Our study showed similarly improved results of WOMAC scales compared to the previous studies reported by Dumais in 2012, Eslamian in 2015, and Rabago in 2012, 2013 and 2015. For disclosing the cytokine markers of early response to hypertonic dextrose prolotherapy, the follow-up period was 10 weeks post first injection in this study, a shorter period than in previous studies. Nevertheless, the clinical outcomes including pain, joint stiffness and physical function were also improved. This finding indicated that the hypertonic dextrose intra-articular injections for OA knee patients have a clinically effect lasting from weeks to months post treatment [20, 21].
Few studies follow the joint space width as an outcome measurement after hypertonic dextrose phototherapy for knee OA. Our study tried to survey the knee-joint cartilage structural change in addition to functional evaluation. Though there was no statistically significant improvement, the medial minimum joint space width increased in most of the knee joints after 5 injections. This result is compatible with the chondrogenic effect of intra-articular hypertonic dextrose in knee OA as published by Topol GA et al. in 2016 [22].
Dextrose prolotherapy is an alternative to surgery for knee osteoarthritis patients. The potential mechanism of dextrose prolotherapy relevant pain-intensity reduction is associated with the hyperpolarization of nociceptive pain fibers by opening the potassium channels [21]. Prolotherapy simulates the normal tissues’ healing and repair response, which includes the three stages of inflammation, proliferation, and tissue remodeling [23]. Hypertonic dextrose solutions induce inflammation and stimulate local healing in injured articular tissue through attracting immune cells. In addition, some in vitro and in vivo studies indicated that dextrose also augments the growth of ligaments and tendons, fibroblastic proliferation, and the restoration of the extracellular matrix and articular cartilage by triggering the production of growth factors [20, 24, 25].
The biological mechanisms of dextrose have been indicated in cell and animal models. However, the question of which proteins that participate in tissue repair and the healing process will be regulated by hypertonic dextrose in human knee joints remains ambiguous. Therefore, we used a human cytokine antibody array to evaluate the expression of dextrose-induced cytokine in synovial fluid. A number of immune cells such as macrophages, mast cells, leucocytes, and T cells, which have been found in the synovial tissues of OA patients, are involved in the pathogenesis of OA [26–29]. RAGE, CA11, and GDF-15 control leucocyte adhesion, mast-cell-modulated inflammation, and macrophage activation, respectively [30–32]. IL-22, the proinflammatory cytokine, has been demonstrated to modulate inflammatory processes in inflamed and non-inflamed synovium from osteoarthritis patients [33]. We found that hypertonic dextrose increased many cytokines, such as RAGE, CA11, GDF-15, TREM-1, and IL-22, which contribute to the recruitment of inflammatory cells and inflammation.
The biology of OA is also influenced by the T cell–mediated immune response. Both CD4+ and CD8+ T cells induce inflammation and cartilage degradation [29, 34, 35]. Regulatory T cells (Treg cells), suppressor T cells, inhibit the proliferation of CD4+ and CD8+ T cells. The Treg cell response is decreased and is involved in the pathogenesis of OA [29, 36]. The accumulation of T cells and the chemoattraction of resting T cells are regulated by cytokines CXCL9,-16, and MPIF-1 [37–39]. Siglec-9 is a critical immunosuppressor that promotes Treg cell differentiation in the pathogenesis of rheumatoid arthritis (RA) [40]. The expected increased inflammation process is induced by hypertonic dextrose. Notably, we found that 10% dextrose not only diminishes the expression of CXCL16 and MPIF-1 but also increases the level of Siglec‐9.
A number of growth factors, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor β (TGF-β1), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), and connective tissue growth factor (CTGF), are essential for the growth and repair of ligaments, tendons, and cartilage [24]. TGF-β has been shown to trigger cartilage matrix synthesis and chondrogenesis of bone-marrow-derived mesenchymal stem cells (MSCs) and to promote the repair of cartilage defects [41, 42]. BMP-6, a member of the TGF-beta superfamily of cytokines, participates in the maintenance/repair of human articular cartilage [43] EGF, a potent mitogen that augments MSCs and fibroblast proliferation, is involved in the development and healing of tendons and ligaments [44]. Thyroid hormone is the important regulator for remodeling and maintaining bone and cartilage repair. The concentration of thyroid hormone is modulated by the negative feedback regulation of thyrotropin-releasing hormone (TRH) and thyroid-stimulating hormone (TSH) [45, 46]. Some studies demonstrated that high glucose concentration induces the growth and repair of normal cells and tissues and the production of growth factors and hormones [24, 47]. The results showed that TGF−β, EGF and TSH are overexpressed with 10% dextrose in synovial fluid.
Matrix metalloproteinases (MMPs), a large group of zinc-dependent endopeptidases, have been classified into six groups: collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9), stromelysins (MMP-3, -10, -11), matrilysins (MMP-7, -26), membrane-type MMPs (MMP-14, -15, 16, -17, -24, -25), and other nonclassified MMPs (MMP-12, -19, 20, -21, -23A/B, -27, -28). MMP-1, -2, -3, -9, and − 13 cleave the components of the extracellular matrix (ECM) and serve as critical mediators of cartilage destruction in OA [48, 49]. The activities of MMPs are downregulated with tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) [50]. MMP-1, -3, -9, and − 13-triggered degradation of ECM are suppressed with TIMP-1 [51]. Furin, proprotein convertase, has been shown to reduce MMP-13 expression in a TGFβ-dependent manner and to restrict osteoarthritis in mice [52]. Besides MMPs, serine proteases also degrade ECM and contribute to articular cartilage destruction in OA. Trappin-2, a small serine protease inhibitor, was found to bind to ECM, resulting in the inhibition of serine protease-mediated degradation of ECM [53, 54]. PAI-1, a serine protease inhibitor, suppresses the degradation of ECM and inhibits osteoclastic bone resorption and subchondral osteopenia after the induction of OA [55, 56]. We found that 10% dextrose enhanced MMP-2 expression, and the protein levels of TIMP-1, EGF, IL-10, and IL-22 were increased in synovial fluid.
Post-injection soreness is common in hypertonic dextrose prolotherapy [57]. During this treatment course, patients needed to receive 5 injections, and most of them felt short-term discomfort after each injection. Those factors decreased the willingness of the elderly with knee OA to participate in this research project. This may mean that the clinical application of this treatment protocol is not acceptable for every patient. Although the hypertonic dextrose prolotherapy is effective, multiple injections and post-injection discomfort are still clinical issues that may lessen patient compliance. This was the reason for our interest, and why we tried to explore the key cytokines related to effective and side-effect mechanisms. For wide clinical application, further studies need to be designed.