Corinne N. Riggin

The (dys)functional extracellular matrix

The extracellular matrix (ECM) is a major component of the biomechanical environment with which cells interact, and it plays important roles in both normal development and disease progression. Mechanical and biochemical factors alter the biomechanical properties of tissues by driving cellular remodeling of the ECM. This review provides an overview of the structural, compositional, and mechanical properties of the ECM that instruct cell behaviors. Case studies are reviewed that highlight mechanotransduction in the context of two distinct tissues: tendons and the heart. Although these two tissues demonstrate differences in relative cell-ECM composition and mechanical environment, they share similar mechanisms underlying ECM dysfunction and cell mechanotransduction. Together, these topics provide a framework for a fundamental understanding of the ECM and how it may vary across normal and diseased tissues in response to mechanical and biochemical cues. This article is part of a Special Issue entitled: Mechanobiology.

The Detrimental Effects of Systemic Ibuprofen Delivery on Tendon Healing Are Time-Dependent

BackgroundCurrent clinical treatment after tendon repairs often includes prescribing NSAIDs to limit pain and inflammation. The negative influence of NSAIDs on bone repair is well documented, but their effects on tendon healing are less clear. While NSAIDs may be detrimental to early tendon healing, some evidence suggests that they may improve healing if administered later in the repair process.Questions/purposesWe asked whether the biomechanical and histologic effects of systemic ibuprofen administration on tendon healing are influenced by either immediate or delayed drug administration.MethodsAfter bilateral supraspinatus detachment and repair surgeries, rats were divided into groups and given ibuprofen orally for either Days 0 to 7 (early) or Days 8 to 14 (delayed) after surgery; a control group did not receive ibuprofen. Healing was evaluated at 1, 2, and 4 weeks postsurgery through biomechanical testing and histologic assessment.ResultsBiomechanical evaluation resulted in decreased stiffness and modulus at 4 weeks postsurgery for early ibuprofen delivery (mean ± SD [95% CI]: 10.8 ± 6.4 N/mm [6.7–14.8] and 8.9 ± 5.9 MPa [5.4–12.3]) when compared to control repair (20.4 ± 8.6 N/mm [16.3–24.5] and 15.7 ± 7.5 MPa [12.3–19.2]) (p = 0.003 and 0.013); however, there were no differences between the delayed ibuprofen group (18.1 ± 7.4 N/mm [14.2–22.1] and 11.5 ± 5.6 MPa [8.2–14.9]) and the control group. Histology confirmed mechanical results with reduced fiber reorganization over time in the early ibuprofen group.ConclusionsEarly administration of ibuprofen in the postoperative period was detrimental to tendon healing, while delayed administration did not affect tendon healing.Clinical RelevanceHistorically, clinicians have often prescribed ibuprofen after tendon repair, but this study suggests that the timing of ibuprofen administration is critical to adequate tendon healing. This research necessitates future clinical studies investigating the use of ibuprofen for pain control after rotator cuff repair and other tendon injuries.

Photocrosslinked alginate with hyaluronic acid hydrogels as vehicles for mesenchymal stem cell encapsulation and chondrogenesis

Ionic crosslinking of alginate via divalent cations allows for high viability of an encapsulated cell population, and is an effective biomaterial for supporting a spherical chondrocyte morphology. However, such crosslinking chemistry does not allow for injectable and stable hydrogels which are more appropriate for clinical applications. In this study, the addition of methacrylate groups to the alginate polymer chains was utilized so as to allow the free radical polymerization initiated by a photoinitiator during UV light exposure. This approach establishes covalent crosslinks between methacrylate groups instead of the ionic crosslinks formed by the calcium in unmodified alginate. Although this approach has been well described in the literature, there are currently no reports of stem cell differentiation and subsequent chondrocyte gene expression profiles in photocrosslinked alginate. In this study, we demonstrate the utility of photocrosslinked alginate hydrogels containing interpenetrating hyaluronic acid chains to support stem cell chondrogenesis. We report high cell viability and no statistical difference in metabolic activity between mesenchymal stem cells cultured in calcium crosslinked alginate and photocrosslinked alginate for up to 10 days of culture. Furthermore, chondrogenic gene markers are expressed throughout the study, and indicate robust differentiation up to the day 14 time point. At early time points, days 1 and 7, the addition of hyaluronic acid to the photocrosslinked scaffolds upregulates gene markers for both the chondrocyte and the superficial zone chondrocyte phenotype. Taken together, we show that photocrosslinked, injectable alginate shows significant potential as a delivery mechanism for cell-based cartilage repair therapies.

Matrix molecule influence on chondrocyte phenotype and proteoglycan 4 expression by alginate‐embedded zonal chondrocytes and mesenchymal stem cells

Articular cartilage resists load and provides frictionless movement at joint surfaces. The tissue is organized into the superficial, middle, deep, and calcified zones throughout its depth, each which serve distinct functions. Proteoglycan 4 (PRG4), found in the superficial zone, is a critical component of the joints lubricating mechanisms. Maintenance of both the chondrocyte and zonal chondrocyte phenotype remain challenges for in vitro culture and tissue engineering. Here we investigate the expression of PRG4 mRNA and protein by primary bovine superficial zone chondrocytes, middle/deep zone chondrocytes, and mesenchymal stem cells encapsulated in alginate hydrogels with hyaluronic acid (HA) and chondroitin sulfate (CS) additives. Chondrogenic phenotype and differentiation markers are evaluated by mRNA expression, histochemical, and immunohistochemical staining. Results show middle/deep cells express no measurable PRG4 mRNA by day 7. In contrast, superficial zone cells express elevated PRG4 mRNA throughout culture time. This expression can be significantly enhanced up to 15‐fold by addition of both HA and CS to scaffolds. Conversely, PRG4 mRNA expression is downregulated (up to 5‐fold) by CS and HA in differentiating MSCs, possibly due to build up of entrapped protein. HA and CS demonstrate favorable effects on chondrogenesis by upregulating transcription factor Sox9 mRNA (up to 4.6‐fold) and downregulating type I collagen mRNA (up to 18‐fold). Results highlight the important relationship between matrix components and expression of critical lubricating proteins in an engineered cartilage scaffold.

Analysis of Collagen Organization in Mouse Achilles Tendon Using High-Frequency Ultrasound Imaging

Achilles tendon ruptures are traumatic injuries, and techniques for assessing repair outcomes rely on patient-based measures of pain and function, which do not directly assess tendon healing. Consequently, there is a need for a quantitative, in vivo measure of tendon properties. Therefore, the purpose of this study was to validate ultrasound imaging for evaluating collagen organization in tendons. In this study, we compared our novel, high-frequency ultrasound (HFUS) imaging and analysis method to a standard measure of collagen organization, crossed polarizer (CP) imaging. Eighteen mouse Achilles tendons were harvested and placed into a testing fixture where HFUS and CP imaging could be performed simultaneously in a controlled loading environment. Two experiments were conducted: (1) effect of loading on collagen alignment and (2) effect of an excisional injury on collagen alignment. As expected, it was found that both the HFUS and CP methods could reliably detect an increase in alignment with increasing load, as well as a decrease in alignment with injury. This HFUS method demonstrates that structural measures of collagen organization in tendon can be determined through ultrasound imaging. This experiment also provides a mechanistic evaluation of tissue structure that could potentially be used to develop a targeted approach to aid in rehabilitation or monitor return to activity after tendon injury.

Nonsurgical treatment and early return to activity leads to improved Achilles tendon fatigue mechanics and functional outcomes during early healing in an animal model

Achilles tendon ruptures are common and devastating injuries; however, an optimized treatment and rehabilitation protocol has yet to be defined. Therefore, the objective of this study was to investigate the effects of surgical repair and return to activity on joint function and Achilles tendon properties after 3 weeks of healing. Sprague–Dawley rats (N = 100) received unilateral blunt transection of their Achilles tendon. Animals were then randomized into repaired or non‐repaired treatments, and further randomized into groups that returned to activity after 1 week (RTA1) or after 3 weeks (RTA3) of limb casting in plantarflexion. Limb function, passive joint mechanics, and tendon properties (mechanical, organizational using high frequency ultrasound, histological, and compositional) were evaluated. Results showed that both treatment and return to activity collectively affected limb function, passive joint mechanics, and tendon properties. Functionally, RTA1 animals had increased dorsiflexion ROM and weight bearing of the injured limb compared to RTA3 animals 3‐weeks post‐injury. Such functional improvements in RTA1 tendons were evidenced in their mechanical fatigue properties and increased cross sectional area compared to RTA3 tendons. When RTA1 was coupled with nonsurgical treatment, superior fatigue properties were achieved compared to repaired tendons. No differences in cell shape, cellularity, GAG, collagen type I, or TGF‐β staining were identified between groups, but collagen type III was elevated in RTA3 repaired tendons. The larger tissue area and increased fatigue resistance created in RTA1 tendons may prove critical for optimized outcomes in early Achilles tendon healing following complete rupture.

Effect of overuse-induced tendinopathy on tendon healing in a rat supraspinatus repair model.

Supraspinatus tears often result in the setting of chronic tendinopathy. However, the typical repair model utilizes an acute injury. In recognition of that distinction, our laboratory developed an overuse animal model; however it is unclear whether induced overuse is necessary in the repair model. We studied the repair properties of overuse‐induced tendons compared to normal tendons. We hypothesized that histological and mechanical properties would not be altered between the overuse‐induced and normal tendons 1 and 4 weeks after repair. Thirty‐one adult male Sprague‐Dawley rats were subjected to either overuse or cage activity for 4 weeks prior to bilateral supraspinatus tendon repair surgery. Rats were sacrificed at 1 and 4 weeks post‐surgery and evaluated for histology and mechanics. Results at 1 week showed no clear histologic changes, but increased inflammatory protein expression in overuse tendons. At 4 weeks, percent relaxation was slightly increased in the overuse group. No other alterations in mechanics or histology were observed. Our results suggest that the effects of the surgical injury overshadow the changes evoked by overuse. Because clinically relevant mechanical parameters were not altered in the overuse group, we conclude that when examining tendons 4 weeks after repair in the classic rat supraspinatus model, inducing overuse prior to surgery is likely to be unnecessary.

Intra-articular tibiofemoral injection of a nonsteroidal anti-inflammatory drug has no detrimental effects on joint mechanics in a rat model.

Administration of intra‐articular medications, including corticosteroids and analgesics, is common clinical practice for knee pathology and dysfunction. Non‐steroidal anti‐inflammatory drugs (NSAIDs) are another category of medication commonly prescribed for their analgesic and anti‐inflammatory properties. Recent studies demonstrated the efficacy of injectable NSAIDs in the treatment of intra‐articular pathology and postoperative analgesia. However, little data exist regarding the safety of intra‐articular injection, despite the increase in its application. Therefore, we investigated the effects of intra‐articular NSAID injection on articular cartilage, the anterior cruciate ligament (ACL), and joint function in the rat. Sixty‐four Sprague‐Dawley rats were divided into either saline (SAL) or ketorolac (NSAID) tibiofemoral single injection treatment groups. Animals were euthanized at 2, 7, 28, and 84 days post‐injection for histological and mechanical analyses. Additionally, a subset of animals underwent longitudinal ambulatory evaluation to determine joint functional properties. We hypothesized that intra‐articular ketorolac injection would result in no detrimental mechanical, histological, or functional changes. No differences were reported between the NSAID and SAL groups in any of the parameters measured at any time point, demonstrating the potential safety of intra‐articular NSAID administration. Therefore, NSAID injection could be further considered for clinical application in humans.

Temporal Healing of Achilles Tendons After Injury in Rodents Depends on Surgical Treatment and Activity

Introduction: Achilles tendon ruptures affect 15 of 100,000 women and 55 of 100,000 men each year. Controversy continues to exist regarding optimal treatment and rehabilitation protocols. The objective of this study was to investigate the temporal effects of surgical repair and immobilization or activity on Achilles tendon healing and limb function after complete transection in rodents. Methods: Injured tendons were repaired (n = 64) or left nonrepaired (n = 64). The animals in both cohorts were further randomized into groups immobilized in plantar flexion for 1, 3, or 6 weeks that later resumed cage and treadmill activity for 5, 3, or 0 weeks, respectively (n = 36 for each regimen), which were euthanized at 6 weeks after injury, or into groups immobilized for 1 week and then euthanized (n = 20). Results: At 6 weeks after injury, the groups that had 1 week of immobilization and 5 weeks of activity had increased range of motion and decreased ankle joint toe stiffness compared with the groups that had 3 weeks of immobilization and 3 weeks of activity. The groups with 6 weeks of immobilization and no activity period had decreased tendon cross-sectional area but increased tendon echogenicity and collagen alignment. Surgical treatment dramatically decreased fatigue cycles to failure in repaired tendons from groups with 1 week of immobilization and 5 weeks of activity. Normalized comparisons between 1-week and 6-week postinjury data demonstrated that changes in tendon healing properties (area, alignment, and echogenicity) were maximized by 1 week of immobilization and 5 weeks of activity, compared with 6 weeks of immobilization and no activity period. Discussion: This study builds on an earlier study of Achilles tendon fatigue mechanics and functional outcomes during early healing by examining the temporal effects of different immobilization and/or activity regimens after initial postinjury immobilization. Conclusion: This study demonstrates how the temporal postinjury healing response of rodent Achilles tendons depends on both surgical treatment and the timing of immobilization/activity timing. The different pattern of healing and qualities of repaired and nonrepaired tendons suggest that two very different healing processes may occur, depending on the chosen immobilization/activity regimen.

Electrospun PLGA Nanofiber Scaffolds Release Ibuprofen Faster and Degrade Slower After In Vivo Implantation

While delayed delivery of non-steroidal anti-inflammatory drugs (NSAIDs) has been associated with improved tendon healing, early delivery has been associated with impaired healing. Therefore, NSAID use is appropriate only if the dose, timing, and mode of delivery relieves pain but does not impede tissue repair. Because delivery parameters can be controlled using drug-eluting nanofibrous scaffolds, our objective was to develop a scaffold for local controlled release of ibuprofen (IBP), and characterize the release profile and degradation both in vitro and in vivo. We found that when incubated in vitro in saline, scaffolds containing IBP had a linear release profile. However, when implanted subcutaneously in vivo or when incubated in vitro in serum, scaffolds showed a rapid burst release. These data demonstrate that scaffold properties are dependent on the environment in which they are placed and the importance of using serum, rather than saline, for initial in vitro evaluation of biofactor release from biodegradable scaffolds.