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All in One Hydrogel Approach: A Translationally-worthwhile Scenario for Rotator Cuff Tendinopathies.

Introduction

Tendon disorders, especially rotator cuff tendinopathies (RCT) are one of the most common musculoskeletal disorders where pain, inflammation, fatty infiltration and ECM disorganization are the major symptoms [1]. Despite the increased incidence of tendinopathies, the exact mechanism behind the etiology and pathogenesis of shoulder tendinopathies are largely unknown. Conventional therapeutic approaches for RCT focus mainly on the management of pain and inflammation [1]. However, the hypovascular and hypocellular tendon-anatomy offers a hurdle for bio availability and bioretention of therapeutics. Moreover, the underlying molecular mechanisms behind RCT pathology especially the interrelationship between inflammation, fatty infiltration, pain and the delayed healing responses remain to be unveiled.

Our preliminary investigations revealed the upregulation of MMP9, AMPK and TREM-1 which were responsible for ECM disorganization fatty infiltration and inflammation respectively that play significant role in sustaining clinical symptoms [2,3]. These findings opened the need of promising strategies for the simultaneous management of fatty infiltration, inflammation and ECM disorganization by site-specific targeting of AMPK, TREM1 and MMP9 respectively which could be an ideal approach to manage RCT. However, such multi-targeting approach is rare in the literature and raises concerns regarding the bio-availability, biostability, bio-retention and side effects of therapeutics/drugs employed. To accomplish this approach, an ideal biomaterial career vehicle/scaffold is required. The polymeric hydrogels have been hailed for various biomedical and tissue engineering applications owing to their ECM mimic nature, and biocompatibility [4,5]. The incorporation of Compound C (AMPK inhibitor), LR-12 (TREM-1 inhibitor) and MMP9-Inhibitor-1 (small compound inhibitor of MMP9) with optimization of their release profile and the subsequent implantation at the injury site can enhance healing responses. Moreover, such vehicles are capable of delivering stem cells, miRNAs, free radical scavenging and other signaling molecules which can orchestrate neo-tissue formation at injury-implant interface. This 'Allin one hydrogel approach can be considered as a combination of drug delivery, tendon tissue engineering and tendon regenerative medicine and the implanted hydrogels are capable of biodegradation and bio-absorption.

Natural polysaccharides like alginate have been considered to be ideal for biomedical applications owing to their excellent biomimetic character, visco-elastic nature and ease of gel formation with divalent metal ions. Fumarate based unsaturated polyesters can reinforce with alginate gels using simple free radical polymerization. Interestingly, such polymers can be designed to scavenge local free radicals at the vicinity of target site. Moreover, the incorporation of FDA approved synthetic polyols like PVA or PEG can impart additional hemo/cytocompatibility [6,7].

Based on this background, we hypothesize the 'All in one hydrogel approach for tendon regeneration that homes cells, therapeutics, signaling molecules and regulatory molecules which elicit simultaneous management of fatty infiltration, inflammation and ECM disorganization by site-specific targeting of AMPK, TREM1 and MMP9 respectively and also act as a cue for the neo-tendon tissue formation. The current study focus on the design, fabrication and optimization of a hybrid hydrogel scaffold synthesized form alginate and unsaturated polyester poly(propylene-fumarate) (PPF) reinforced with the polyol, poly-vinyl alcohol (PVA). Ultimate aim of the study is to examine the translational aspects of the 'All in one hydrogel approach for the management of shoulder tendinopathies by exploiting the molecular mechanisms behind the pathology.

Methodology

Establishment of surgically induced RCT rat model and elucidation of pathological mechanisms

Sprague Dawley rats of 8-10 weeks old were used for establishing the RCT animal model. Briefly, the rotator cuff tendons of the rats were exposed and a suture was placed through each of the supraspinatus and infraspinatus at the musculotendinous junction to control the tendon stumps after detachment. The supraspinatus and infraspinatus tendons were sharply detached from the greater tuberosity and tendon stump was resected further in order to prevent the healing of the tendon stump back to the greater tuberosity. Contra lateral controls were used for comparison.

Hematoxylin and eosin (H & E) staining was performed for determining the tissue morphology using standard protocols [8]. The organization and arrangement of collagen fibers were monitored by Masson-trichrome and pentachrome staining [9]. The tissue sections were analyzed for the expression of Collagen I, Collagen III, MMP-9, TREM-1, AMPK and downstream signals and cell phenotype biomarkers by immune-staining [11].

Synthesis, characterization, and optimization of PVA-interpenetrated-Alginate-co-poly(propylene-fumarate) (PAP) hydrogel

Hydroxyl-terminated PPF was synthesized by the condensation polymerization of maleic anhydride and 1,2-propylene-glycol and was co-polymerized to sodium alginate using acid catalyst. The PPF-alginate co-polymer was interpenetrated with PVA. The alginate fraction was cross linked with [Ca.sup.2+] ions and the doubles bonds with free radical activated cross linking of poly-ethylene glycoldiacrylate (PEGDA) to form PAP hydrogel. The physiochemical characterization of the hydrogel was performed by IR spectral analysis, water content and holding capacity, surface morphology, surface hydrophilicity, biodegradation, mechanical and thermal properties following the standard protocols. The biocompatibility was assessed by standard direct contact assay, test on extract assay, live dead assay and cell infiltration assays.

Translational significances of PAP hydrogels for the management of RCT--All in one approach

The PAP hydrogels were loaded with the inhibitor molecules (MMP9-I, Compound C and LR-12) and the release profile were determined by HPLC analysis. The drugs were loaded individually and in combinations. The effect of drug release from PAP hydrogels in the recovery of tenocytes under hypoxic and oxidative stress were monitored using immunofluorescence analysis. The growth and survival of human tenocytes in the interstices of MMP9-I, Compound C and LR-12 loaded PAP hydrogels under adverse conditions like metabolic, hypoxic and oxidative stress were assessed by cell penetration assay and immunofluorescence. The inherent ROS scavenging activity of PAP hydrogel was determined by the health status and mitochondrial activity of seeded cells grown under oxidative stress. The cyto-protective effects of drug (combination of MMP9-I, Compound C and LR-12) loaded PAP hydrogel was determined by the expression of mRNA transcripts of MMP9, Collagen type I, TREM1 and AMPK with respect to control cells.

Results

Surgically induced RCT rat model defines the pathological mechanisms

Histo-morphology determination by H and E revealed altered physiological architecture of tendon tissue in surgery group when compared to the other. The presence of immune cells and the clustering of tenocytes were evident at the vicinity of inflamed areas. On the other hand the tendon cells of control tissues were characterized by their less dense distribution of nuclei in the intact ECM. The RCT rats showed massive fatty infiltration after 5 days and 10 days which got minimized after 24 days. ECM disorganization was noticeable in the RC tendons of RCT Group while the control displayed intact ECM without the symptoms of disorganization. The expression of MMP9, TREM1 and AMPK was found to be higher in RCT group suggesting their role in ECM disorganization, inflammation and fatty infiltration respectively. The downstream signaling of genes (HMGB1, RAGE, ACC1, etc.) associated with ECM disorganization, inflammation and fatty infiltration and highly altered histomorphology of the tendons revealed the severity of damage in the RCT group.

PAP hydrogel exhibited promising characteristics tendon applications

PAP hydrogel was successfully synthesized from PPF, Alginate and PVA. The IR spectral analysis revealed the surface functionalization of hydrogel and the hydrogels possessed appreciable water content and holding capacity. The surface morphology of hydrogel revealed interconnecting pores. The surface hydrophilicity, biodegradation, and mechanical properties of the hydrogels were appreciable for the drug delivery and tissue engineering applications of tendon tissues. The thermal properties by DSC analysis revealed the water transition status of the hydrogels which is necessary for the release of loaded drugs to be released. The hydrogels were biocompatible and cytocompatible as determined by direct contact assay, test on extract assay, live dead assay and cell infiltration assays.

Translational significances of PAP hydrogels for the management of RCT--All in one approach

The PAP hydrogel loaded with the inhibitor molecules (MMP9-I, Compound C and LR-12) revealed appreciable release profile both in individually and in combinations. The release of was optimized to be 7ig/ml for LR-12, ~10iM for Compound C and 0.05ig/ml for MMP9-I. The drug released from PAP hydrogels accelerated the recovery of tenocytes under hypoxic as well as oxidative stress. The inherent ROS scavenging activity of PAP hydrogel was superior as determined by health status and mitochondrial activity of seeded cells grown under oxidative stress. The cyto-protective effects of drug (combination of MMP9-I, Compound C and LR-12) loaded PAP hydrogel was determined by the expression of mRNA transcripts of MMP9, Collagen type I, TREM1 and AMPK with respect to control cells.

Discussion and Future Aspects

PAP hydrogels were found to be appreciable for tendon tissue regeneration owing to their superior physiochemical, mechanical and biological features. Also, the elucidation of underlying mechanisms of initiation and progression of RCT is ideal to apply the principles of biomaterials sciences to the therapeutic arena. Our preliminary evaluations from RCT patients and surgically RCT-rat model revealed the upregulation of Collagen type-3 with a concomitant downregulation of type-1 collagen. Since collagen type1 constitute to more than 85% of the dry weight of RC tendons, the reduction in collagen type-1 to type-3 ratio could be the major reason for ECM disorganization. The upregulation of MMP9 in these specimens unveiled the enhanced degradation of collagenous matrisome by digesting collagen fragments. The inflammatory responses can be defined by upregulation of Triggering Receptors Expressed on Myeloid cells-1 (TREM-1). In addition, the upregulation of AMPK was found to be the major cause for increased fat tissue infiltration and fat deposition in RC tendons following the pathology. Moreover, the micro array analysis revealed the alteration of a school of miRNAs revealing the epigenetic regulation of RCT [3]. In a nutshell, ECM disorganization due to altered collagen composition and MMP9 hyperactivity, TREM-1 mediated inflammation and AMPK mediated fatty infiltration and epigenetic alterations can be considered to be basis of RCT where the persistence of which delays the healing responses.

Simultaneous targeting strategy of MMP9 hyperactivity, TREM-1 mediated inflammation and AMPK mediated fatty infiltration using a biocompatible hydrogel is a promising approach for the management of RCT. Such a multi-functional hydrogel system has not been tried so far for the management of musculoskeletal disorders. The in vitro responses offered by the PAP hydrogel were encouraging especially in terms of the physiochemical properties, drug release, tenocyte survival, ROS scavenging and neoorganogenesis. The ultimate goal of the study is to formulate, design and utilize the PAH hydrogel to s atis fy all therapeutic aspects of RCT and tendon regeneration. Such and 'All in one hydrogel approach should be capable of integrating drug delivery, tendon tissue engineering, tendon regeneration, tenocye programing/ reprograming, epigenetic regulations, stem cell differentiation, triggering healing responses, host responses and translation to therapeutic arena for the management of RCT and other musculoskeletal disorders.

References

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Finosh G. Thankam, Matthew F. Dilisio, Devendra K. Agrawal

Departments of Clinical & Translational Science and Orthopedic Surgery, Creighton University School of Medicine, Omaha, NE 68178, USA
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Title Annotation:Original Article
Author:Thankam, Finosh G.; Dilisio, Matthew F.; Agrawal, Devendra K.
Publication:Trends in Biomaterials and Artificial Organs
Date:Jan 1, 2018
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