Long-term local PDGF delivery using porous microspheres modified with heparin for tendon healing of rotator cuff tendinitis in a rabbit model
Introduction
The rotator cuff (RC) is a group of four tendons including supraspinatus, infraspinatus, subscapularis, and teres minor that covers the humeral head and controls arm rotation and elevation. RC tendinitis is the inflammation to any or all of the RC tendons due to impingement or overuse (Cools, Declercq, Cagnie, Cambier, & Witvrouw, 2008; Marcondes, de Jesus, Bryk, de Vasconcelos, & Fukuda, 2013). Degeneration as well as healing of the RC occur in three stages including inflammatory phase, repairing phase, and remodelling phase (Carpenter, Thomopoulos, Flanagan, DeBano, & Soslowsky, 1998). During the inflammatory phase, inflammatory cells invade the healing site and they produce the cytokines that cause recruitment and proliferation of macrophages as well as resident tendon fibroblasts. Inflammatory cytokines attract fibroblasts to the repair site. However, the excessive inflammation may lead to tendon ruptures and poor clinical results (Lichtnekert, Kawakami, Parks, & Duffield, 2013; Sugg, Lubardic, Gumucio, & Mendias, 2014).
Conservative treatments including ice, rest, pain medication, physiotherapy, local injection of corticosteroid, and surgery could be considered for the treatment of RC tendinopathy. Among these treatments, local injection of corticosteroid is most commonly performed for the treatment of RC tendinopathy to reduce inflammation. However, repetitive steroid injections induced several side effects (i.e., post-injection pain, tendon rupture, ecchymosis, cellulitis, skin depigmentation, and atrophy) and undesirable complications (Brinks, Koes, Volkers, Verhaar, & Bierma-Zeinstra, 2010). As a second-line treatment, platelet-rich plasma (PRP) has been suggested to expedite tendon healing or remodeling (Nourissat et al., 2015) because the PRP contains a host of soluble growth factors including transforming growth factor-β, platelet-derived growth factor (PDGF), insulin-like growth factor-1, vascular endothelial growth factor, epidermal growth factor, and hepatocyte growth factor (Dhillon, Schwarz, & Maloney, 2012; Middleton, Barro, Muller, Terada, & Fu, 2012). However, there are controversial arguments considering the effectiveness of PRP in clinical trials due to an unclear understanding of the biological properties as well as the lack of optimized and standardized production procedures (Chahla et al., 2017).
PDGF plays a significant role in promoting chemotaxis, cell proliferation, extracellular matrix (ECM) production, surface integrin expression, and revascularization in fibroblasts (Galatz et al., 2007; Nakamura et al., 1998). Previous studies demonstrated that PDGF also improved the biochemical, structural, and biomechanical properties of animal tendons or ligament healing (Chan, Fu, Qin, Rolf, & Chan, 2006; Hildebrand et al., 1998). Furthermore, PDGF combined with type I bovine collagen matrix enhanced biomechanical function and anatomic appearance compared to a suture-only in an acute ovine model of RC repair (Hee et al., 2011). Uggen et al. showed that PDGF-coated sutures induced sheep RC repairs according to gross examination, histologic analysis, and biomechanical function compared to sutures alone (Uggen et al., 2010). Although PDGF is a promising material for tendon or ligament healing, its therapeutic effects have not been effective in the clinics due to its short half-life in the blood (Hollinger, Hart, Hirsch, Lynch, & Friedlaender, 2008; Uggen et al., 2010).
To overcome these limitations described above, the local injection of PDGF combined with microspheres- or nanoparticles-based delivery systems may be useful for treating RC diseases. These delivery systems have been used to achieve extended action time, toxicity reduction, and efficacy improvement. Recently, we have developed porous microspheres using a simple fluidic device and modified with negatively charged heparin for the long-term delivery of small molecular drugs and growth factors (Kim et al., 2015, 2018; Kim, Yun, Shim et al., 2014; Lee et al., 2017; Park et al., 2016). These drug-loaded porous microspheres have better anti-inflammatory and more beneficial tendon healing effects in vivo compared to small molecular drugs or growth factors (Jeong et al., 2018; Kim et al., 2018).
In this study, we fabricated the porous poly(lactic-co-glycolic acid) (PLGA) microspheres (PMSs) immobilized with heparin-dopamine (Hep-DOPA), and PDGF subsequently immobilized on their surfaces. Furthermore, we investigated whether PDGF-immobilized Hep-PMSs (PDGF/Hep-PMSs) suppress in vitro and in vivo inflammatory responses and enhance tendon healing and restorative effects on RC tendinitis rabbit model.
Section snippets
Materials
PLGA (50:50, Mw: 30,000–60,000), polyvinyl alcohol (PVA, Mw: 13,000–23,000, 98% hydrolysed), dichloromethane (DCM), gelatin from porcine skin, dopamine (DOPA), 2-(N-morpholino)-ethanesulfonic acid (MES), and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human PDGF and the PDGF ELISA kit were purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). Heparin sodium (Mw: 12,000–15,000 g/mol) was from Acros Organics (Geel, Belgium).
Characterization of the microspheres
In this study, PMSs and Hep-PMSs were used as the PDGF delivery systems. These PMSs were produced using a fluidic device, which is very useful to control the size, porosity, and shape of the porous microspheres (Jeong et al., 2018; Kim et al., 2015, 2018; Kim, Yun, Shim et al., 2014; Lee et al., 2017; Park et al., 2016). PDGF was immobilized on the surfaces of PMSs or Hep-PMSs. Our previous studies demonstrated that Hep-DOPA molecules were easily anchored on the surface of PMSs, hydroxyapatite
Conclusion
In this study, we prepared the Hep-PMSs using a fluidic device for a long-term local delivery of PDGF to treat tendon tissues in RC tendinitis rabbit model. Due to the strong electrostatic interactions between heparin and PDGF, PDGF/Hep-PMSs showed the sustained and long-term PDGF release. The PDGF/Hep-PMSs had a potent anti-inflammatory effects by suppressing the mRNA levels of pro-inflammatory cytokines (i.e., MMP-3, MMP-13, COX-2, IL-1, IL-6, and TNF-α) in inflamed tenocytes. Furthermore,
Acknowledgements
This work is funded by a Korea University Guro Hospital Grant (O1600151) and grants from the Korea Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI13C1501 and HI15C1665).
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