NSAID and Their Effect on Bone Remodeling and Repair

Non-Steroidal Anti-Inflammatories and Their Effect on Bone Remodeling and RepairBy: Andrew Beardsall

Introduction

Non-steroidal anti-inflammatory drugs (NSAID’s) are the most widely prescribed

drugs in the world, with 30 million people consuming prescription NSAIDs every day

[1]. Due to NSAID’s general anti-inflammatory properties, they are used for a range of

physical ailments including, but not limited to, osteoarthritis, rheumatoid arthritis, and

musculoskeletal injuries. NSAID’s effectiveness as analgesics, anti-inflammatories, and

anti-pyretics is well documented [2]. However, the mechanism of action for this class of

drugs is still being disputed, specifically in regards to the effects of the metabolites

produced by the involved pathway [8].

The metabolic pathway targeted by NSAIDs is the arachidonic acid (AA) pathway.

Within this pathway, catabolism of AA leads to the formation of inflammatory and non-

inflammatory metabolites. NSAIDs are able to modify the breakdown of AA through

inhibition of the cyclooxygenase (COX) enzymes. There are 2 COX enzymes of

importance, which are named COX-1 and COX-2, respectively. NSAIDs are grouped into

categories depending on selectivity to either enzyme. [3]. The selectivity of a particular

NSAID towards either COX enzyme plays an integral role in both the function and side

effects of the drug [4].

Of particular importance within the medical community is the effect that NSAIDs

have on bone remodeling and repair. Inconclusive evidence surrounds the acute and

chronic effects of NSAID ingestion on bone health [13, 14, 15]. Due to this, physicians

avoid prescribing NSAIDs for traumatic bone injuries [16]. The aim of this literature

review is to summarize current scientific knowledge related to NSAID ingestion and

bone health. Current research is performed using mostly single drug trials in animals and

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in-vitro studies using human bone grafts. In light of the current evidence surrounding the

AA pathway and COX inhibition it is unreasonable to generalize single drug trials to the

entire grouping of NSAIDs. Therefore, the effects that commonly prescribed NSAID’s

have on bone health will be investigated with no attempt to generalize current findings to

NSAIDs as a whole. In this literature review drugs will be grouped based on enzyme

interactions to evaluate their efficacy and side effects.

AA Pathway

The AA pathway is a fatty acid metabolism pathway that is triggered by a variety

of stimuli; one of which is cell injury or death. This pathway is essential for the creation

of inflammation within the human body as AA is the precursor to many inflammatory

metabolites. Inflammation is a local immune response created by the body to initialize the

local healing process through increased blood flow. The increased blood flow supplies

the injured tissue with nutrients and growth factors necessary for tissue repair [5].

Once phospholipids are released from local cells they are quickly broken down

into AA. AA then binds with either a COX-1 enzyme, which is continuously expressed,

or a COX-2 enzyme, which is inducible. COX-1 is responsible for the production of

Thromboxane A2 and other prostaglandins, which are largely responsible for thrombosis

formation. COX-2 is responsible for the production of prostaglandins (PGD2, PGE2, PGI2,

PGF2), which have a variety of functions. The COX enzyme byproducts are called

eicosanoids, which are a group of lipid signaling molecules. The following is an

illustration of the AA pathway:

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Figure 1. AA pathway. Source: (Knights, Mangoni, Miners 2010) [4]

Although it has been found that both COX-1 and COX-2 produce prostaglandins,

COX-2 has been suggested to produce the prostaglandins that are responsible for the

body’s pain perception and inflammation response [6,7]. Each prostaglandin produced by

COX-2 has a separate function depending on how it is used as a signaling molecule.

PGE2 and PGI2 are responsible for vasodilation around the inflammatory site [7].

Prostaglandins are hypothesized to play a vital role in the healing process of bone though

a variety of interacting mechanisms [8, 37]. However, the mechanisms of action of these

prostaglandins are beyond the scope of this literature review.

NSAIDs: Classification and Description of Action

The creation of NSAIDs dates back to 1899 when Felix Hoffman, a chemist

working for a company in Germany called Bayer discovered the drug commonly known

today as aspirin. The mechanism action of Aspirin remained unknown until the 1960’s

when Dr. John Vane discovered that it had significant effect on the production of

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prostaglandins. The enzymes responsible were later discovered to be the COX-1 and

COX-2 enzymes [9]. Since this discovery, research in the field of NSAID therapy has

progressed significantly in the form of creation of new drug therapies and a greater

understanding of the underlying mechanisms.

The classification of NSAIDs is very important in understanding how the drug

interacts with an organism. The selectivity and binding characteristics of an NSAID

largely determine both the efficacy of the drug and its side effects. Below is a table used

to classify most NSAIDs.

Table 1. Classification of NSAIDs on the basis of their COX inhibition activityGroup Group Description Drugs Within GroupsGroup 1: nsNSAID

Poorly selective NSAIDs that fully inhibit both COX-1 and COX-2 (<5-fold COX-2 selectivity)

Ibuprofen, diclofenac, aspirin, piroxicam, naproxen, ketorlac [20]

Group 2:sNSAID(semi-selective)

NSAIDs capable of inhibiting both COX-1 and COX-2 with a preferential selectivity toward COX-2 (5 to 50 fold COX-2 selectivity)

Celecoxib, meloxicam, nimesulide, etodolac, indomethacin [12]

Group 3:sNSAID

NSAIDs that strongly inhibit COX-2 but only weakly inhibit COX-1 (,50-fold COX-2 selectivity)

Rofecoxib, NS-398

Group 4 NSAIDs that seem to be only weak inhibitors of both COX-1 and COX-2

Sodium salicylate, nabumetone

Source (Bacchi, Palumbo, Sponta & Coppolino 2012)

The method of classification of NSAIDs is through the use of an IC50 value, which

is the concentration of the drug that is required to cause 50% inhibition of the COX-1 or

COX-2 enzyme. This method however has been proven to be unreliable due to

considerable variation in assay methods, leading to variation in IC50 numbers ranging

from 0.0015 to 16 µM for a single NSAID [10].

It has been proven that within NSAID groupings there is a significant variation of

COX enzyme affinities. It has also been proven that the methods used to classify these

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drugs and to measure each drugs specific affinity towards one COX enzyme is unreliable.

For this reason, the first conclusion of this literature review is that results from

investigating the effects of specific NSAIDs on bone remodeling and repair are not

generalizable to larger grouping of NSAIDs. Results should also not be generalized

within specific subgroupings (based on COX enzyme affinity) of NSAIDs. However,

knowledge of the drugs COX affinity and its binding properties can act as a starting point

for an investigation. For the above reasons, the review will now focus on current drug

specific research for commonly prescribed NSAIDs.

Group 1: Non-Selective Non-Steroidal Anti-Inflammatories (nsNSAID)

Ibuprofen and other similar drugs such as Dicolfenac, Aspirin, and Naproxen are

within a subcategory of NSAIDs called non-selective NSAIDs (nsNSAIDs)[3]. This

means that the extent they inhibit both COX-1 and COX-2 is nearly equal. The binding

characteristics of these drugs with the subcategory nsNSAID differ significantly.

Ibuprofen exhibits binding properties that are rapid, competitive, and reversible with both

COX enzymes. Diclofenac differs as it demonstrates slow initial binding speed but has a

strong binding strength to both COX enzymes. Aspirin differs from the other two

mentioned as it exhibits rapid reversible binding [11,17]. Within the nsNSAID subgroup,

due to minor differences in binding affinity to COX-1 or COX-2, as well as different

binding characteristics, the interaction with bone remodeling and repair should vary

significantly within the nsNSAID subgroup [4].

In-vivo studies of rabbits and rats make up a significant portion of early research

regarding the effects of nsNSAIDs on bone remodeling and repair. The methodology

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used in these studies was variable, yet fairly simple. Fractures were manually induced to

the femur, the tibia, or the ulna; in most cases followed by medullary reaming. The

fracture limb was then pinned and set to heal. Once the treatment period was over the

animals were euthanized so biomechanical, histological, and radiographical data could be

collected.

Biomechanical data was collected through torsional bending by Ho, Chang and

Wang (1995) [18] and Huo et al. (2005)[23], as well as through three-point bending by

Beck et al. (2003) [19]. In Chang et al. (1995) drug treatment lasted 6 weeks, leading to a

finding that torsional stiffness was decreased in a dose dependent manner whereas

maximal torque and energy absorption only saw a decline with higher doses of Ketorolac

(4mg/kg/day). Huo et al (2005) found no significant difference in torsional strength and

rigidity with higher doses of Ibuprofen (30mg/kg/day) contradicting the results to those

found in Chang et al. (1995). Beck et al. (2003) in a three-week trial found that given a

therapeutic dose of Diclofenac (2mg/kg/day) bone stiffness was only significantly

depleted at 7-day evaluation.

Histological and radiographical data was also presented for the above in-vivo

animal studies. Chang et al. found no histological changes when looking at PGE2 levels

with Ketorolac drug therapy; these results agreed with those found by Huo et al. (2005).

Shahriari et al. (2011) [22] recently contradicted the results found by Chang et al. and

Huo et al. in a human trial, finding that a similar nsNSAID had a statistically significant

negative effect on PGE2 synthesis. Radiographical analysis looked at cross sectional area

(CSA) and bone mineral density (BMD). With Diclofenac drug therapy, Beck et al. found

that BMD was significantly decreased with therapeutic doses. Two out of the three

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animal studies investigated concluded that NSAIDs should not be used during the healing

process after traumatic bone injuries or surgeries. Huo et al. (2005) found no

radiographical measurement differences in bone post NSAID treatment. A possible

explanation for this is that Huo et al. (2005) delayed the administration of NSAIDs by 3

days. This is hypothesized to be a crucial period of healing that is significantly affected

by NSAIDs ingestion; a hypothesis supported by the findings of Beck et al. (2003). The

conclusion that NSAIDs have a negative effect on histological and radiographical

measurements of bone was also supported by an earlier study performed by Altman et al.

(1995) [24]

In-vitro studies have also been used to study the effect of NSAID on cell function

and proliferation. These studies use bone cells from human bone grafts from multiple

hosts [28] or specific laboratory cell lines from a single host [29, 32]. The dosages range

from 5-3000 uM of Ibuprofen with equivalent dosages, based on recommended adult

dosages [25, 26] in other drugs such as Ketorolac, Diclofenac, Piroxicam. The typical

incubation period for these drugs is 24 to 48 hours, which is a sufficient exposure time to

cause cellular changes [28, 29, 31].

Cell counting was done as a marker of cell proliferation in all of the studies

mentioned above. Therapeutic doses of Ibuprofen (under 100 uM) and equivalent doses

of other drugs have been shown to decrease cell proliferation [31, 28] but it requires

larger doses to cause a decrease in osteoblast precursor differentiation [30]. In both the

Chang et al. (2009) and Garcia-Martinez (2011) studies, no antigen profile, or phagocytic

cell abnormalities were reported [28, 31]. However a study performed by Diaz-

Rodriguez, Garcia-Martinez, Luna-Bertos, Ramos-Torrecillas & Ruiz (2012) was

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recently published that refuted these findings using an MG-63 cell line. This cell line

however has been proven to be a poor representation of osteoblast activity [32]. Studies

using the control drug of acetaminophen (an analgesic) showed smallest negative effect

of NSAID on cell proliferation [28]. Due to decrease osteoblast differentiation with

therapeutic doses of NSAID it is recommended that usage of these drugs be avoided in

situations requiring rapid bone growth such as a traumatic bone injury or surgery [27,

28].

There is sparse research investigating NSAID therapy and bone remodeling on

human participants. In orthodontic surgery performed in a study by Sakka and Hanouneh

(2013) [33], they found that post surgery ibuprofen administration did not significantly

affect implant integration through bone growth; however, they were not able to test bone

strength. The only study addressing bone strength through the investigation of re-fracture

rate with Ibuprofen administration was performed by Drendel et al. (2009). The study

was performed on children but was to underpowered to yield a significant result [34]. It

has been shown in human trials that prostaglandin levels are significantly affected by

NSAID administration [22]. Given our current knowledge of prostaglandin action these

results seem to contradict each other.

Other human in-vivo research exists that addresses the topic of ibuprofen

administration timing pre and post exercise. A study performed by Kohrt. et al. (2010)

had premenopausal women participate in a weight bearing exercise regime[35, 46]. These

women were separated into 3 groups: Ibuprofen before exercise, Ibuprofen after exercise,

and a double placebo group. Adaptations to exercise were measured using DXA scan in

multiple bony regions. Ibuprofen administered post exercise saw the highest BMD

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increase of three subgroups whereas Ibuprofen administered before exercise saw the least

favourable adaptations. The positive effects of Ibuprofen on bone mineral density were

also seen in an in-vivo rat study performed by Jain et al. (2014) [36]. However, this study

was performed under constant administration of Ibuprofen; timing of dosage was not a

controlled variable.

Data has been presented from; in-vivo animal studies addressing biomechanical,

radiographical and histological characteristic: In-vitro studies performed with human

bone grafts looking at osteoblast proliferation: and in-vivo human studies addressing

exercise and post traumatic administration of nsNSAIDs. These studies have been mostly

conclusive in that nsNSAIDs have a negative effect on bone health. However, more

research is needed in areas related to chronic use, timing of dosage, and re-fracture rates

as an indication of ultimate bone strength.

Group 2 and 3: Selective NSAID (sNSAID)

Until the latter half of the 20th century, the mechanisms through which NSAID

interacted with the body remained completely unknown. In that time an effort was made

to develop drugs to selectively target COX-2 due to the negative gastric side effects of

COX-1 inhibition. In 1998 the United States Food and Drug Administration (USFDA)

approved the first COX-2 specific drug; Celexocib [2, 38]. Due to the differential action

of COX-1 and COX-2, it is crucial that an effort is made to separately explore the effects

on bone growth within this subgroup. In doing so, a comparison of nsNSAIDs to

selective NSAID (sNSAID) will be established enabling an analysis of the original

hypothesis that these NSAID subgroups should be evaluated separately.

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The in-vivo animal studies that have been performed to date have been set up

much the same way as the nsNSAID studies. They involve an induced fracture with

medullary reaming and present data on histological, radiographical, and biomechanical

aspects of bone health. The studies looked at to evaluate the effects of sNSAID on bone

health involved rabbits, rats, and mice. Drug administration periods lasted from 3 days to

12 weeks in length, with data being collected intermittently.

Studies looking at the mechanical properties in bone using torsional or 3 point

bending converge on one conclusion; with prescription of sNSAID the bone remodeling

and repair process that occurs after a bone fracture is delayed. Evidence of this delay is

clear when comparing studies of different lengths. Regardless of the duration or method

of administration, there is a significant decrease in strength and stiffness of healing bone

for studied periods of 2-8 weeks with therapeutic doses of indomethacin or other

sNSAIDs [14, 24, 39, 40, 41, 42]. O-Connor et al. (2009) evaluated the mechanical

properties of bone in rabbit fibula and found that the difference in mechanical properties

to the control disappeared by 12 weeks [40]. O’Connor et al. (2009) and Brown et al.

(2004) evaluated the differences between nsNSAIDs and sNSAIDs and found that

nsNSAIDs, such as ibuprofen, were less deleterious on bone remodeling then sNSAIDs

when looking at therapeutic dosages [39, 40]. Brown et al. (2004) found that within the

subgroup of sNSAIDs, when one compares the effects of a less selective COX-2 inhibitor

(indomethacin) to a COX-2 specific inhibitors (celecoxib) there is a greater deleterious

effect in mechanical properties with a more selective COX-2 inhibitor [39]. These

findings support the need for a segregated comparison of NSAIDs.

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Histological and radiographical data can be used to help explain the reason for

these mechanical findings of decreased bone strength and stiffness. O’Connor et al.

(2009) found that 6 weeks post fracture it was evident that indomethacin treated rabbits

had poorer mineralization of callus bone compared to controls [40]. Brown et al. (2004)

in a previous study had also reported a poorer mineralization of bone in both

indomethacin and celecoxib treated rats compared with no drug controls [39]. It was

shown by O’Connor et al. that there is a compensatory increase in callus CSA to make up

for poorer bone quality and mineralization [40].

In-vivo human studies directly addressing the effects of sNSAID on bone

remodeling and repair are very rare due to the relative youth of this class of drugs. One

retrospective study performed by Reuben, Ablett and Kaye (2005) looked at spinal fusion

operation success with the administration of NSAIDs [43]. Data was analyzing from 434

patients taking either a nsNSAID (ketorolac) or a sNSAID (rofecoxib). Upon analysis of

the data it was found that high doses of the nsNSAID showed a significantly higher non-

union rate then both the sNSAID and the control group. It would be expected based on

data collected from both in-vitro studies and in-vivo animal studies that the sNSAID

would have the largest increase in non-union incidence indicating a bone healing failure

[31, 44, 45]. The study also illustrated that duration of drug administration could play a

large role in recovery of bone health in chronic usage (more than 3 months) compared to

acute usage (less than 5 days).

Conclusion

Both nsNSAID and sNSAID have been shown to have a significant effect on bone

health. These effects have been demonstrated through in-vivo animal studies on rats,

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mice and rabbits as well as in in-vitro studies using human bone grafts. Human studies,

although the most relevant to this topic, are infrequently investigated and published. The

generalizability of both in-vivo animal studies and in-vitro human cell studies is

sometimes considered questionable. This is stated in many studies as both previously

mentioned study designs are considered an imperfect representation of a clinical setting.

These studies concluded that further research is needed [2, 19].

Although previous studies are an imperfect representation their clinical relevance

is important in many ways. The few studies that have highlighted the differences in

finding between nsNSAID and sNSAID are generally draw inconclusive results as they

are inconsistent in both methods and findings [31, 40, 41, 45, 46]. This however does not

contraindicate the need for separation of nsNSAID and sNSAID. The current research

only highlights the need for a standardized procedure and method of evaluation.

To truly evaluate the hypothesized theories supporting the segregation of NSAIDs

into their respective subgroupings, one would need to conduct a randomized control trial

(RCT) in human participants. It is considered ethical to perform RCT by many physicians

including Dr. J. Bertoia, practicing Orthopaedic Surgeon and previous Chief of Surgery

at Southlake Regional Health Center, however funding for a major study such as this, is

considered a significant barrier. Current Orthopaedic Surgeons evaluate the prescription

of NSAIDs, regardless of their respective subgroupings, on a case to case basis.

Preference to drug treatment and personal evaluation of current literature plays a large

role in a physician’s prescription patterns for NSAIDs[8].

Research proposal

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In light of the current lack of a standard RCT performed in human participants

and the sufficient grounds to prove such a study is ethical, I believe that the only way to

move the literature forward is to design a study that directly addresses this issue. The

current knowledge of the AA pathway and how prostaglandins interact with the human

body provide enough evidence to justify a human trial. Differential effects of NSAIDs

between their subgroupings seen in in-vivo animal studies support current knowledge of

the AA pathways. Although these results are inconclusive there has never been a large

RCT targeting this specific aspect of the literature.

Funding for a large RCT that can render statistically significant results would be

difficult to secure. There is little need for drug companies to support expensive RCT

projects, as they are not without inherent risk; with the potential to damage company

reputation. NSAID companies are weary of investing heavily in large projects such as

this as they have resulted in massive lawsuits in the past; an example being the

ADVANTAGE trial performed by the company Merck with the drug Vioxx. This trial

was actually a marketing tool that Merck used to launch their drug and gather much

needed clinical usage data. Unfortunately, the study revealed a large negative effect on

cardiovascular health and was subsequently disapproved by the FDA, forcing its

withdrawal [47].

If funding were secured, the RCT would involve the use of four NSAIDs. There

would be two commonly used drugs representative of each of their respective

subcategories and an analgesic control. The two drugs representing the nsNSAID

category would be ibuprofen and naproxen, as they are two very commonly prescribed

drugs in a clinical setting. The two drugs representative of the sNSAID category would

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be indomethacin and celecoxib as both are prescribed as an anti-inflammatory within a

clinical setting. The analgesic control would be Tramadol to allow all participants pain

relief during the healing process.

The population targeted in this study would be men aged 20-35 as this subset of

the population display similar BMD and the rapid growth and remodeling due to the

onset of puberty is no longer a confound [48]. Female participant exclusion is justified as

estrogen plays a large role in creating fluctuations in bone remolding markers throughout

the menstrual cycle [49]. We would be looking to recruit and study men who had

experienced lower body long bone fractures within the last 24 hours. The short time

period post fracture would eliminate pre-evaluation healing effects. All participants

would be required to be non-smokers to avoid the inhibitory effects of smoking on bone

healing as a study confound [54].

Therapeutic dosages based on reference material [34, 44].Drug Dosage instructionIbuprofen 400-800 mg taken up to 4 times per dayNaproxan 275-550 mg taken two times per dayIndomethacin 25-50 mg taken three times per dayCelecoxib 200-600 mg/day taken in one dosageTramadol 50-100mg taken 4 ties per day

When patients enter into the study presenting a fracture they would receive a

DXA scan to analyze BMD, CSA, number of cortices spanned by bridging callus, and

total callus formation [19, 40, 41]. All of these variables can be calculated using the

software that is present in the DXA machines. After DXA scan completion they would

proceed with fracture treatment as prescribed by the physician depending on fracture

type. Regardless of fracture type participants would be randomly selected and placed into

the five drug groups. Patients would be informed of the drug dosages and instructions; all

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drugs would be prescribed at normal therapeutic dosages. Length of drug treatment

would be prescribed as needed to manage pain and swelling.

Follow up evaluations would be conducted for all participants in the study at the

four week mark as this has been shown in rat studies to yield the largest difference [14,

24, 39, 40, 41, 42]. These evaluations would include a DXA scan to evaluate BMD, CSA,

number of cortices spanned by bridging callus, and total callus formation; all markers of

bone maturity and healing progress. These values would be compared to the original

DXA scan values to evaluate healing progress. Each group would be compared to the 4

others groups to evaluate comparative bone maturity and healing rate. At this time

physicians would also conduct an Enzyme-Linked Immunorbent Assay (ELISA) to

evaluate the drugs effects on prostaglandin levels; a variable hypothesized to be the main

contributor to the delayed healing process [3, 22, 55]. This test is not the most cost

effective measure but it has been shown to be effective at measuring prostaglandin levels

in humans in a previous study by Shahriari et al. (2011) [25]. The measurement of

prostaglandins would be compared statistically to bone maturity in an attempt to confirm

the underlying mechanism causing delayed healing in bone.

Fracture delayed unions will be followed up on via telephone interview and

analysis of patient medical charts. The evaluation for delayed union will be based on a

definition provided by Furlong et al. (1999). Furlong et al. (1999) describes delayed

union as “any fracture that takes longer then 12 weeks to heal” and also states “fracture

union was defined as painless full weight bearing in the presence of circumferential

callus in two planes on radiographs”.

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Given that definition, a patient presenting pain in the location of the fracture will

be re-evaluated at the 12-week mark and will have a further DXA scan performed.

Delayed or non-union rates vary from 1-2% [34, 52], depending on fracture type, with

NSAID administration retrospectively evaluated to increase risk of non-union by 5 fold

[54]. To statistically evaluate the rate of delayed or non-union a large sample group is

needed. Given this low delayed union rate the study population would need to include

approximately 1500 participants at a study cost of $667,275.00; a complete break-down

of which can be viewed in Appendix A.

If a significant difference were shown between NSAID drug subgroupings and a

control within a human trial then there would be significant clinical implications.

Currently orthopedic surgeons avoid the usage of NSAID in most cases involving bone

trauma including but not limited to joint replacement, fractures, and spinal fusion. As an

alternative analgesic to NSAIDs they prescribe either Tylenol or narcotics, both having

significant side effects. If either subgroup of drugs were found to have a more limited

effect on bone healing then it would significantly impact the drugs that are prescribed.

Given current knowledge it would be expected that nsNSAID would have little or no

impact in bone healing within a RCT. This finding would have a significant impact on

quality of life for a very large subset of the patient population in Canada and the world.

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Appendix A.

Cost analysisTest Approximate

numberApproximate cost per

Total cost

DXA pre treatment 1500 $157 [51] $235,500ELISA pre treatment

1500 $60 [50] $90,000

DXA at 4 weeks 1500 $157 $235,500ELISA at 4 weeks 1500 $60 $90,000DXA at 12 weeks for non-union

75 [52, 53] $157 $11,775

ELISA at 12 weeks for non-union

75 [52, 53] $60 $4,500

Total cost: $667,275.00

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Documents

NSAID and Their Effect on Bone Remodeling and Repair