morphology (Figure 4B,D).

**Figure 2.** *Cont*.

**Figure 2.** The impact of 2nd and 3rd generation ECVC on human primary osteoblast function. (**A**) Effect of 24 h exposure to 2nd and 3rd generation ECVC at concentrations from 0.25–2.5% on human primary osteoblast OPG secretion. (**B**) Effect of 24 h exposure to 2nd and 3rd generation ECVC

at concentrations from 0.25–2.5% on human primary osteoblast OPG secretion expressed as fold change from untreated control. (**C**) Effect of 24–168 h (7d) exposure to 2nd generation ECVC at concentrations from 0.25–0.5% on human primary osteoblast OPG secretion. (**D**) Effect of 24–168 h (7d) exposure to 3rd generation ECVC at concentrations from 0.25–0.5% on human primary osteoblast OPG secretion. (**E**,**F**) Representative images of primary human osteoblasts treated with 0–0.5% 2nd and 3rd generation ECVC for up to 7 days. *n* = 3 patient replicates, with 3 biological replicates performed per patient. \* *p* < 0.05, \*\*\*\* *p* < 0.0001 denoting a significant difference to relevant untreated control. # *p* < 0.05, denoting a significant difference between treatment groups.

**Figure 3.** Pharmacological rescue of ECVC induced reductions in human osteoblast viability. (**A**) Effect of 1 mM NAC on human primary osteoblast viability in the absence or presence of 2.5% 2nd generation ECVC for 24 h. (**B**) Effect of 1 mM NAC on human primary osteoblast viability in the absence or presence of 2.5% 3rd generation ECVC for 24 h. (**C**) Representative images of human primary osteoblasts following 24 h exposure to 2nd or 3rd generation ECVC in the presence or absence of 1 mM NAC. Images captured at 20× magnification. Viability was inferred by 4 h incubation with cell titre aqueous assay. *n* = 3 patient replicates, with 3 biological replicates performed per patient. \*\*\*\* *p* < 0.0001 denoting a significant difference to relevant untreated control. #### *p* < 0.05 denoting a significant difference between treatment groups.

**Figure 4.** The effect of treatment with e-cigarette constituents on human osteoblast viability. (**A**,**B**) Effect of 24 h treatment of increasing dosages of PG and VG human primary osteoblast viability. (**C**,**D**) Representative images of human primary osteoblasts following 24 h exposure to increasing dosages of PG and VG, images captured at 20× magnification. *n* = 4 Patient replicates with 4 biological replicates performed per patient. \* signifies *p* < 0.05 denoting a significant treatment effect by a Kruscal-Wallace test.

#### **4. Discussion**

This is the first study to demonstrate that e-cigarette condensate exposure reduces human primary osteoblast viability and function in a dose-dependent manner, utilising a model system that accounts for changes in the chemical composition of e-cigarette liquids that occur during vaping.

Treatment of osteoblasts with concentrations of 2.5% ECVC and above, was cytotoxic from both 2nd generation and 3rd generation devices, reducing viability to less than 30% compared to untreated controls. Although there is a lack of data regarding the concentration of nicotine and other vapour constituents delivered to bone following e-cigarette

usage, a concentration of 2.5% ECVC (15.5 μM, 3rd generation) as used here is within a physiologically relevant systemic concentration based on reported levels after e-cigarette usage [32]. In concordance with these results, previous studies have demonstrated that cigarette smoke is toxic to human osteoblasts, reducing osteoblast viability in both a concentration and time-dependent manner [33]. More recently, a toxic effect of e-cigarette vapour on a variety of cell types, including alveolar macrophages and epithelial cells has been reported [23,34]. Additionally, Shaito et al. observed both reduced proliferation and impaired osteoblastic differentiation of bone marrow derived mesenchymal stem cells (MSCs) following exposure to e-cigarette aerosol extract [34]. Therefore, e-cigarette use may not only reduce osteoblast viability directly, as observed in this study, but could also reduce MSC-mediated bone repair. Together, these data suggest long-term e-cigarette use could have significant implications for individuals following chronic use and especially individuals with disorders of the skeletal system such as osteoarthritis [35,36], osteoporosis [36] and scoliosis [37], where there is evidence of abnormal bone and/or osteoblast pathology. In addition, this is also likely to impact skeletal remodelling during bone healing following injury, orthopaedic surgery and oral surgery such as dental implants, where exposure to e-cigarette vapour will be in very close proximity to the wounded site. Furthermore, it is also very important to consider the impact of e-cigarettes on adolescent and young adults, who comprise one of the largest cohort of e-cigarette users. Sustained e-cigarette use in such individuals may impair ongoing bone development, leading to reduced bone mineral density into adulthood. Harmful effects of conventional cigarettes and e-Cigarettes have been attributed, at least in part, to nicotine [38]. Our data supports a role for nicotine in driving osteoblast dysfunction, as the impact of nfECVC on osteoblast viability was significantly less than following exposure to ECVC of the same concentration. This effect was particularly apparent for second-generation device condensate. However, it should be noted that although osteoblast viability was greater following 10% 2nd gen nfECVC exposure in comparison to the 3rd generation device, cellular morphology appeared abnormal. This could be explained due to the viability assay fundamentally being based on cellular metabolism, therefore it is possible that although stressed and so losing typical morphology, the cells treated with the 2nd generation condensate were still more metabolically active. Whereas those treated with the 3rd gen condensate, quickly began to die after treatment. The considerable sustained impact of 3rd generation nfECVC on osteoblast viability may be attributable to differences in vapour constituent content. In addition to nicotine, e-Cigarette vapour also contains carrier agents/humectants including propylene glycol and vegetable glycerine. Thermal degradation of these carrier compounds generates reactive carbonyl species at similar concentrations to those seen in cigarettesmoke (~5 μg·puff−1) [39] and in some cases, in excess of cigarette smoke (200 ug·puff−1) [21]. Importantly, reactive carbonyl species have been demonstrated to reduce proliferation, increase cell death and inhibit both osteoblast alkaline phosphatase activity and mineralisation [40–42]. Additionally, e-cigarette liquids also generate a considerable amount of short-lived, highly reactive free radicals (>1013 molecules/puff) [21,43–45] upon vapourising. ROS are reported to induce apoptosis of osteoblasts, as well as inhibit osteoblastic differentiation, reducing osteoblast number and impairing function [45–47]. Furthermore, Bai et al. present evidence that increased intracellular ROS can stimulate the expression of RANKL in human osteoblast-like cells, which would be expected to promote osteoclast activity and bone resorption [48]. Collectively, these findings suggest that ROS not only reduces osteoblast mediated bone formation, but may also increase bone resorption through activation of osteoclasts, ultimately resulting in reduced bone density. Critically, increasing battery size and decreased coil resistance result in increased amounts of both ROS and RCS being generated per puff by newer generation e-cigarette devices, such as the 3rd generation device used in this study [20,22,45]. Unlike previous studies that treat cells with e-cigarette liquids directly from the bottle, our model system accounts for changes in chemical composition that occur upon vaping. Therefore, it is possible that the nicotine free ECVC from the 3rd generation device used in this study contained a greater amount of reactive species compared to 2nd

generation devices, in turn mediating a greater impact on osteoblasts. To investigate this possibility, we treated osteoblasts with ECVC in the presence of NAC, an antioxidant that has been reported to protect against ROS and reactive aldehydes [49]. Following treatment with 3rd generation ECVC, NAC provided a significant protective effect, restoring viability to the level of control osteoblasts. This suggests that the toxic effects of ECVC may indeed be mediated in part by increased levels of reactive species. We also demonstrate that components of e-cigarette condensate other than nicotine, including the humectant PG, also significantly impaired osteoblast viability and altered cellular morphology. Emerging data in gingival and airway epithelial cells has also shown cytotoxic, inflammatory and metabolic effects of such compounds, widely regarded as inert carrier agents [50,51]. It should be noted that repeated exposure to vapour generated from humectants alone over a 6 month period had no significant effect on bone morphology in mice [52]. However, the potential impact of such humectant exposure on osteoblast/osteoclast function and bone turnover remains to be determined and therefore potential implications to processes such as bone healing still need to be considered. Indeed, although we observed no reduction in viability following VG treatment, recent work has demonstrated that VG exposure can impact chloride channel expression [53]. Therefore, non-toxic doses of VG and PG may still have considerable effects on osteoblast function, similarly to the effect non-toxic doses of ECVC had on OPG secretion as we report in this study. Collectively these data emphasise that further studies are necessary to understand the effect of chronic exposure to humectants and other components of e-cigarettes on human bone, especially following chronic use. Additionally the need for such studies is paramount, as e-cigarette devices are continually innovating, leading to ever greater power output and therefore increasing burden of RCS per puff and so in turn potential harm is only likely to increase

In line with the detrimental effect of ECVC on osteoblast viability, we also found that ECVC impaired the functional ability of osteoblasts by reducing their secretion of OPG, the decoy receptor for RANK ligand and a key regulator of bone turnover. Previous studies have examined the impact of conventional cigarette smoking on serum OPG levels, concluding that levels were significantly reduced in smokers [54]. In addition, OPG:RANKL is significantly reduced in patients who smoke, suggesting smoking may drive bone resorption [54]. Here, we also observed that OPG production by osteoblasts was significantly reduced following treatment with ECVC at 24 h, suggesting that the effects of e-Cigarette use and conventional cigarette smoking on osteoblast function maybe comparable. We did attempt to measure RANKL protein content in osteoblast supernatants by ELISA, however protein concentrations were below the lower limit of detection of our assay. Critically, as e-cigarette use is typically chronic, we also found that stimulation using concentrations of ECVC from either 2nd or 3rd generation devices, that did not impair osteoblast viability (0.25% and 0.50%) was sufficient to elicit a sustained reduction in OPG secretion for up to 7 days. This suggests long term use of e-cigarettes could lead to chronic suppression of OPG, promoting greater bone resorption Such non-toxic effects could also extend to suppression of other critical cellular functions of osteoblasts, such as alkaline phosphatase activity, or expression of genes such as COL1A1, in turn reducing extracellular matrix secretion. This carries clear clinical implications for a number cohorts including; adolescent users still undergoing bone development, in addition to individuals with bone associated disease such as osteoporosis, and those recovering from injury and surgery as discussed above. In line with this, it may also be important to consider chronic e-cigarette use in the context of DNA damage and ageing. Although e-cigarettes are widely thought to produce fewer carcinogenic compounds relative to cigarettes, nicotine nitrosation does indeed occur following e-cigarettes use, inducing the formation of the DNA adducts O(6)-methyl-deoxyguanosines and cyclic γ-hydroxy-1, *N2*–propano-dG [55]. Additionally, the presence of DNA adducts associated with reactive carbonyl species, such as acrolein, have also been reported following e-cigarette use in humans [56–58]. Therefore, it seems reasonable to assume that chronic systemic delivery of such compounds following e-cigarette use could reduce DNA repair in bone, in turn driving an accelerated

bone ageing phenotype as described by Chen et al. [59]. Consequently, this may compound dysfunctional bone remodelling and further contribute to reduced bone mass and increased fracture risk, particularly in older users.

Due to the difficulty in replicating e-cigarette use in vitro, this study does have limitations. Firstly, although we have used a range of does in this study, concentrations of nicotine and other metabolites in the vapour condensate may not represent localised interstitial concentrations. Smoker and vaper plasma nicotine will of course vary greatly dependent on personal addiction level. However, ex-smokers will vape to meet their individual nicotine addiction needs [18] and as such, it is likely tissue exposure will remain comparable between these groups. Smoker urinary nicotine and nicotine metabolites (assessing chronic exposure) have been quantified in a range from 7–338 μM [60]. Here, we have delivered non-toxic nicotine doses of 0.82 μM (0.5% 2nd generation challenge); and 3.1 μM (0.5% 3rd generation challenge) [32]. Whilst lacking definitive data for comparison, these low dose challenges are well within a feasible physiological range [32]. Secondly, osteoblasts were only stimulated with one dose of ECVC, and therefore we can only speculate that repeated daily usage of chronic e-cigarette use would have a similarly detrimental effect on osteoblast viability.

#### **5. Conclusions**

In summary, we have demonstrated that ECVC has a negative effect on both osteoblast viability and function, with these effects being mediated, in part, by nicotine-dependent mechanisms and also reactive carbonyl species derived from e-liquid humectants as summarised in Table 1. Reduced osteoblast viability, coupled with a reduction in OPG secretion as observed following ECVC treatment, may lead to increased bone resorption following chronic exposure, in turn potentially impacting bone development in younger users, while increasing bone associated disease progression and negatively impacting orthopaedic and dental surgeryoutcomes.

**Table 1.** A summary of key findings.

**Author Contributions:** Conceptualization, S.W.J., A.S., M.N.E. and T.N. Methodology T.N., L.D., A.S. and S.W.J. Formal analysis, T.N.; S.W.J. and A.S. Investigation T.N., L.D., S.W.J. and A.S. Resources, S.W.J., A.S., M.N.E. and E.T.D. Data curation, T.N., L.D. and S.W.J. Writing—original draft preparation, T.N., L.D., A.S. and S.W.J. Writing—review and editing, T.N., S.W.J. and A.S. Visualization, T.N., S.W.J. and A.S. Supervision, S.W.J. and A.S. project administration, S.W.J. and A.S. Funding acquisition, S.W.J. and A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** AS is Supported by Asthma + Lung UK (MCFPHD20F\2), the NIHR Health technology assessment (NIHR129593), NIHR EME programme (NIHR131600) and Medical Research Council (MR/L002736/1). SWJ is supported by Versus Arthritis, UK (21530; 21812).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study (NRES 16/SS/0172).

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We gratefully acknowledge the contributions of Grace Shattock and Hollie Cooke to the collection of data. We also wish to acknowledge the research nurses and research healthcare practitioners at the Royal Orthopaedic Hospital, Birmingham, UK, for their efforts in recruiting patients and collecting tissue samples. Finally, we thank the patients who participated in this study, without which, this work would not have been possible.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**

**Figure A1.** Low dose stimulation of Primary human osteoblasts for up to 7 days does not affect viability. Effect of treating primary human osteoblasts either 2nd (**A**) or 3rd generation (**B**) ECVC at either 0.25% or 0.5% for 24 h-7d on primary human osteoblasts. *n* = 3 patient replicates with 4 biological replicates performed per patient.

#### **References**