Various types of cellular injection have become a popular and costly treatment option for patients with knee osteoarthritis despite a paucity of literature establishing relative efficacy to each other or corticosteroid injections. Here we aimed to identify the safety and efficacy of cell injections from autologous bone marrow aspirate concentrate, autologous adipose stromal vascular fraction and allogeneic human umbilical cord tissue-derived mesenchymal stromal cells, in comparison to corticosteroid injection (CSI). The study was a phase 2/3, four-arm parallel, multicenter, single-blind, randomized, controlled clinical trial with 480 patients with a diagnosis of knee osteoarthritis (Kellgren–Lawrence II–IV). Participants were randomized to the three different arms with a 3:1 distribution. Arm 1: autologous bone marrow aspirate concentrate (n = 120), CSI (n = 40); arm 2: umbilical cord tissue-derived mesenchymal stromal cells (n = 120), CSI (n = 40); arm 3: stromal vascular fraction (n = 120), CSI (n = 40). The co-primary endpoints were the visual analog scale pain score and Knee injury and Osteoarthritis Outcome Score pain score at 12 months versus baseline. Analyses of our primary endpoints, with 440 patients, revealed that at 1 year post injection, none of the three orthobiologic injections was superior to another, or to the CSI control. In addition, none of the four groups showed a significant change in magnetic resonance imaging osteoarthritis score compared to baseline. No procedure-related serious adverse events were reported during the study period. In summary, this study shows that at 1 year post injection, there was no superior orthobiologic as compared to CSI for knee osteoarthritis. ClinicalTrials.gov Identifier: NCT03818737
Human induced pluripotent stem cells (hiPSCs) hold immense promise in regenerative medicine as they can differentiate into various cell lineages, including adipocytes, osteoblasts, and chondrocytes. Precisely guiding hiPSC-derived mesenchymal progenitor cells (iMSCs) towards specific differentiation pathways is crucial for harnessing their therapeutic potential in tissue engineering, disease modeling, and regenerative therapies. To achieve this, we present a comprehensive and reproducible protocol for effectively differentiating iMSCs into adipocytes and osteoblasts. The differentiation process entails culturing iMSCs in tailored media supplemented with specific growth factors, which act as cues to initiate adipogenic or osteogenic commitment. Our protocol provides step-by-step guidelines for achieving adipocyte and osteoblast differentiation, ensuring the generation of mature and functional cells. To validate the success of differentiation, key assessment criteria are employed. For adipogenesis, the presence of characteristic lipid droplets within the iMSC-derived cells is considered indicative of successful differentiation. Meanwhile, Alizarin Red staining serves as a marker for the osteogenic differentiation, confirming the formation of mineralized nodules. Importantly, the described method stands out due to its simplicity, eliminating the need for specialized equipment, expensive materials, or complex reagents. Its ease of implementation offers an attractive advantage for researchers seeking robust and cost-effective approaches to derive adipocytes and osteoblasts from iMSCs. Overall, this protocol establishes a foundation for exploring the therapeutic potential of hiPSC-derived cells and advancing the field of regenerative medicine.
Age is a patient-specific factor that can significantly delay fracture healing and exacerbate systemic sequelae during convalescence. The basis for this difference in healing rates is not well-understood, but heightened inflammation has been suggested to be a significant contributor. In this study, we investigated the systemic cytokine and intestinal microbiome response to closed femur fracture in 3-month-old (young adult) and 15-month-old (middle-aged) female wild-type mice. Middle-aged mice had a serum cytokine profile that was distinct from young mice at days 10, 14, and 18 post-fracture. This was characterized by increased concentrations of IL-17a, IL-10, IL-6, MCP-1, EPO, and TNFα. We also observed changes in the community structure of the gut microbiota in both young and middle-aged mice that was evident as early as day 3 post-fracture. This included an Enterobacteriaceae bloom at day 3 post-fracture in middle-aged mice and an increase in the relative abundance of the Muribaculum genus. Moreover, we observed an increase in the relative abundance of the health-promoting Bifidobacterium genus in young mice after fracture that did not occur in middle-aged mice. There were significant correlations between serum cytokines and specific genera, including a negative correlation between Bifidobacterium and the highly induced cytokine IL-17a. Our study demonstrates that aging exacerbates the inflammatory response to fracture leading to high levels of pro-inflammatory cytokines and disruption of the intestinal microbiota.
Induced pluripotent stem cells (iPSCs) generated from human sources are valuable tools for studying skeletal development and diseases, as well as for potential use in regenerative medicine for skeletal tissues such as articular cartilage. To successfully differentiate human iPSCs into functional chondrocytes, it is essential to establish efficient and reproducible strategies that closely mimic the physiological chondrogenic differentiation process. Here, we describe a simple and efficient protocol for differentiation of human iPSCs into chondrocytes via generation of an intermediate population of mesenchymal progenitors. These methodologies include step-by-step procedures for mesenchymal derivation, induction of chondrogenic differentiation, and evaluation of the chondrogenic marker gene expression. In this protocol, we describe the detailed procedure for successful derivation of mesenchymal progenitor population from human iPSCs, which are then differentiated into chondrocytes using high-density culture conditions by stimulating with bone morphogenetic protein-2 (BMP-2) or transforming growth factor beta-3 (TGFβ-3). The differentiated iPSCs exhibit temporal expression of cartilage genes and accumulation of a cartilaginous extracellular matrix in vitro, indicating successful chondrogenic differentiation. These detailed methodologies help effective differentiation of human iPSCs into the chondrogenic lineage to obtain functional chondrocytes, which hold great promise for modeling skeletal development and disease, as well as for potential use in regenerative medicine for cell-based therapy for cartilage regeneration.
Treatments for congenital and acquired craniofacial (CF) bone abnormalities are limited and expensive. Current reconstructive methods include surgical correction of injuries, short-term bone stabilization, and long-term use of bone grafting solutions, including implantation of (i) allografts which are prone to implant failure or infection, (ii) autografts which are limited in supply. Current bone regenerative approaches have consistently relied on BMP2 application with or without addition of stem cells. BMP2 treatment can lead to severe bony overgrowth or uncontrolled inflammation, which can accelerate further bone loss. Bone marrow-derived mesenchymal stem cell-based treatments, which do not have the side effects of BMP2, are not currently FDA approved, and are time and resource intensive. There is a critical need for novel bone regenerative therapies to treat CF bone loss that have minimal side effects, are easily available, and are affordable. In this study we investigated novel bone regenerative therapies downstream of JAGGED1 (JAG1).
We previously demonstrated that JAG1 induces murine cranial neural crest (CNC) cells towards osteoblast commitment via a NOTCH non-canonical pathway involving JAK2-STAT5 (1) and that JAG1 delivery with CNC cells elicits bone regeneration in vivo. In this study, we hypothesize that delivery of JAG1 and induction of its downstream NOTCH non-canonical signaling in pediatric human osteoblasts constitute an effective bone regenerative treatment in an in vivo murine bone loss model of a critically-sized cranial defect. Using this CF defect model in vivo, we delivered JAG1 with pediatric human bone-derived osteoblast-like (HBO) cells to demonstrate the osteo-inductive properties of JAG1 in human cells and in vitro we utilized the HBO cells to identify the downstream non-canonical JAG1 signaling intermediates as effective bone regenerative treatments. In vitro, we identified an important mechanism by which JAG1 induces pediatric osteoblast commitment and bone formation involving the phosphorylation of p70 S6K. This discovery enables potential new treatment avenues involving the delivery of tethered JAG1 and the downstream activators of p70 S6K as powerful bone regenerative therapies in pediatric CF bone loss.
Traumatic joint injuries are common, leading to progressive tissue degeneration and the development of osteoarthritis. The post-traumatic joint experiences a pro-inflammatory milieu, initiating a subtle but deteriorative process in cartilage tissue. To prevent or even reverse this process, our group previously developed a tissue-penetrating methacrylated hyaluronic acid (MeHA) hydrogel system, crosslinked within cartilage to restore and/or protect the tissue. In the current study, we further optimized this approach by investigating the impact of biomaterial molecular weight (MW; 20, 75, 100 kDa) on its integration within and reinforcement of cartilage, as well as its ability to protect tissue degradation in a catabolic state. Indeed, the low MW MeHA integrated and reinforced cartilage tissue better than the high MW counterparts. Furthermore, in a 2 week IL-1β explant culture model, the 20 kDa MeHA demonstrated the most protection from biphasic mechanical loss, best retention of proteoglycans (Safranin O staining), and least aggrecan breakdown (NITEGE). Thus, the lower MW MeHA gels integrated better into the tissue and provided the greatest protection of the cartilage matrix. Future work will test this formulation in a preclinical model, with the goal of translating this therapeutic approach for cartilage preservation.
Background
Dimerization of the myeloid differentiation primary response 88 protein (MyD88) plays a pivotal role in the exacerbated response to innate immunity-dependent signaling in rheumatoid arthritis (RA). ST2825 is a highly specific inhibitor of MyD88 dimerization, previously shown to inhibit the pro-inflammatory gene expression in peripheral blood mononuclear cells from RA patients (RA PBMC). In this study, we elucidated the effect of disrupting MyD88 dimerization by ST2825 on the pathological properties of synovial fibroblasts from RA patients (RA SFs).
Methods
RA SFs were treated with varying concentrations of ST2825 in the presence or absence of bacterial lipopolysaccharides (LPS) to activate innate immunity-dependent TLR signaling. The DNA content of the RA SFs was quantified by imaging cytometry to investigate the effect of ST2825 on different phases of the cell cycle and apoptosis. RNA-seq was used to assess the global response of the RA SF toward ST2825. The invasiveness of RA SFs in Matrigel matrices was measured in organoid cultures. SFs from osteoarthritis (OA SFs) patients and healthy dermal fibroblasts were used as controls.
Results
ST2825 reduced the proliferation of SFs by arresting the cells in the G0/G1 phase of the cell cycle. In support of this finding, transcriptomic analysis by RNA-seq showed that ST2825 may have induced cell cycle arrest by primarily inhibiting the expression of critical cell cycle regulators Cyclin E2 and members of the E2F family transcription factors. Concurrently, ST2825 also downregulated the genes encoding for pain, inflammation, and joint catabolism mediators while upregulating the genes required for the translocation of nuclear proteins into the mitochondria and members of the mitochondrial respiratory complex 1. Finally, we demonstrated that ST2825 inhibited the invasiveness of RA SFs, by showing decreased migration of LPS-treated RA SFs in spheroid cultures.
Conclusions
The pathological properties of the RA SFs, in terms of their aberrant proliferation, increased invasiveness, upregulation of pain and inflammation mediators, and disruption of mitochondrial homeostasis, were attenuated by ST2825 treatment. Taken together with the previously reported anti-inflammatory effects of ST2825 in RA PBMC, this study strongly suggests that targeting MyD88 dimerization could mitigate both systemic and synovial pathologies in a variety of inflammatory arthritic diseases.
Background: The clinical healing environment after a posterior spinal arthrodesis surgery is one of the most clinically challenging bone-healing environments across all orthopedic interventions due to the absence of a contained space and the need to form de novo bone. Our group has previously reported that sclerostin in expressed locally at high levels throughout a developing spinal fusion. However, the role of sclerostin in controlling bone fusion remains to be established. Methods: We computationally identified two FDA-approved drugs, as well as a single novel small-molecule drug, for their ability to disrupt the interaction between sclerostin and its receptor, LRP5/6. The drugs were tested in several in vitro biochemical assays using murine MC3T3 and MSCs, assessing their ability to (1) enhance canonical Wnt signaling, (2) promote the accumulation of the active (non-phosphorylated) form of β-catenin, and (3) enhance the intensity and signaling duration of BMP signaling. These drugs were then tested subcutaneously in rats as standalone osteoinductive agents on plain collagen sponges. Finally, the top drug candidates (called VA1 and C07) were tested in a rabbit posterolateral spine fusion model for their ability to achieve a successful fusion at 6 wk. Results: We show that by controlling GSK3b phosphorylation our three small-molecule inhibitors (SMIs) simultaneously enhance canonical Wnt signaling and potentiate canonical BMP signaling intensity and duration. We also demonstrate that the SMIs produce dose-dependent ectopic mineralization in vivo in rats as well as significantly increase posterolateral spine fusion rates in rabbits in vivo, both as standalone osteogenic drugs and in combination with autologous iliac crest bone graft. Conclusions: Few if any osteogenic small molecules possess the osteoinductive potency of BMP itself - that is, the ability to form de novo ectopic bone as a standalone agent. Herein, we describe two such SMIs that have this unique ability and were shown to induce de novo bone in a stringent in vivo environment. These SMIs may have the potential to be used in novel, cost-effective bone graft substitutes for either achieving spinal fusion or in the healing of critical-sized fracture defects.
Intervertebral disc (IVD) disease (IDD) is a complex, multifactorial disease. While various aspects of IDD progression have been reported, the underlying molecular pathways and transcriptional networks that govern the maintenance of healthy nucleus pulposus (NP) and annulus fibrosus (AF) have not been fully elucidated. We defined the transcriptome map of healthy human IVD by performing single-cell RNA-sequencing (scRNA-seq) in primary AF and NP cells isolated from non-degenerated lumbar disc. Our systematic and comprehensive analyses revealed distinct genetic architecture of human NP and AF compartments and identified 2,196 differentially expressed genes. Gene enrichment analysis showed that SFRP1, BIRC5, CYTL1, ESM1 and CCNB2 genes were highly expressed in the AF cells; whereas, COL2A1, DSC3, COL9A3, COL11A1, and ANGPTL7 were mostly expressed in the NP cells. Further, functional annotation clustering analysis revealed the enrichment of receptor signaling pathways genes in AF cells, while NP cells showed high expression of genes related to the protein synthesis machinery. Subsequent interaction network analysis revealed a structured network of extracellular matrix genes in NP compartments. Our regulatory network analysis identified FOXM1 and KDM4E as signature transcription factor of AF and NP respectively, which might be involved in the regulation of core genes of AF and NP transcriptome.
Purpose of Review: Growing evidence supports the critical role of transcriptional mechanisms in promoting the spatial and temporal progression of bone healing. In this review, we evaluate and discuss new transcriptional and post-transcriptional regulatory mechanisms of secondary bone repair, along with emerging evidence for epigenetic regulation of fracture healing. Recent Findings: Using the candidate gene approach has identified new roles for several transcription factors in mediating the reactive, reparative, and remodeling phases of fracture repair. Further characterization of the different epigenetic controls of fracture healing and fracture-driven transcriptome changes between young and aged fracture has identified key biological pathways that may yield therapeutic targets. Furthermore, exogenously delivered microRNA to post-transcriptionally control gene expression is quickly becoming an area with great therapeutic potential. Summary: Activation of specific transcriptional networks can promote the proper progression of secondary bone healing. Targeting these key factors using small molecules or through microRNA may yield effective therapies to enhance and possibly accelerate fracture healing.