Autologous micro-fragmented adipose tissue in the treatment of atherosclerosis patients with knee osteoarthritis in geriatric population: A systematic review and meta-analysis

Background Atherosclerosis and osteoarthritis are closely related. However, no high-quality studies have investigated the potential of micro-fragmented adipose tissue to treat patients with atherosclerosis accompanied by osteoarthritis. Methods PubMed, Embase, the Cochrane Library, Web of Science, China National Knowledge Infrastructure, WANFANG DATA, and CQVIP were searched for potentially eligible studies published before October 13, 2022. Due to the statistical limitations of the existing relevant literature, it is not possible to make direct statistics on the patients with osteoarthritis accompanied by atherosclerosis treated by micro-fragmented adipose tissue. The primary outcome consisted of two parts: 1) Correlation between atherosclerosis and osteoarthritis; 2) Scores of the Knee injury and Osteoarthritis Outcome Score (KOOS). And secondary outcomes were pain assessed by visual analog scale (VAS) or numeric rating scale (NRS), quality of life (QoL) (assessed using tools apart from the KOOS), and adverse events (AEs). Random meta-analysis was conducted using STATA 14.0. Results Nineteen studies were included. The metaanalysis evidenced a positive association between atherosclerosis and osteoarthritis (OR 1.17, CI 1.01–1.36). The mean absolute difference in KOOS subscale scores between pre- and post-treatment (mean with 95% confidence interval [CI]) was 19.65 (13.66, 25.63), 14.20 (4.64, 23.76), 19.95 (13.02, 26.89), 25.23 (14.80, 35.66), and 26.01 (13.68, 38.35) for pain, symptoms, activities of daily living (ADL), sports/recreation, and quality-of-life (QOL), respectively. The mean differences in VAS, resting VAS, activity VAS, and NRS between pre- and post-treatment was -8.24 (-10.66, —5.82), -3.61 (-4.49, -2.72), -4.17 (-4.89, -3.44), and -2.17 (-4.06, and -0.28), respectively. The mean difference in score of Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), EQ-5D, and University of California in Los Angeles (UCLA) between pre- and post-treatment was -24.81 (-40.80, -8.82), 0.07 (0.02, 0.12), and 0.30 (-0.42, 1.02), respectively. The mean difference in Tegner score and the International Knee Documentation Committee (IKDC) score between pre- and post-treatment was 0.67 (-0.62, 1.97) and 13.70 (6.35, 21.04), respectively. The use of micro-fragmented adipose tissue was associated with risk of bruising, bleeding, hematoma, drainage, infection, soreness, swelling, pain, and stiffness in harvest and injection sites. Conclusion Atherosclerosis and osteoarthritis share common risk factors and comorbidity. And the use of micro-fragmented adipose tissue may benefit for improving symptoms of knee osteoarthritis accompanied by atherosclerosis although may lead to some mild adverse events. Randomized controlled trials with long-term follow-up are necessary for further evaluation because many limitations of this meta-analysis cannot be ignored.


Introduction
Knee osteoarthritis (OA) is one of the most prevalent musculoskeletal disorders, with a significant impact on one's ability to carry out daily activities (ADL) [1].Knee OA is characterized by articular cartilage loss, bone remodeling, and periarticular muscle weakness, resulting in knee joint pain, swelling, deformity, and instability [2,3].Over half of the persons suffering from symptomatic knee OA experience significant disabilities daily [4].It cannot be denied that knee OA has risen to become the leading cause of disability among the elderly [5].In United States of America Studies, 38% to 47% of elderly aged more than 60 years being affected by knee OA [6,7].In addition, according to recent studies, 18% of elderly males and 27% of elderly females were estimated to have KOA, and this ratio is estimated to increase over the coming decades [8].Therefore, it is crucial to early use effective therapies to treat knee OA.
Cardiovascular disease (CVD) remains the leading cause of death worldwide, and atherosclerosis is the primary pathophysiological substrate of the disease process.Osteoarthritis (OA) is the most common cause of joint disease worldwide and its incidence is increasing as the population ages, affecting one in five people worldwide.As two very common diseases, their frequent co-existence is not surprising and increases the likelihood that they will interact with each other.Not surprisingly, OA has been identified as a credible risk factor for non-classical CVD.The association between osteoarthritis and cardiovascular disease is complex, difficult to direct, and stems from a common mechanism in the origin of chronic low-grade inflammation, compounded risk factors, and lifestyle.The prevalence of these two diseases in the general population is strongly related to age.For osteoarthritis, the prevalence increases in people over the age of 40.However, the increase in prevalence was different between the sexes.After the age of menopause, prevalence increases twice as fast in women as in men.Similar sex-specific differences were seen in atherosclerotic related deaths.The leading cause of death in men after the age of 35 is associated with atherosclerosis, while the number of atherosclerotic deaths in women begins to increase mainly after the age of menopause and becomes the leading cause of death after the age of 60.In old age, women have an even higher risk of death from atherosclerosis than men.In addition to the common risk factors for atherosclerosis and OA such as age and BMI, systematic reviews show that typical risk factors for atherosclerosis are most likely also risk factors for OA, such as high blood pressure for OA of the knee and high cholesterol levels for OA of the knee.
The primary goals of treatment for knee OA are to relieve pain and inflammation, reducing stiffness, optimizing mobility, function, and range of motion, and improving quality of life (QoL) [3,9].Currently, non-surgical and surgical therapies are available for the treatment of knee OA [10].Non-surgical therapies include physical therapy, oral medicines, and intra-articular injections of steroids, hyaluronic acid, and platelet-rich plasma (PRP); nevertheless, these are only capable of providing limited therapeutic benefits, with an effect that is frequently not totally satisfactory and even decreasing over time, and varies between patients [11][12][13].For more advanced diseases, joint-preserving and joint-replacing surgeries can be considered [3,9].As the ultimate therapeutic option for knee OA, primary and revision total knee arthroplasties (TKA) carry a non-negligible risk of failure and the need for later revision arthroplasty, especially in younger patients [14].Therefore, it is imperative to find new effective treatment options for knee OA in order to delay or prevent invasive surgery [15].
Recently, the use of mesenchymal stromal cells (MSCs) has been introduced as an attractive therapeutic option for knee OA due to their immunomodulatory, anti-inflammatory, and paracrine properties [16,17].Bone marrow and adipose tissue were considered as the most common sources of MSCs [18], and adipose tissue-derived MSCs have similar properties to bone marrow-derived MSCs [19]; however, adipose-derived MSCs are easier to collect for clinical applications and have a higher isolation rate than bone marrow-derived MSCs [19][20][21].Furthermore, micro-fragmented adipose tissue (MFAT) has been described as one of the smartest and most straightforward ways to employ adipose tissue in regenerative techniques in a variety of clinical applications [22,23].Specifically, MFAT contains MSCs that originate from the adipose blood vessels as pericytes and are released and primed during the extraction process through sheer stress and microfiltration of the adipose tissue [24,25].MFAT would be beneficial in providing an optimal biological environment for healing because its advantages in providing a high number of cells and growth factors without expansion or enzymatic treatment, thus preserving the integrity of cells and tissue microarchitecture [26].To date, several studies have shown promising results when using autologous MFAT injection to treat knee OA [27,28].In animal experiments, it was found that MFAT produced by Lipogems achieved the best cartilage repair effect in the treatment of knee osteoarthritis with enzymatic digestion or cell expansion [29].Zeira showed that MFAT injection in 130 dogs with spontaneous osteoarthritis is safe, feasible, and beneficial [30].In clinical treatments, Russo evaluated the 1-year safety and outcome of a single intra-articular injection of autologous microfragment adipose tissue in 30 patients with knee osteoarthritis.An overall median improvement of 20 points was observed in IKDC subjective and total KOOS, and higher success rates were found in VAS pain and Tegner Lysholm knee joints, of which 22 patients maintained good outcomes at follow-up 3 years later with no associated complications or clinical deterioration documented [31,32].And another study showed significant improvements in pain, quality of life, and function for at least 12 months in patients with refractory severe (grade 3 or 4) knee osteoarthritis [33].A multi-centric, international study show that seventy-five patients, 120 primary treatments, with KL grade 2 to 4 knee OA treated with a single MFAT injection.Patients with KL grade 2 disease had the best results in KOOS-Pain.Including advanced KL grade 3 and 4 osteoarthritis patients, significant functional and quality of life success was seen in 106/120 treatments at all follow-up time points.Fourteen treatments failed prior to the study endpoint [34].In addition, a number of literature studies have found that MFAT has a good effect on knee/hip osteochondral injury and knee/hip osteoarthritis [35,36].And the clinical efficacy is better than repeated doses of leucocyte-poor platelet-rich plasma plus hyaluronic acid [37].
Still, to date, no high-level studies have comprehensively investigated the therapeutic efficacy and safety of autologous MFAT in the treatment of knee OA.Therefore, we conducted the current meta-analysis to systematically evaluate the therapeutic efficacy and safety of autologous MFAT for the treatment of knee OA, with the aim to help increase the focus on this strategy for performing additional studies to improve patient prognosis.

Literature search
This systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines [38,39].The study was designed based on the PICOS principle [40].Two investigators independently searched PubMed, Embase, the Cochrane Library, Web of Science, China National Knowledge Infrastructure (CNKI), WANFANG DATA, and CQVIP for potentially eligible studies published before October 13, 2022, using the medical subject heading (MeSH) terms of 'Osteoarthritis', 'Osteoarthritis, Knee', and 'Autologous Micro-fragmented Fat Tissue' as well as relevant analogs.The retrieved records were screened according to the eligibility criteria.The detailed search strategies of target databases are summarized in S1 Table .Any discrepancies between the two investigators were resolved by discussion until a consensus was reached.Due to the statistical limitations of the existing relevant literature, it is not possible to make statistics on the patients with OA accompanied by AT treated by MFAT.So we made the primary outcome consist of two parts: 1) Correlation between atherosclerosis and osteoarthritis; 2) Scores of the Knee injury and Osteoarthritis Outcome Score (KOOS).

Eligibility criteria
The inclusion criteria were as follow: (1) population: knee OA, (2) intervention: surgery combined with autologous MFAI, (3) comparison: not specifically defined, (4) outcomes: the Knee injury and Osteoarthritis Outcome Score (KOOS), pain, quality of life (QoL) (assessed using tools apart from the KOOS), and adverse events (AEs), and (5) full texts published in English and Chinese.

Exclusion criteria
The exclusion criteria were (1) in vitro study, (2) animal experiment, or (3) studies with ineligible designs, such as meta-analysis, review, case report, commentary, letter to the editor, or conference abstract.

Data extraction and synthesis
Data extraction was conducted by two investigators.The extracted data included study characteristics (authors, year of publication, country where the study was performed, and study design), patient characteristics (sex, sample size, and knee OA degree), intervention characteristics (procedure, donor site for adipose tissue, and device), and outcome data.The primary outcome was the KOOS, which includes five subscales: pain, symptoms, activities of daily living (ADL), sports/recreation, and QoL.The continuous variables in the form of mean ± standard deviation (SD) at the last follow-up were extracted.The secondary outcomes were pain assessed by visual analog scale (VAS) or numeric rating scale (NRS), QoL (assessed using tools apart from the KOOS), and AEs.Disagreements between the two investigators were resolved by discussion until reaching a consensus.

Quality of the evidence
The level of evidence of all studies was assessed independently by two investigators according to the Methodological Index for Non-Randomized Studies (MINORS) [41].The evaluation is based on (1) a clearly stated aim, (2) the inclusion of consecutive patients, (3) a prospective collection of data, (4) appropriate endpoints for the study aim, (5) unbiased assessment of the study endpoint, (6) follow-up period appropriate for the study aim, (7) <5% loss to follow-up, and (8) prospective calculation of the sample size.For comparative studies, additional criteria were available: (9) adequate control group, (10) contemporary groups, (11) baseline equivalence of the groups, and ( 12) adequate statistical analyses.Discrepancies between the two investigators were resolved through discussion until a consensus was reached.

Statistical analysis
For continuous variables, the changing value between pre-and post-treatment was used for meta-analysis, and estimate was expressed using mean difference (MD) and 95% confidence interval (CI).For dichotomous variable, the number of event and total sample size were used for meta-analysis, and estimate was expressed using rate and 95% CI.Statistical heterogeneity between the included studies was evaluated using Cochrane Q and Higgins' I 2 .Statistical heterogeneity was deemed as significant if I 2 >50% and P<0.1 [42].Nevertheless, the randomeffects model was used for meta-analysis because it is not rational to ignore the variations between studies in the real settings [43,44].Subgroup analyses were conducted according to the subscales of tools.The possible publication bias was not assessed using funnel plots and Egger's test because the numbers of included studies were <10 in all analyses, in which case the funnel plots and Egger's test could yield misleading results [45].Two-sided P-values <0.05 were considered statistically significant.All statistical analyses were conducted using STATA 14.0.

Basic characteristics and quality of the included studies
There were eleven prospective studies [27,46,48,49,[52][53][54][55][56][57][58] and eight retrospective studies [47,50,51,[59][60][61][62][63].Other basic characteristics of the fourteen included studies to investigate the therapeutic efficacy and safety of autologous MFAT for the treatment of knee OA can be found in Table 1.And the basic characteristics of the five included studies to explore the similarity and correlation between the comorbidity of OA and AS can be found in Table 2.

Knee injury and osteoarthritis outcome score
The mean absolute differences in KOOS subscale scores between pre-and post-treatment were 19.65 (13.66, 25.63) .In addition, the results of individual studies which were included in the meta-analysis of KOOS subscale scores are displayed in S2 Fig.

Tegner and IKDC
The mean difference in Tegner scoring system and International Knee Documentation Committee (IKDC) between pre-and post-treatment was 0.67 (-062, 1.97) and 13.70 (6.35, 21.04), respectively.The pooled results are depicted in S5 Fig.

Osteoarthritis and atherosclerosis
The metaanalysis evidenced a positive association between AT and OA (OR 1.17, CI 1.01-1.36).The pooled results are depicted in Fig 3.

Discussion
Both myocardial infarction and ischemic stroke are most often caused by atherosclerosis.To date, three systematic reviews have been published on the increased risk of OA and CVD [64][65][66].The first systematic review by Hall et al. [64]   heterogeneity.All studies included in the meta-analysis were longitudinal studies and measured confounding factors were taken into account.The second systematic review by Wang et al. [65], retrieved May 2016, also included 15 studies.In this review, cardiovascular disease risk from 10 prospective studies and 5 retrospective studies are presented.Overall, there was an increased risk of CVD for OA in prospective studies (RR 1.24; 95 CI 1.12 to 1.37), but not in retrospective studies (RR 1.15; 95% CI 0.95 ~1.38).Veronese et al. [66] reported the risk of cardiovascular death due to the presence of any joint osteoarthritis from studies published prior to November 2015, with a hazard ratio (HR) of 1.21 (95% CI 1.10 to 1.34) and a high degree of homogeneity in results.In short, most studies show cross-sectional and longitudinal associations, although not for all joints and all measures of atherosclerosis.Evidence from most studies suggests that atherosclerosis is associated with hand and knee osteoarthritis, but never hip osteoarthritis.Moreover, in studies analyzing gender differences, these associations were stronger in women than in men.
MFAT combined with surgery may be a promising therapeutic strategy for the early management of knee OA, but strong evidence for supporting the clinical application of MFAT is lacking.Therefore, the present meta-analysis was conducted to comprehensively investigate the therapeutic efficacy and safety of autologous MFAT for the treatment of knee OA.The pooled results reveal that MFAT may benefit to improve symptoms of knee OA although may also lead to some mild adverse events.
The present meta-analysis showed that post-operative application of MFAT significantly improve the five subscale -scores of the KOOS, pain scores, QoL scores, and Tegner score.It is consistent with the fact that all included studies reported some degree of improvements after the use of MFAT in their patients with knee OA [27,[46][47][48][49][50][51][52][53][54][55][56][57][58].Still, because most included studies have positive results, a publication bias is possible but could not be examined in the present study because of the insufficient number of included studies for individual analysis [45].Nevertheless, other studies that were not eligible for the present meta-analysis support the results of the present meta-analysis, suggesting that the use of MFAT may be promising for the treatment of knee OA [67,68].MFAT also showed benefits in other indications, such as menopausal vaginal atrophy, perianal fistula repair, and diabetic foot [48,[69][70][71].
Conventional treatments of knee OA is corticosteroid injection, but an inflammatory flare occurs in 2%-25% of the cases [7].In addition, hyaluronic acid injection carries a risk of flares and granulomatous inflammation [72].In the present meta-analysis, the meta-analysis of AEs reveals that the use of MFAT may lead to the occurrence of bruising, bleeding, hematoma, drainage, infection, soreness, pain, and stiffness; however, all adverse events were mild and relieved in the short term without specific treatment.In addition, whether the safety profile of MFAT is better than with other therapies (e.g., platelet-rich plasma) remains to be investigated in comparative trials.
Five studies [49][50][51][52]58] included outcomes that could not be pooled in the present metaanalysis.These studies showed improvements in Oxford Knee Score (OKS) and Hospital for Special Surgery Knee Score (HSS), but not in the Emory Quality of Life (EQoL) score and delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) index.Of course, different assessment tools have different degrees of subjectivity/objectivity and measure different outcomes.Therefore, it might highlight the need to use multiple assessment tools and to use similar tools among studies to improve the comparability of the results.
This meta-analysis has limitations.The number of included studies was relatively small because the autologous MFAT strategy is relatively novel.Therefore, our findings should be interpreted with caution due to limited statistical power.All included studies used the same technique for harvest; however, there are rules or at least recommendations for the conduction of clinical and/or surgical trials in OA, which were not followed by the studies included in this systematic revie and meta-analysis.Indeed, the MINORS scores of the included studies were moderate.Significant heterogeneity was observed in several analyses, which would inevitably compromise the robustness and reliability of the pooled results.However, such heterogeneity was inevitable considering the differences among trials regarding severity of knee OA, donor sites for adipose tissue, techniques of intra-articular injection, and follow-up duration.We cannot perform subgroup analysis to explore which factors may contribute to significant heterogeneity due to limited number of eligible studies.Therefore, the results must be interpreted with caution.At the same time, most studies included in this systematic review and meta-analysis were retrospective studies, which had lower statistical power for assessing efficacy of a certain.Therefore, more prospective comparative or even randomized controlled trials with highquality and large sample size are needed.Furthermore, these eligible studies did not assess changes in joint range of motion, a finding that is important for knee OA.Therefore, future studies should consider this result.Finally, there are many therapeutic options for the treatment of knee OA, such as sodium hyaluronate injection.However, it was impossible to quantitatively compared the efficacy and safety of intra-articular MFAT versus other therapeutic options due to lack of eligible studies.So, we recommend future studies to directly compared intra-articular MFAT to other therapeutic options.

Conclusions
In conclusion, based on the currently available limited evidence, there is a positive association between atherosclerosis and osteoarthritis; and the use of micro-fragmented adipose tissue may benefit for improving symptoms of knee osteoarthritis accompanied by atherosclerosis although may lead to some mild adverse events.However, more studies are necessary for further validating our findings because some limitations such as limited number of eligible studies, significant statistical heterogeneity, and potential publication bias may significantly compromise the reliability of the pooled results.

Fig 1 .
Fig 1. Forest plot of subscales scores of the Knee injury and Osteoarthritis Outcome Score (KOOS).ADL, activities of daily living; QoL, quality of life; CI, confidence interval.https://doi.org/10.1371/journal.pone.0289610.g001 Fig presents those outcomes that could not be pooled for meta-analysis.As shown in S6 Fig, the use of MFAT could be significantly associated with changes in OKS and HSS but not EQoL and dGEMRIC index.