Single-cell RNA-sequencing atlas of bovine caudal intervertebral discs: Discovery of heterogeneous cell populations with distinct roles in homeostasis.

Back and neck pain are significant healthcare burdens that are commonly associated with pathologies of the intervertebral disc (IVD). The poor understanding of the cellular heterogeneity within the IVD makes it difficult to develop regenerative IVD therapies. To address this gap, we developed an atlas of bovine (Bos taurus) caudal IVDs using single-cell RNA-sequencing (scRNA-seq). Unsupervised clustering resolved 15 unique clusters, which we grouped into the following annotated partitions: nucleus pulposus (NP), outer annulus fibrosus (oAF), inner AF (iAF), notochord, muscle, endothelial, and immune cells. Analyzing the pooled gene expression profiles of the NP, oAF, and iAF partitions allowed us to identify novel markers for NP (CP, S100B, H2AC18, SNORC, CRELD2, PDIA4, DNAJC3, CHCHD7, and RCN2), oAF (IGFBP6, CTSK, LGALS1, and CCN3), and iAF (MGP, COMP, SPP1, GSN, SOD2, DCN, FN1, TIMP3, WDR73, and GAL) cells. Network analysis on subpopulations of NP and oAF cells determined that clusters NP1, NP2, NP4, and oAF1 displayed gene expression profiles consistent with cell survival, suggesting these clusters may uniquely support viability under the physiological stresses of the IVD. Clusters NP3, NP5, oAF2, and oAF3 expressed various extracellular matrix (ECM)-associated genes, suggesting their role in maintaining IVD structure. Lastly, transcriptional entropy and pseudotime analyses found that clusters NP3 and NP1 had the most stem-like gene expression signatures of the NP partition, implying these clusters may contain IVD progenitor cells. Overall, results highlight cell type diversity within the IVD, and these novel cell phenotypes may enhance our understanding of IVD development, homeostasis, degeneration, and regeneration.

Adjusting to Your Surroundings: An Inquiry-Based Learning Module to Teach Principles of Mechanobiology for Regenerative Medicine.

Mechanobiology is an interdisciplinary field that aims to understand how physical forces impact biological systems. Enhancing our knowledge of mechanobiology has become increasingly important for understanding human disease and developing novel therapeutics. There is a societal need to teach diverse students principles of mechanobiology so that we may collectively expand our knowledge of this subject and apply new principles to improving human health. Toward this goal, we designed, implemented, and evaluated a hands-on, inquiry-based learning (IBL) module to teach students principles of cell-biomaterial interactions. This module was designed to be hosted in two 3-h sessions, over two consecutive days. During this time, students learned how to synthesize and mechanically test biomaterials, culture bacteria cells, and assess effects of matrix stiffness on bacteria cell proliferation. Among the 73 students who registered to participate in our IBL mechanobiology module, 40 students completed both days and participated in this study. A vast majority of the participants were considered underrepresented minority (URM) students based on race/ethnicity. Using pre/post-tests, we found that students experienced significant learning gains of 33 percentage points from completing our IBL mechanobiology module. In addition to gaining knowledge of mechanobiology, validated pre/post-surveys showed that students also experienced significant improvements in scientific literacy. Instructors may use this module as described, increase the complexity for an undergraduate classroom assignment, or make the module less complex for K-12 outreach. As presented, this IBL mechanobiology module effectively teaches diverse students principles of mechanobiology and scientific inquiry. Deploying this module, and similar IBL modules, may help advance the next generation of mechanobiologists.

Cyr61 delivery promotes angiogenesis during bone fracture repair.

Compromised vascular supply and insufficient neovascularization impede bone repair, increasing risk of non-union. Cyr61, Cysteine-rich angiogenic inducer of 61kD (also known as CCN1), is a matricellular growth factor that is regulated by mechanical cues during fracture repair. Here, we map the distribution of endogenous Cyr61 during bone repair and evaluate the effects of recombinant Cyr61 delivery on vascularized bone regeneration. In vitro, Cyr61 treatment did not alter chondrogenesis or osteogenic gene expression, but significantly enhanced angiogenesis. In a mouse femoral fracture model, Cyr61 delivery did not alter cartilage or bone formation, but accelerated neovascularization during fracture repair. Early initiation of ambulatory mechanical loading disrupted Cyr61-induced neovascularization. Together, these data indicate that Cyr61 delivery can enhance angiogenesis during bone repair, particularly for fractures with stable fixation, and may have therapeutic potential for fractures with limited blood vessel supply.

Moesin activation controls bone resorption and tunneling nanotube-dependent osteoclast fusion.

Osteoclasts are multinucleated cells unique in their ability to resorb bone. Osteoclastogenesis involves several steps of actin-driven rearrangements that participate not only in the cell-cell fusion process, but also in the formation of the sealing zone, the adhesive structure determining the resorption area. Despite the importance of these actin cytoskeleton-based processes, their precise mechanisms of regulation are still poorly characterized. Here, we found that moesin, a member of the Ezrin/Radixin/Moesin (ERM) protein family, is activated during osteoclast maturation and plays an instrumental role for both osteoclast fusion and function. In mouse and human osteoclast precursors, moesin is negatively regulated to potentiate their ability to fuse and degrade bone. Accordingly, we demonstrated that moesin depletion decreases membrane-to-cortex attachment and enhances formation of tunneling nanotubes (TNTs), F-actin-containing intercellular bridges that we revealed to trigger osteoclast fusion. In addition, via a ß3-integrin/RhoA/SLK pathway and independently of its role in fusion, moesin regulates the number and organization of sealing zones in mature osteoclast, and thus participates in the control of bone resorption. Supporting these findings, we found that moesin-deficient mice are osteopenic with a reduced density of trabecular bones and increased osteoclast abundance and activity. These findings provide a better understanding of the regulation of osteoclast biology, and open new opportunities to specifically target osteoclast activity in bone disease therapy.

Teaching Tissue Repair Through an Inquiry-Based Learning Bioadhesives Module.

Bioadhesives are an important class of biomaterials for wound healing, hemostasis, and tissue repair. To develop the next generation of bioadhesives, there is a societal need to teach trainees about their design, engineering, and testing. This study designed, implemented, and evaluated a hands-on, inquiry-based learning (IBL) module to teach bioadhesives to undergraduate, master's, and PhD/postdoctoral trainees. Approximately 30 trainees across three international institutions participated in this IBL bioadhesives module, which was designed to last approximately 3 h. This IBL module was designed to teach trainees about how bioadhesives are used for tissue repair, how to engineer bioadhesives for different biomedical applications, and how to assess the efficacy of bioadhesives. The IBL bioadhesives module resulted in significant learning gains for all cohorts; whereby, trainees scored an average of 45.5% on the pre-test assessment and 69.0% on the post-test assessment. The undergraduate cohort experienced the greatest learning gains of 34.2 points, which was expected since they had the least theoretical and applied knowledge about bioadhesives. Validated pre/post-survey assessments showed that trainees also experienced significant improvements in scientific literacy from completing this module. Similar to the pre/post-test, improvements in scientific literacy were most significant for the undergraduate cohort since they had the least amount of experience with scientific inquiry. Instructors can use this module, as described, to introduce undergraduate, master's, and PhD/postdoctoral trainees to principles of bioadhesives.

Combining adhesive and nonadhesive injectable hydrogels for intervertebral disc repair in an ovine discectomy model.

Background: Intervertebral disc (IVD) disorders (e.g., herniation) directly contribute to back pain, which is a leading cause of global disability. Next-generation treatments for IVD herniation need advanced preclinical testing to evaluate their ability to repair large defects, prevent reherniation, and limit progressive degeneration. This study tested whether experimental, injectable, and nonbioactive biomaterials could slow IVD degeneration in an ovine discectomy model. Methods: Ten skeletally mature sheep (4-5.5 years) experienced partial discectomy injury with cruciate-style annulus fibrosus (AF) defects and 0.1 g nucleus pulposus (NP) removal in the L1-L2, L2-L3, and L3-L4 lumbar IVDs. L4-L5 IVDs were Intact controls. IVD injury levels received: (1) no treatment (Injury), (2) poly (ethylene glycol) diacrylate (PEGDA), (3) genipin-crosslinked fibrin (FibGen), (4) carboxymethylcellulose-methylcellulose (C-MC), or (5) C-MC and FibGen (FibGen + C-MC). Animals healed for 12 weeks, then IVDs were assessed using computed tomography (CT), magnetic resonance (MR) imaging, and histopathology. Results: All repaired IVDs retained ~90% of their preoperative disc height and showed minor degenerative changes by Pfirrmann grading. All repairs had similar disc height loss and Pfirrmann grade as Injury IVDs. Adhesive AF sealants (i.e., PEGDA and FibGen) did not herniate, although repair caused local endplate (EP) changes and inflammation. NP repair biomaterials (i.e., C-MC) and combination repair (i.e., FibGen + C-MC) exhibited lower levels of degeneration, less EP damage, and less severe inflammation; however, C-MC showed signs of herniation via biomaterial expulsion. Conclusions: All repair IVDs were noninferior to Injury IVDs by IVD height loss and Pfirrmann grade. C-MC and FibGen + C-MC IVDs had the best outcomes, and may be appropriate for enhancement with bioactive factors (e.g., cells, growth factors, and miRNAs). Such bioactive factors appear to be necessary to prevent injury-induced IVD degeneration. Application of AF sealants alone (i.e., PEGDA and FibGen) resulted in EP damage and inflammation, particularly for PEGDA IVDs, suggesting further material refinements are needed.

Genipin-crosslinked fibrin seeded with oxidized alginate microbeads as a novel composite biomaterial strategy for intervertebral disc cell therapy.

Discectomy procedures alleviate disability caused by intervertebral disc (IVD) herniation, but do not repair herniation-induced annulus fibrosus (AF) defects. Cell therapy shows promise for IVD repair, yet cell delivery biomaterials capable of sealing AF defects and restoring biomechanical function have poor biological performance. To balance the biomechanical and biological demands of IVD cell delivery biomaterials, we engineered an injectable composite biomaterial using cell-laden, degradable oxidized alginate (OxAlg) microbeads (MBs) to deliver AF cells within high-modulus genipin-crosslinked fibrin (FibGen) hydrogels (FibGen + MB composites). Conceptually, the high-modulus FibGen would immediately stabilize injured IVDs, while OxAlg MBs would protect and release cells required for long-term healing. We first showed that AF cells microencapsulated in OxAlg MBs maintained high viability and, upon release, displayed phenotypic AF cell morphology and gene expression. Next, we created cell-laden FibGen + MB composites and demonstrated that OxAlg MBs functionalized with RGD peptides (MB-RGD) minimized AF cell apoptosis and retained phenotypic gene expression. Further, we showed that cell-laden FibGen + MB composites are biomechanically stable and promote extracellular matrix (ECM) synthesis in long-term in vitro culture. Lastly, we evaluated cell-laden FibGen + MB-RGD composites in a long-term bovine caudal IVD organ culture bioreactor and found that composites had low herniation risk, provided superior biomechanical and biological repair to discectomy controls, and retained anabolic cells within the IVD injury space. This novel injectable composite hydrogel strategy shows promise as an IVD cell delivery sealant with potentially broad applications for its capacity to balance biomechanical and biological performance.

Hydrogel-Embedded Poly(Lactic-co-Glycolic Acid) Microspheres for the Delivery of hMSC-Derived Exosomes to Promote Bioactive Annulus Fibrosus Repair.

OBJECTIVE: Intervertebral disk degeneration is a prevalent postoperative complication after discectomy, underscoring the need to develop preventative and bioactive treatment strategies that decelerate degeneration and seal annulus fibrosus (AF) defects. Human mesenchymal stem cell-derived exosomes (MSC-Exos) hold promise for cell-free bioactive repair; however, their ability to promote AF repair is poorly understood. The objective of this study was to evaluate the ability of MSC-Exos to promote endogenous AF repair processes and integrate MSC-Exos within a biomaterial delivery system. DESIGN: We characterize biophysical and biochemical properties of normoxic (Nx) and hypoxic (Hx) preconditioned MSC-Exos from young, healthy donors and examine their effects on AF cell proliferation, migration, and gene expression. We then integrate a poly(lactic-co-glycolic acid) microsphere (PLGA µSphere) delivery platform within an interpenetrating network hydrogel to facilitate sustained MSC-Exo delivery. RESULTS: Hx MSC-Exos led to a more robust response in AF cell proliferation and migration than Nx MSC-Exos and was selected for a downstream protection experiment. Hx MSC-Exos maintained a healthy AF cell phenotype under a TNFα challenge in vitro and attenuated catabolic responses. In all functional assays, AF cell responses were more sensitive to Hx MSC-Exos than Nx MSC-Exos. PLGA µSpheres released MSC-Exos over a clinically relevant timescale without affecting hydrogel modulus or pH upon initial embedment and µSphere degradation. CONCLUSIONS: This MSC-Exo treatment strategy may offer benefits of stem cell therapy without the need for exogenous stem cell transplantation by stimulating cell proliferation, promoting cell migration, and protecting cells from the degenerative proinflammatory microenvironment.