Johnna S. Temenoff

Purpose: Following rotator cuff tendon injury, the corresponding muscle undergoes degeneration, which has been found to occur asymmetrically. Thus, the goals of this study were 1) to develop an injection strategy for spatially localized Stromal-Derived Factor 1α (SDF-1α) loaded fragment delivery to two muscle regions in a rat model of severe rotator cuff tear and 2) to determine the degree of spatial cellular recruitment and subsequent muscle healing in response to these localized injections. Methods: Hydrolytically-degradable heparin-containing hydrogel fragments were optimized for sustained release of SDF-1α over 7 + days in vivo. Fragments were then injected into either the muscle near the myotendinous junction (MTJ) or the muscle belly (MB) region of injured supraspinatus muscle. The presence of inflammatory cells (neutrophils, macrophage and T cell subsets) and stem cells (fibroadipogenic progenitor cells, satellite cells, mesenchymal stromal cells) in each region was assessed via flow cytometry 3- and 7-days following treatment. Muscle tissue was evaluated via immunohistochemical staining for regenerating muscle fibers (eMHC), fibrosis (collagen) and fatty infiltration (perilipin) 7 days post treatment. Results/Conclusion: Delivery of SDF-1α to injured rotator cuff muscle caused alterations in the amount of adaptive and innate immune cells and stem cells, and resulted in increased muscle regeneration in the MTJ region. Regional differences in cell populations observed after local treatment demonstrated the technical feasibility of spatial material delivery and subsequent spatial cell recruitment in injured muscle. Thus, localized delivery using this biomaterial platform may be a useful tool to probe the effects of regional cell recruitment in spatially-complex orthopaedic injuries. Lay Summary: We developed a spatially targeted injection strategy to study regional treatment effects in injured rotator cuff muscle. Our hydrogel delivery system provides sustained SDF-1α release over 10 days in vivo. Cellular analysis suggests that localized SDF-1α delivery modulates immune responses in both adaptive and innate cells. Regional variations in cell populations following treatment highlight the feasibility of spatial material delivery and targeted cell recruitment. However, spatial SDF-1α delivery did not induce regional differences in early muscle regeneration, despite these variations in cell populations. This approach offers a customizable platform for investigating regional tissue responses and developing treatments for complex injuries.

Purpose Degeneration of the human supraspinatus muscle after rotator cuff tendon tear varies by location within the muscle; however, the localization of changes to muscle fiber size, amount of regenerating fibers, and cellular immune response have not been previously characterized. Due to the proximity to the injured tendon, we hypothesized that these processes are more pronounced near the lateral myotendinous junction region of the muscle and the intramuscular tendon. Methods Spatially-defined immunohistochemical analysis was used to evaluate lengthwise and radial differences within the supraspinatus muscle in a rat model of full supraspinatus and infraspinatus tendon transection and subscapular denervation. Muscle fiber diameter was quantified, regeneration was evaluated by myofiber central nuclei and embryonic myosin heavy chain, and the immune environment was assessed by the presence of macrophage subsets and T cells. Results Degeneration (smaller fiber diameter), regeneration (embryonic myosin heavy chain-positive fibers), and macrophage infiltration are more substantial in the lateral muscle near the myotendinous junction and radially near the intramuscular tendon. The muscle adjacent to the myotendinous junction had a balanced M1-like and M2-like macrophage response up to 2 weeks post-injury followed by an M1-dominant inflammation. The medial muscle belly maintained an M1-dominant response throughout 3 weeks. T cells were not highly abundant, however, were most elevated one week after injury near the intramuscular tendon. Fibrous infiltration was observed 1-week post-injury throughout the muscle, while fatty infiltration was elevated after 3 weeks only in the lateral muscle region. Conclusions Together, these findings suggest that the supraspinatus muscle undergoes regional changes (particularly muscle fiber size, markers of regeneration, and macrophage infiltration) after supraspinatus tendon injury. Lay Summary and Future Work Rotator cuff tendon tear in the rat shoulder leads to a greater inflammatory response and a greater early pro-regenerative response in the supraspinatus muscle spatially near the muscle–tendon attachment compared with areas deeper in the muscle in the first two weeks after the tendon is injured. Both fatty and fibrous muscle degeneration were observed over three weeks throughout the muscle, with less spatial localization. Together, these data indicate that there are regional differences in damage and repair of the supraspinatus muscle early post-injury. These findings may help interpret results from clinical studies of muscle degeneration after rotator cuff tear and may lead to improved, spatially-targeted approaches for delivery of therapeutics to muscle after rotator cuff tendon tear.

Mesenchymal stromal cells (MSCs) show great promise as a clinical treatment for a variety of diseases, but their susceptibility to senescence during culture reduces the therapeutic potential and limits cell expansion. In this study, we explored how MSC lipid metabolism is altered in culture over time using ultrahigh-performance liquid chromatography mass spectrometry. The proportion of cells with senescence-associated β-galactosidase (SA-β-gal) activity was evaluated during 12 days of culture expansion of MSCs from two human donors. Lipid profiles were evaluated in parallel using exact mass and tandem mass spectrometry spectral database matching to generate 237 unique lipid annotations. Lipid abundance generally increased across most lipid classes over serial culture; however, many changes were heterogeneous between donors. Despite donor differences, 12 lipids, including 4 triglycerides (TG), provided discrimination between cultures with less than 10% SA-β-gal+, those with 10-20% SA-β-gal+, and greater than 20% SA-β-gal+ senescence proportion regardless of donor. More specifically, TG composed of long-chain, highly unsaturated fatty acids was strongly associated with higher MSC senescence. These changes in bulk lipid profiles may inform future strategies to monitor early culture senescence during the expansion of MSCs.

Hydrogels offer a promising solution for sustained and controlled drug delivery and cell‐tissue biocompatibility. In the intervertebral disc (IVD), delivering growth factors faces challenges due to the antagonistic inflammatory environment and continuous mechanical stress, which can degrade biological agents and may reduce their local activity. To address this, we investigated the prolonged release of platelet‐derived growth factor isoforms BB (PDGF‐BB) and AB (PDGF‐AB) by using N‐desulfated heparin methacrylamide (Hep^‐N) crosslinked within matrix‐metalloproteinase sensitive poly(ethylene glycol) (PEG) hydrogels. Using electrostatic interactions between the heparin derivative and PDGF, we optimized a sustained release dose of PDGF‐BB from the hydrogel in the presence of collagenase to mimic the in vivo environment. We then assessed the effects of PDGF released from PEG‐hydrogel on human nucleus pulposus (NP) Cells. The MTT assay confirmed that 100 and 200 ng doses significantly increased cell viability by 2.52‐fold and 2.46‐fold on Day 3, respectively. RT‐qPCR analysis revealed that PDGF‐AB and PDGF‐BB upregulated the expression of proliferation marker Ki‐67 (MKI67) on both Day 3 and Day 5. Additionally, collagen type II alpha 1 chain (COL2A1) was significantly upregulated in the PDGF‐AB group on Day 5, indicating potential anabolic effects. These findings could pave the way for long‐term in vivo studies on sustainable PDGF treatment for IVD degeneration.

Muscle degeneration after rotator cuff tendon tear is a significant clinical problem. In these experiments, we developed a poly(ethylene glycol)-based injectable granular hydrogel containing two heparin derivatives (fully sulfated [Hep] and fully desulfated [Hep−]) as well as a matrix metalloproteinase-sensitive peptide to promote sustained release of tumor necrosis factor–stimulated gene 6 (TSG-6) over 14+ days in vivo in a rat model of rotator cuff muscle injury. The hydrogel formulations demonstrated similar release profiles in vivo , thus facilitating comparisons between delivery from heparin derivatives on the level of tissue repair in two different areas of muscle (near the myotendious junction [MTJ] and in the muscle belly [MB]) that have been shown previously to have differing responses to rotator cuff tendon injury. We hypothesized that sustained delivery of TSG-6 would enhance the anti-inflammatory response following rotator cuff injury through macrophage polarization and that release from Hep would potentiate this effect throughout the muscle. Inflammatory/immune cells, satellite cells, and fibroadipogenic progenitor cells were analyzed by flow cytometry 3 and 7 days after injury and hydrogel injection, while metrics of muscle healing were examined via immunohistochemistry up to day 14. Results showed controlled delivery of TSG-6 from Hep caused heightened macrophage response (day 7 macrophages, 4.00 ± 1.85% single cells, M2a, 3.27 ± 1.95% single cells) and increased markers of early muscle regeneration (embryonic heavy chain staining) by day 7, particularly in the MTJ region of the muscle. This work provides a novel strategy for localized, controlled delivery of TSG-6 to enhance muscle healing after rotator cuff tear.

Cell therapies, potent populations of cells used to treat disease and injury, can be strategically manufactured with biomaterial intervention to improve clinical translation. In this viewpoint, we discuss biomaterial design and integration into cell manufacturing steps to achieve three main goals: scale-up, phenotype control, and selection of potent cells. Material properties can be engineered to influence the cell-biomaterial interface and, therefore, impart desirable cell behavior such as growth, secretory activity, and differentiation. Future directions for the field should capitalize on the combinatorial design of biomaterial properties to yield highly specific and potent cell populations. Furthermore, future biomaterials could contribute to novel high-throughput cell separation technologies that can individually select the most therapeutically relevant cells within a produced batch.

Muscle degeneration after rotator cuff tendon tear is a significant clinical problem. In these experiments, we developed a poly(ethylene glycol)-based injectable granular hydrogel containing two heparin derivatives (fully sulfated (Hep) and fully desulfated (Hep-)) as well as a matrix metalloproteinase-sensitive peptide to promote sustained release of Tumor Necrosis Factor Stimulated Gene 6 (TSG-6) over 14+ days in vivo in a rat model of rotator cuff muscle injury. The hydrogel formulations demonstrated similar release profiles in vivo , thus facilitating comparisons between delivery from heparin derivatives on level of tissue repair in two different areas of muscle (near the myotendious junction (MTJ) and in the muscle belly (MB)) that have been shown previously to have differing responses to rotator cuff tendon injury. We hypothesized that sustained delivery of TSG-6 would enhance the anti-inflammatory response following rotator cuff injury through macrophage polarization, and that release from a fully sulfated heparin derivative (Hep) would potentiate this effect throughout the muscle. Inflammatory/immune cells, satellite cells, and fibroadipogenic progenitor cells, were analyzed by flow cytometery 3 and 7 days after injury and hydrogel injection, while metrics of muscle healing were examined via immunohistochemistry up to Day 14. Results showed controlled delivery of TSG-6 from Hep caused heightened macrophage response (Day 14 macrophages, 4.00 ± 1.85% single cells, M2a, 3.27 ± 1.95% single cells) and increased markers of early muscle regeneration (embryonic heavy chain staining) by Day 7, particularly in the MTJ region of the muscle, compared to release from desulfated heparin hydrogels. This work provides a novel strategy for localized, controlled delivery of TSG-6 to enhance muscle healing after rotator cuff tear. IMPACT STATEMENT: Rotator cuff tear is a significant problem that can cause muscle degeneration. In this study, a hydrogel particle system was developed for sustained release of an anti-inflammatory protein, Tumor Necrosis Factor Stimulated Gene 6 (TSG-6), to injured muscle. Release of the protein from a fully sulfated heparin hydrogel-based carrier demonstrated greater changes in amount inflammatory cells and more early regenerative effects than a less-sulfated carrier. Thus, this work provides a novel strategy for localized, controlled delivery of an anti-inflammatory protein to enhance muscle healing after rotator cuff tear.

Regeneration of cartilage and bone tissues remains challenging in tissue engineering due to their complex structures, and the need for both mechanical support and delivery of biological repair stimuli. Therefore, the goal of this study was to develop a composite scaffold platform for anatomic chondral and osteochondral repair using heparin-based hydrogels to deliver small molecules within 3D-printed porous scaffolds that provide structure, stiffness, and controlled biologic delivery. We designed a mold-injection system to combine hydrolytically degradable hydrogels and 3D-printed scaffolds that could be employed rapidly (< 30 min) in operating room settings (~23 °C). Micro-CT analysis demonstrated the effectiveness of our injection system through homogeneously distributed hydrogel within the pores of the scaffolds. Hydrogels and composite scaffolds exhibited efficient loading (~94%) of a small positively charged heparin-binding molecule (crystal violet) with sustained release over 14 days and showed high viability of encapsulated porcine chondrocytes over 7 days. Compression testing demonstrated nonlinear viscoelastic behavior where tangent stiffness decreased with scaffold porosity (porous scaffold tangent stiffness: 70%: 4.9 MPa, 80%: 1.5 MPa, and 90%: 0.20 MPa) but relaxation was not affected. Lower-porosity scaffolds (70%) showed stiffness similar to lower ranges of trabecular bone (4–8 MPa) while higher-porosity scaffolds (80% and 90%) showed stiffness similar to auricular cartilage (0.16–2 MPa). Ultimately, this rapid composite scaffold fabrication method may be employed in the operating room and utilized to control biologic delivery within load-bearing scaffolds.

Rotator cuff tear is a significant problem that leads to poor clinical outcomes due to muscle degeneration after injury. The objective of this study was to synergistically increase the number of pro-regenerative cells recruited to injured rotator cuff muscle through a novel dual treatment system, consisting of a bone marrow mobilizing agent (VPC01091), hypothesized to “push” pro-healing cells into the blood, and localized delivery of Stromal cell-derived factor 1α (SDF-1α), to “pull” the cells to the injury site. Immediately after rotator cuff tendon injury in rat, the mobilizing agent was delivered systemically, and SDF-1α-loaded heparin based microparticles were injected into the supraspinatus muscle. Regenerative and degenerative changes to supraspinatus muscle, and the presence of inflammatory/immune cells, mesenchymal stem cells, and satellite cells, were assessed via flow cytometry and histology for up to 21 days. After dual treatment, significantly more mesenchymal stem cells (31.9 ± 8.0% single cells) and T lymphocytes (6.7 ± 4.3 per 20X field of view) were observed in supraspinatus muscle 7 days after injury and treatment compared to Injury alone (14.4 ± 6.5% single cells, 1.2 ± 0.7 per 20X field of view), in addition to an elevated M2:M1 macrophage ratio (3.0 ± 0.5), an indicator of a pro-regenerative environment. These pro-regenerative cellular changes were accompanied by increased nascent fiber formation (indicated by embryonic myosin heavy chain staining) at day 7 compared to SDF-1α treatment alone, suggesting that this method may be a promising strategy to influence the early cellular response in muscle and promote a pro-regenerative micro-environment to increase muscle healing after severe rotator cuff tear.

In order to scale up culture therapeutic cells, such as mesenchymal stromal cells (MSCs), culture in suspension bioreactors using microcarriers (mu Cs) is preferred. However, the impact of microcarrier type on the resulting MSC secretory activity has not been investigated. In this study, two poly(ethylene glycol) hydrogel formulations with different swelling ratios (named "stiffer" and "softer") were fabricated as mu C substrates to culture MSCs and MSCs genetically modified to express the interleukin-1 receptor antagonist (IL-1Ra-MSCs). Changes in cell number, secretory and angiogenic activity, and changes in MAPK signaling were evaluated when cultured on hydrogel mu Cs, as well as on tissue culture plastic-based Synthemax mu Cs. We demonstrated that culture on stiffer mu Cs increased secretion of IL-1Ra compared to culture on Synthemax mu Cs by IL-1Ra-MSCs by 1.2- to 1.6-fold, as well as their in vitro angiogenic activity, compared to culture on Synthemax mu Cs, while culture on both stiffer and softer mu Cs altered the secretion of several other factors compared to culture on Synthemax mu Cs. Changes in angiogenic activity corresponded with increased gene expression and secretion of hepatocyte growth factor by MSCs cultured on softer mu Cs by 2.5- to 6-fold compared to MSCs cultured on Synthemax mu Cs. Quantification of phosphoprotein signaling with the MAPK pathway revealed broad reduction of pathway activation by IL-1Ra-MSCs cultured on both stiffer and softer mu Cs compared to Synthemax, where phosphorylated c-Jun, ATF2, and MEK1 were reduced specifically on softer mu Cs. Overall, this study showed that mu C surfaces can influence the secretory activity of genetically modified MSCs and identified associated changes in MAPK pathway signaling, which is a known central regulator of cytokine secretion.