Efficacy of hypertonic dextrose injection (prolotherapy) in temporomandibular joint dysfunction: a systematic review and meta-analysis

Hypertonic dextrose prolotherapy (DPT) has been reported to be effective for temporomandibular disorders (TMDs) in clinical trials but its overall efficacy is uncertain. To conduct a systematic review with meta-analysis of randomized controlled trials (RCTs) to synthesize evidence on the effectiveness of DPT for TMDs. Eleven electronic databases were searched from their inception to October, 2020. The primary outcome of interest was pain intensity. Secondary outcomes included maximum inter-incisal mouth opening (MIO) and disability score. Studies were graded by “Cochrane risk of bias 2” tool; if data could be pooled, a meta-analysis was performed. Ten RCTs (n = 336) with some to high risk of bias were included. In a meta-analysis of 5 RCTs, DPT was significantly superior to placebo injections in reducing TMJ pain at 12 weeks, with moderate effect size and low heterogeneity (Standardized Mean Difference: − 0.76; 95% CI − 1.19 to − 0.32, I2 = 0%). No statistically significant differences were detected for changes in MIO and functional scores. In this systematic review and meta-analysis, evidence from low to moderate quality studies show that DPT conferred a large positive effect which met criteria for clinical relevance in the treatment of TMJ pain, compared with placebo injections. Protocol registration at PROSPERO: CRD42020214305.

www.nature.com/scientificreports/ were characterized by small sample size, short study period, lack of methodologic rigor and inconsistent results, which limit the ability to draw consistent recommendations in for clinical practice 5,13 . Hypertonic dextrose prolotherapy (DPT) is an injection therapy used to treat chronic painful musculoskeletal conditions 14,15 . The mechanism of action is not well understood; the historical understanding posits that DPT facilitates healing and subsequent pain control through initiation of a temporary inflammatory reaction with related tissue proliferation [16][17][18][19] . Recent literature also suggests the mechanism is multifactorial and may include direct sensorineural effects 20 . Recently, a growing number of methodologically higher quality clinical trials have evaluated the use DPT for TMDs, which reported beneficial effects on pain and dysfunction using standardized outcomes 21,22 . However, the findings were not included in the previous systematic review 23 . Patients, clinicians and health care systems benefit from ongoing review of changing medical literature to assist clinical decision making informed by the best available evidence 24 .
The aim of this study was to conduct a systematic review of randomized control trials (RCTs) to assess and analyze the overall efficacy of DPT in TMDs. We hypothesized that DPT would reduce pain and improve function of TMJs, compared to placebo interventions, among patients with TMDs.
Characteristics of included trials. Characteristics of 10 included trials was summarized in Table 1. The sample sizes of the studies ranged from 12 to 72, with a total of 336 individuals. The study period ranged from 4 weeks to 1 year post-enrollment. The injection protocols consisted of intra-articular injection only, or a combined approach of intra and extra-articular injections. The injection frequency ranged from single injection to 4 injections, weekly to 4 weeks apart, with dextrose concentration varying from 10 to 30% (Table 1) .
DPT versus placebo on TMJ dysfunction at 12 weeks. Two RCTs (n = 71) were eligible for pooling; an NRS was used in both trials to assess TMJ dysfuction 21,22 . Although pooled results suggested a potential positive effect of DPT on reducing jaw disability, it was not statistically significant, with the weighted mean difference (WMD − 1.43; 95% CI − 2.89 to 0.03, P = 0.06, I 2 = 43%) (Fig. 3).
DPT versus other active interventions. Pooling of results was not possible due to the use of different control interventions, different assessment time-points, and absence of raw figures in the publications. In Mahmoud et al., the use of platelet rich plasma demonstrated a statistically significant reduction in MIO compared to DPT and hyaluronic acid at 12 weeks, though no between-group differences were detected for pain scores 29 . In Hassanein et al., the use of laser therapy also resulted in a statistically significant reduction in MIO compared to DPT at 4 weeks; similarly, there were no between-group differences for pain scores 32 . In Arafet et al., the use of autologous blood was superior to DPT in reducing MIO at 2 and 4 weeks (P < 0.001), though longer term data was lacking 31 . Effectiveness of DPT at 12 months. In Kilic et al., no statistically significant improvement was observed between DPT and placebo groups at 12 months 26 . In Louw et al. and Zarate et al., DPT was offered to the control groups after participants were un-blinded at 12 weeks. The intra-group improvement in pain and function scores was sustained at 1 year, and inter-group difference was statistically significant in Louw  www.nature.com/scientificreports/ of longer term effectiveness 21,22 . However, the un-blinding and subsequent injection of DPT upon participant request, prevented us from including 12-month outcomes data in our meta-analysis.
Adverse events. Adverse event-related outcomes were reported in 3 of the 10 included trials. One trial reported painful and burning sensations among 18 participants, with temporary paralysis of temporal branch of the facial nerve in 4 participants 27 . One trial reported one participant had worsening of jaw pain and swelling 2 months after study enrolment, and was subsequently diagnosed with an actinic cell tumor of the parotid gland unrelated to therapy 21 . One trial reported no adverse event reported throughout the study period 22 .

Discussion
This study showed that DPT is superior to placebo injections in reducing TMJ pain intensity, with a moderate to large effect size and low heterogeneity at 12 weeks 33,34 . Although the findings do not demonstrate a statistically significant improvement in the disability score of DPT compared to placebo injections, the positive trend suggests that even in the context of meta-analysis, the comparison may be underpowered and that a larger sample size may be able to detect a difference. Comparison with other injection therapies such as corticosteroids and hyaluronic acid was not possible due to the absence of effect sizes in relevant TMJ reviews 35,36 . Because different injection approaches were used in the included studies, special attention is needed in the interpretation of MIO findings. The normal values of MIO have been reported as 51.00 mm for male and   37 . In the four included RCTs, Refai et al. and Mustafa et al. used the standard protocol of DPT consisting of intra-articular and extra-articular (capsular) injections. Participants in these trials had painful subluxation or dislocation of the TMJ; therefore, reducing MIO was expected to improve the overall joint stability through a "whole" joint treatment 25,28 . The finding was consistent with other prospective case-series, when extra-articular injections were found to reduce jaw motion 38,39 . Conversely, participants in the other two trials had painful clicking TMJ, without subluxation or dislocation; in these studies the effect of intra-articular DPT injection on joint stability was less consistent. Louw el at., reported an increase in MIO in the DPT group; Zarate et al., reported an increase in MIO in both groups 21,22 . We suggest that extra-articular injections, with multiple needling and the tissue proliferative effects of dextrose, may have recruited the inflammatory cascades leading to capsular strengthening 20 . Previous rodent studies of medial collateral ligaments injected with dextrose have reported increased levels of inflammatory markers in healthy tissue and an increased cross-sectional area in strain-injured tissue 16,17 . In rabbit models, injection of DPT into the connective tissue in the carpal tunnel produced thickening of the collagen bundles when compared with saline controls 18,19 . Although, we have not detected a statistically significant effect size on MIO, it appears possible that different protocols may be optimal for different sets of symptoms and signs. This view is supported by Fouda et al., who suggested that the selection of the injection site is the most important part of treatment, and that hypermobility should be treated with injection into the outer capsule, whereas pain is best treated with injection into the joint space 27 .
The mechanism by which DPT may decrease musculoskeletal pain, including TMD pain, is not well understood. Recruitment of the inflammatory cascade noted above may contribute to pain control through indirect, downstream wound healing effects. In addition, several models have been proposed which feature the direct effect of dextrose on nerve and other tissues. First, dextrose (d-glucose) is a crucial nutrient for functioning of cartilage and is the precursor for synthesis of glycosaminoglycans, glycoproteins, and glycolipids 40 . A recent in vitro study by Wu et al. showed that dextrose upregulates expression of aggrecan in chondrocytic ATDC5 cells and downregulates microRNA-14103-3p (miT141-3p). The resulting high local concentration of aggrecan may provide a favourable osmotic environment for growth and function of cartilage 41 Second, dextrose solution hyperpolarises nerves by opening their potassium channels, thereby decreasing signal transmission in nociceptive pain fibres 42 . Third, glucose solutions may work by blocking transient receptor potential vanilloid type 1 (TRPV 1), a membrane cation channel that allows influx of sodium and calcium. Sodium influx is thought to result in an action potential and nociception, whereas calcium results in the release of substance P and calcitonin generelated peptide 43 . Hence, blocking the influx of both cations may theoretically minimise neuropathic pain 44 . This mechanisms is consistent with recent preclinical and clinical data which strongly support a role for various TRP channels 45 . Clinically, a potential sensorineural analgesic mechanism of dextrose is suggested by its apparent effects in several clinical studies, including epidural injection of dextrose in the treatment of chronic low back pain 46 , intra-articular DPT injections for knee pain 47 , and significant pain reduction after perineural injection of DPT in patients with carpal tunnel syndrome or Achilles tendinitis 48,49 .
Strengths of the current study included timely conduct of study to review an area that is rapidly emerging, clinically important, and has disparate findings. Besides, we used rigorous methodology that conforms to best practice guidelines. There are several limitations in the current study. First, the number of included studies and total participant sample size were small. Second, raw data were missing in some articles as they were reported by plots and histograms; therefore, not all the data could be synthesized [29][30][31] . Third, changes in the diagnostic criteria of TMD resulted in a lack of diagnostic specificity across RDC/TMD categories in some studies, and some trials recruited participants with TMJ pain and others with hypermobility or subluxations. It is likely that patients in different diagnostic categories respond optimally to different injection protocols. Finally, the 12-week time frame available for data pooling was short. Therefore, longer term effects remain uncertain.

Conclusion
In this systematic review and meta-analysis, evaluation of best available evidence shows that DPT conferred a large positive effect which met criteria for clinical relevance in the treatment of TMJ pain, compared with placebo injections. Therefore, in carefully selected patients, especially those with functional derangement of the TMJs and who are refractory to more conventional care, DPT can be considered an appropriate non-surgical treatment option. Selection of specific injection sites may best be informed by the presenting symptoms. Future rigorous research should include studies of longer-term follow-up. Direct comparison with other injection therapies, cost-effective analysis and a better understanding of mechanism of action will further inform the role of DPT in TMDs.

Methods
We followed the statement on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses for RCTs 50 . The protocol has been registered in the PROSPERO registry (CRD42020214305).
Eligibility criteria. This review included parallel or cross-over RCTs that assessed the efficacy or effectiveness of DPT regardless of blinding or type of reporting 51 . For cross-over RCTs, only data before the wash-out period was used 52 . We excluded complex interventions in which DPT was not a sole treatment. Dissertations and conference abstracts were included if they contained sufficient details 53  RoB judegement SOME SOME LOW SOME SOME SOME SOME SOME LOW SOME Types of participants. This study included participants with TMD diagnosed by any pre-defined or specified diagnostic criteria, which fulfilled the Diagnostic Criteria/TMD Axis 1 (physical symptoms), regardless of age, race and gender 6 . Our study excluded patients with TMDs found to be caused by psychogenic or autoimmune inflammatory causes, which multidisciplinary care had been the core disease management 54,55 . RoB judegement SOME SOME HIGH SOME LOW SOME SOME SOME SOME SOME

Reponses of RCTs
Overall bias SOME HIGH HIGH HIGH SOME HIGH SOME SOME SOME SOME Table 2. Details of signaling questions in each domain of risk of bias assessment for 10 randomized controlled trials. HIGH high risk of bias, LOW low risk of bias, N no, NA not applicable, NI no information, PN probably no, PY probably yes, RCTs randomized controlled trials, RoB risk of bias, SOME some concerns, Y yes.   Consistent with the clinical practice of DPT, at least part of the injection protocol had to include an intra-articular injection, with or without additional injections to the peri-articular soft tissues.
Types of comparison controls. Comparison groups could include saline, free water, any kind of active injections or interventions, or exercise. Co-interventions were allowed as long as they were uniform across all groups such that the net effect of DPT could be estimated.
Outcome measures. The primary outcome of interest was pain intensity or pain relief in TMJ, measured by visual analogue scale (VAS), numerical rating scale (NRS), or algometry. Secondary outcomes included functional score, maximum inter-incisal mouth opening (MIO), frequency of locking or luxation, and number of adverse events.
Study selection and data extraction. Two reviewers (RWWS, KDR) independently screened electronic retrieved titles and abstracts, evaluated potential relevant full texts and determined study eligibility. Copies of all articles of RCTs were obtained and read in full, and data from the articles were validated and extracted according to pre-defined criteria 56 . For eligible studies, data were extracted independently using a piloted data extraction form. For each eligible study, the following data were extracted: study design, participant characteristics, features of interventions, outcomes, duration of follow up and adverse events. An attempt was made to contact study authors regarding these methodological elements if not reported. Discrepancies in study selection and data extraction were resolved by third reviewer (DR).
Risk of bias assessment. The Cochrane risk of bias (RoB) assessment tool 2 was used to evaluate the following 5 RoB domains: bias arising from randomization process; deviation from intended interventions; missing outcome data; measurement of outcome and selection of the reported results 57 . The RoB was assessed by two independent reviewers (CHLW, RWWS); any discrepancy was resolved by a 3rd reviewer (VCHC).

Statistical analysis.
All meta-analyses were conducted using the using Revman version 5.3 58 . A random effect model was used to pool study results, taking into account possible variations in effect sizes across trials 59 . Changes in continuous outcomes were pooled as standardized mean differences (SMD), with 95% confidence intervals (CI). Magnitude of the SMD was determined using standard approach: small, SMD = 0.2; medium, SMD = 0.5; and large, SMD = 0.8 33 . Weighted mean difference (WMD) was used to measure outcomes sharing the same unit of measure, and its potential clinical impact was interpreted according to the minimal clinical important difference (MCIDs) for TMD 60 . The I square (I 2 ) statistic was calculated to estimate heterogeneity across studies. An I 2 level of less than < 25%, 25-50% and greater than 50% were regarded as indicators of low, moderate and high levels of heterogeneity respectively 34 .