Search

Category: Basic Sciences/Biologics; Ankle Arthritis Introduction/Purpose: Arthrodesis stands as the primary method for treating advanced osteoarthritis in the foot and ankle [1]. However, a significant hurdle in this procedure is non-union occurrence, emphasizing the need for adjuncts to enhance osteogenesis and ensure successful fusion. Platelet-Derived Growth Factor (PDGF) emerges as the only clinically approved bone graft substitute for ankle and hindfoot fusion. Bone marrow stimulation, crucial for recruiting cells to aid healing in arthrodesis, is a standard step in all fusions. While preclinical studies on PDGF have mainly focused on human cell lines, the in vitro impact of PDGF on marrow-derived cells, crucial for arthrodesis sites, remains unexplored. This study aims to assess the osteogenic potential of PDGF on heterogeneous marrow-derived cells in both 2D and 3D environments. Methods: Marrow-derived cells (MDCs) from young bovine femoral condyle were utilized without sorting or filtering, to maintain heterogeneity. For 2D assays, MDCs were plated at 10,000 cells per well and cultured in osteogenic media. Both alizarin staining and alkaline phosphatase activity were assessed at 14 days. For 3D assays, we cultured MDCs in fibrin gels at 2M/mL. in osteogenic media for 28 days. For 3D analysis, micro CT and alizarin red staining were performed on fibrin gels. Results: In 2D in vitro analysis, PDGF demonstrated significant increase in alizarin red staining and ALP activity [Figure 1A, 1B]. In 3D analysis, PDGF showed no significant increase in and has trended toward a decrease in alizarin red staining and in micro CT [Figure 1C, 1D]. Conclusion: While many studies have examined the impact of PDGF on human cell lines, this is the first work to analyze the effects of PDGF in a representative distal extremity fusion environment. In our current study, in 2D PDGF demonstrates significant osteoinducton, while in 3D, no increase is seen and a trend towards decreased osteogenic function was observed. Our findings reveal that the differential effects of PDGF in 2D and 3D which may vary depending on the surrounding environment. This work highlights the need for further studies to fully grasp how PDGF functions in an arthrodesis.

Category: Basic Sciences/Biologics; Ankle Introduction/Purpose: One of the most common procedures for treating end-stage osteoarthritis of the foot and ankle is arthrodesis [1]. However, a significant complication with arthrodesis is the development of non-union. Thus, identification of adjuvants to enhance osteogenesis are paramount to fusion success. One possible molecule is the immunosuppressant FK506 (Tacrolimus). Previous research [2] demonstrated FK506 as a stand-alone small molecule capable of initiating osteogenesis and bone formation. The purpose of this study was to evaluate the osteogenic potential of FK506 alone and in combination with other bioactive factors to provide a foundation for future in vivo and clinical applications. Methods: Marrow-derived cells (MDCs) were isolated from juvenile bovine femoral condyles, to represent cells present at the ankle fusion site. MDCs (P1) were seeded at 50k cells per well (24-well plate). After 14 days, the wells were stained for calcium deposits using Alizarin Red S, and imaged with brightfield microscopy to visualize calcium deposits. Drug combinations of potentially osteogenic small molecules were tested (simvastatin, platelet derived growth factor [PDGF], tamoxifen, triiodothyronine). The highest alizarin red absorbance values were used to choose the best drug combination. These combinations were further evaluated for gene expression by RT-qPCR [Osteocalcin (OCN), Osteopontin (OPN), and Bone Sialoprotein (IBSP)]. To simulate a 3D osteogenic environment, cells were seeded into fibrin gels (to mimic the “fusion clot”), cultured for 14 days, and sectioned/stained with Alizarin red. Results: FK506 and PDGF produced the highest level of calcium staining compared to other bioactive factors, highlighted via heatmap of alizarin red absorbance values (Figure 1A). Compared to the control, FK506, PDGF, and the combination of both all resulted in enhanced alizarin red staining (Figure 1A/B). Overall, FK506 exhibited an increased expression across all genes. PDGF and the combination of FK506 and PDGF had a more varied expression within the genes queried (Figure 1C). Finally, in a fibrin gel system, variability was observed between gels of the same group. Displaying the best, middle, and worst sections give a comprehensive view of the explained variability. Across the sections, the FK506-PDGF combination showed the densest mineralization, highlighted by the dark maroon regions of the section (Figure 1D). Conclusion: This study confirms that FK506 demonstrates osteogenesis within marrow-derived cells (representative of a fusion site). The combination of FK506 to PDGF may enhance PDGF’s known healing potential within arthrodesis applications. This work provides the foundation for future clinical applications of FK506. Ongoing studies are focused on the effect of FK506, and potentially in combination with PDGF, in an rabbit ankle fusion model to evaluate efficacy and bone formation. The authors thank the American Orthopaedic Foot and Ankle Society (AOFAS) and Emory Department of Orthopaedics for their support and funding. References: [1] Park 2022. [2] Sangadala 2019

Abstract Environmental cues and internal states such as mood, reward, or aversion directly influence feeding behaviors beyond homeostatic necessity. The hypothalamus has been extensively investigated for its role in homeostatic feeding. However, many of the neural circuits that drive more complex, non-homeostatic feeding that integrate valence and sensory cues (such as taste and smell) remain unknown. Here, we describe a basal forebrain (BF)-to-lateral habenula (LHb) circuit that directly modulates non-homeostatic feeding behavior. Using viral-mediated circuit mapping, we identified a population of glutamatergic neurons within the BF that project to the LHb, which responds to diverse sensory cues, including aversive and food-related odors. Optogenetic activation of BF-to-LHb circuitry drives robust, reflexive-like aversion. Furthermore, activation of this circuitry suppresses the drive to eat in a fasted state. Together, these data reveal a role of basal forebrain glutamatergic neurons in modulating LHb-associated aversion and feeding behaviors by sensing environmental cues.

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.

Meniscal tearing in the knee increases the risk of post-traumatic osteoarthritis (OA) in patients. The therapeutic application of tissue-specific mesenchymal progenitor cells is currently being investigated as an emerging biologic strategy to help improve healing of musculoskeletal tissues like meniscal fibrocartilage and articular hyaline cartilage. However, many of these approaches involve isolating cells from healthy tissues, and the low yield of rare progenitor populations (< 1% of total cells residing in tissues) can make finding a readily available cell source for therapeutic use a significant logistical challenge. In the present study, we investigated the therapeutic efficacy of using expanded cartilage-derived and bone marrow-derived progenitor cell lines, which were stabilized using retroviral SV40, for repair of meniscus injury in a rodent model. Our findings indicate that these cell lines express the same cell surface marker phenotype of primary cells (CD54+, CD90+, CD105+, CD166+), and that they exhibit improved proliferative capacity that is suitable for extensive expansion. Skeletally mature male athymic rats treated with 3.2 million cartilage-derived progenitor cell line exhibited approximately 79% greater meniscal tear reintegration/healing, compared to injured animals that left untreated, and 76% greater compared to animals treated with the same number of marrow-derived stromal cells. Histological analysis of articular surfaces also showed that cartilage-derived progenitor cell line treated animals exhibited reduced post-traumatic OA associated articular cartilage degeneration. Stable cell line treatment did not cause tumor formation or off-target engraftment in animals. Taken together, we present a proof-of-concept study demonstrating, for the first time, that intra-articular injection of a stable human cartilage-derived progenitor cell line stimulates meniscus tear healing and provide chondroprotection in an animal model. These outcomes suggest that the use of stable cell lines may help overcome cell source limitations for cell-based medicine.

Standard assessment of cartilage repair progression by visual arthroscopy can be subjective and may result in suboptimal evaluation. Visible–near infrared (Vis-NIR) fiber optic spectroscopy of joint tissues, including articular cartilage and subchondral bone, provides an objective approach for quantitative assessment of tissue composition. Here, we applied this technique in the 350–2,500 nm spectral region to identify spectral markers of osteochondral tissue during repair with the overarching goal of developing a new approach to monitor repair of cartilage defects in vivo. Full thickness chondral defects were created in Yucatan minipigs using a 5-mm biopsy punch, and microfracture (MFx) was performed as a standard technique to facilitate repair. Tissues were evaluated at 1 month (in adult pigs) and 3 months (in juvenile pigs) post-surgery by spectroscopy and histology. After euthanasia, Vis-NIR spectra were collected in situ from the defect region. Additional spectroscopy experiments were carried out in vitro to aid in spectral interpretation. Osteochondral tissues were dissected from the joint and evaluated using the conventional International Cartilage Repair Society (ICRS) II histological scoring system, which showed lower scores for the 1-month than the 3-month repair tissues. In the visible spectral region, hemoglobin absorbances at 540 and 570 nm were significantly higher in spectra from 1-month repair tissue than 3-month repair tissue, indicating a reduction of blood in the more mature repair tissue. In the NIR region, we observed qualitative differences between the two groups in spectra taken from the defect, but differences did not reach significance. Furthermore, spectral data also indicated that the hydrated environment of the joint tissue may interfere with evaluation of tissue water absorbances in the NIR region. Together, these data provide support for further investigation of the visible spectral region for assessment of longitudinal repair of cartilage defects, which would enable assessment during routine arthroscopy, particularly in a hydrated environment.