Citations

Stem cell-derived β (SC-β) cells are a promising therapy for type 1 diabetes (T1D), but their long-term efficacy is limited by immune-mediated graft rejection. Platelet-derived exosome product (PEP) has emerged as a novel immunomodulatory agent, although its effect on SC-β cell xenograft rejection remains unclear. Here, we evaluated PEP in stimulated human peripheral blood mononuclear cells (PBMCs) and in an immunocompetent mouse model of SC-β cell transplantation under the kidney capsule. Immune-related gene expression was assessed by quantitative PCR, immune cell infiltration by immunofluorescence, and graft function by circulating human insulin. In vitro, PEP reduced NK-cell-associated gene expression, including NK1.1, EOMES, and KLRK1, and increased IL-10 expression. In vivo, untreated SC-β cell xenografts showed progressive immune infiltration and loss of detectable human insulin by day 14, whereas PEP-treated grafts retained detectable insulin through day 14 across co-transplantation, pretreatment, and systemic administration strategies. PEP co-transplantation delayed, but did not prevent, xenograft rejection, with graft loss observed by day 21. This delay was associated with reduced NK1.1+ cell infiltration and lower expression of selected inflammatory and rejection-associated markers, including NK1.1, Nos2, and Nlrp3. These findings suggest that PEP modulates graft-associated immune responses and may serve as an adjunct immunomodulatory strategy for stem cell-based therapies for type 1 diabetes. Further studies are needed to evaluate sustained graft durability, safety, and translational efficacy.

Objective This proof-of-concept study investigated an improved cell-based injection therapy combining mesenchymal stem cells (MSCs) and meniscus cells (MCs) to support superior meniscus allograft repopulation and early revival compared to injecting MSCs alone. Design In this controlled laboratory study, frozen meniscus allograft samples were injected vertically with a cell suspension containing different ratios of MSCs and MCs or control (lactated ringers) and cultured for 28 days. Samples were analyzed weekly for cell viability, migration, and metabolism using histological and biochemical assays. Tissue medium was analyzed for matrix metalloproteinase (MMP) expression using zymography. Results Cellular repopulation of frozen allografts injected with different cell suspensions was validated by immunohistochemistry. Significant higher DNA content was evidenced in grafts treated with suspensions of MCs or MC:MSC (1:4 ratio). Cell metabolic activity was significantly different between all treated groups and control group after 1 week. Allografts injected with MCs showed significantly more cell proliferation than injections with MSCs. MMP2 activity was detected in medium of all grafts cellularized with MCs with or without MSCs. Scanning electron microscopy (SEM) analysis showed resolution of the needle puncture, but not in the control group. Cell labeling of MCs upon injection of mixed MC:MSC suspensions revealed a gradual increase in the cell ratio. Conclusions The findings of this study establish that injection of MCs with or without MSCs enhances the cellularity of meniscus allograft to support early graft revival and remodeling.

AbstractArthrofibrosis, which causes joint motion restrictions, is a common complication following total knee arthroplasty (TKA). Key features associated with arthrofibrosis include myofibroblast activation, knee stiffness, and excessive scar tissue formation. We previously demonstrated that adiponectin levels are suppressed within the knee tissues of patients affected by arthrofibrosis and showed that AdipoRon, an adiponectin receptor agonist, exhibited anti‐fibrotic properties in human mesenchymal stem cells. In this study, the therapeutic potential of AdipoRon was evaluated on TGFβ1‐mediated myofibroblast differentiation of primary human knee fibroblasts and in a mouse model of knee stiffness. Picrosirius red staining revealed that AdipoRon reduced TGFβ1‐induced collagen deposition in primary knee fibroblasts derived from patients undergoing primary TKA and revision TKA for arthrofibrosis. AdipoRon also reduced mRNA and protein levels of ACTA2, a key myofibroblast marker. RNA‐seq analysis corroborated the anti‐myofibrogenic effects of AdipoRon. In our knee stiffness mouse model, 6 weeks of knee immobilization, to induce a knee contracture, in conjunction with daily vehicle (DMSO) or AdipoRon (1, 5, and 25 mg/kg) via intraperitoneal injections were well tolerated based on animal behavior and weight measurements. Biomechanical testing demonstrated that passive extension angles (PEAs) of experimental knees were similar between vehicle and AdipoRon treatment groups in mice evaluated immediately following immobilization. Interestingly, relative to vehicle‐treated mice, 5 mg/kg AdipoRon therapy improved the PEA of the experimental knees in mice that underwent 4 weeks of knee remobilization following the immobilization and therapy. Together, these studies revealed that AdipoRon may be an effective therapeutic modality for arthrofibrosis.

The bone marrow microenvironment acts as a protective niche for malignant cells. Within the bone marrow microenvironment is the endosteal niche, which is the area where the ossified surface of the bone and marrow cavity interact. The endosteal niche provides the opportunity for hematological malignancies such as acute myeloid leukemia (AML) of the bone marrow to interact with osteoblasts from the bone. To study the interaction between AML cells and osteoblasts, our lab developed a co‐culture model of osteoblasts (MC3T3 osteoblast cell line) and AML cells (KG1a cell line or patient samples from bone marrow aspirates). AML cells expressing high levels of CXCR4 were cultured alone or with osteoblasts and then challenged with SDF‐1, the ligand to CXCR4 and a chemokine readily abundant in the bone marrow microenvironment that our lab has previously shown actually induces apoptosis in AML cells expressing high levels of CXCR4 and that osteoblasts are able to protect AML cells from SDF‐1 induced apoptosis. Our lab now shows that treating osteoblasts with cyclosporine (CSA) prevents their ability to effectively protect AML cells from SDF‐1 induced cell death as assayed by annexin‐V staining of AML cells by flow cytometry. CSA treatment of osteoblasts decreased protein levels of tissue non‐specific alkaline phosphatase (ALPL), a phosphatase necessary for osteoblast mineralization, compared to vehicle control. As such, osteoblasts were treated with control siRNA or ALPL siRNA. CXCR4 expressing AML cells were then co‐cultured with the transfected osteoblasts and challenged with SDF‐1. siRNA knockdown of ALPL in osteoblasts was sufficient to prevent protection of AML cells from the apoptotic stimuli. These studies indicate that osteoblast differentiation plays a role in mediating the protection of AML cells. Therefore, targeting specific differentiation factors in osteoblasts may help to make to bone marrow microenvironment a more hostile environment for AML cells and aid in better malignancy eradication. Support or Funding Information This study was supported by NIH R21 CA194217 (to K.E.H.). R.M.S. is supported by the Mayo Clinic Medical Scientist Training Program Robert L. Howell Physician‐Scientist Scholarship.

Background Superimposition of the metabolic syndrome (MetS) on renovascular disease (RVD) amplifies intrarenal microvascular loss. Extracellular vesicles (EVs) released from adipose tissue‐derived mesenchymal stem/stromal cells (MSCs) may mediate their paracrine effect, but their efficacy to protect the renal microcirculation in RVD complicated by MetS remains unclear. Using a novel swine model, we tested the hypothesis that EVs would attenuate microvascular loss in MetS+RVD. Methods Four groups of pigs (n=7 each) were studied after 16 weeks of diet‐induced MetS and unilateral RVD (MetS+RVD), MetS+RVD treated 4 weeks earlier with a single intra‐renal delivery of pre‐labeled EVs harvested from autologous MSCs, and Lean and MetS Sham controls. Stenotic‐kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were measured in‐vivo (fast CT), whereas EV characteristics, renal microvascular architecture (micro‐CT), and injury pathways ex‐vivo. Results mRNA sequencing of EVs and their parent MSCs revealed that EVs were enriched with pro‐angiogenic genes, including vascular endothelial growth factor (VEGF)‐A, VEGF–C, and Von Willebrand Factor (vWF)‐A Domain Containing‐1 (VWA)‐1 (). In addition, protein expression of VEGF and vWF was higher in EVs compared to MSCs (). EVs were detected in the stenotic‐kidney ex‐vivo internalized by tubular and endothelial cells. EVs restored renal expression of the pro‐angiogenic factors VEGF, Notch‐1, and Notch ligand delta‐like‐4 (DLL4) and improved cortical microvascular density (). Tubulointerstitial fibrosis increased and RBF and GFR decreased in MetS+RVD vs. MetS, but were restored in MetS+RVD+EV (). Conclusions Intra‐renal delivery of MSC‐derived EVs bearing pro‐angiogenic properties in swine MetS+RVD restored stenotic‐kidney microcirculation, and in turn its hemodynamics and function. Our study suggests a novel therapeutic potential for MSC‐derived EVs in restoring renal function in chronic experimental MetS+RVD. Support or Funding Information This study was partly supported by NIH grant numbers DK73608, DK104273, HL123160, and DK102325, DK106427, the Mayo Clinic Center for Regenerative Medicine, and the Mayo Clinic Mary Kathryn and Michael B. Panitch Career Development Award.

ABSTRACT Vitamin C (VitC) is essential for bone health, and low VitC serum levels increase the risk for skeletal fractures. If and how VitC affects bone mineralization is unclear. Using micro-computed tomography (μCT), histologic staining, as well as quantitative backscattered electron imaging (qBEI), we assessed the effects of VitC on femoral structure and microarchitecture, bone formation, and bone mineralization density distribution (BMDD) in the VitC incompetent Gulo−/− mouse model and wild-type mice. In particular, VitC-supplemented, 20-week-old mice were compared with age-matched counterparts where dietary VitC intake was excluded from week 15. VitC depletion in Gulo−/− mice severely reduced cortical thickness of the diaphyseal shaft and bone volume around the growth plate (eg, bone volume of the primary spongiosa −43%, p < 0.001). Loss of VitC also diminished the amount of newly formed bone tissue as visualized by histology and calcein labeling of the active mineralization front. BMDD analysis revealed a shift to higher calcium concentrations upon VitC supplementation, including higher average (~10% increase in female VitC deficient mice, p < 0.001) and peak calcium concentrations in the epiphyseal and metaphyseal spongiosa. These findings suggest higher bone tissue age. Importantly, loss of VitC had significantly more pronounced effects in female mice, indicating a higher sensitivity of their skeleton to VitC deficiency. Our results reveal that VitC plays a key role in bone formation rate, which directly affects mineralization. We propose that low VitC levels may contribute to the higher prevalence of bone-degenerative diseases in females and suggest leveraging this vitamin against these conditions. © 2023 American Society for Bone and Mineral Research (ASBMR). Abstract Vitamin C deficiency disrupts bone microarchitecture, as well as bone formation and mineralization in Gulo−/− mice, which emphasizes the importance of this micronutrient for the skeleton. Importantly, the female skeleton is significantly more sensitive to vitamin C deficiency than the male skeleton, indicating sex-specific differences in vitamin C requirements for bone.

AbstractBackgroundFractional carbon dioxide (CO2) laser resurfacing is used successfully for facial rejuvenation. Post procedure skincare is a variable that influences downtime caused by pain/tenderness, erythema, crusting, and bruising.AimsThe primary objective of this pilot study was to demonstrate the benefits of human platelet extract (HPE) (plated)™ CALM Serum, a new topical cosmetic product, following fractionated CO2 ablative laser resurfacing treatment to the entire face versus standard of care.MethodsIn a single‐center, randomized, evaluator‐blinded pilot study, a total of 18 subjects were randomized into two groups, CO2 facial resurfacing followed by post‐procedural standard of care (Stratacel silicone gel) or CO2 facial resurfacing with the addition of HPE renewosomes in the CALM Serum.ResultsCALM Serum demonstrated statistically significant less crusting at Day 10 compared to the control group (p = 0.0193) with less downtime in the first 14 days (p = 0.03). Subjects treated with CALM Serum had statistically significant brighter appearing skin at 14 days (p = 0.007) and more youthful looking skin on Days 14 and 30 (p = 0.003 and 0.04, respectively).ConclusionsThis study demonstrates that Renewosome™ technology provides statistically significant post‐laser clinical recovery over silicone gel for reducing crusting, and downtime. Subjects reported less diary days of symptoms of pain/tenderness, redness, crusting/flaking, bruising, and itching in the first 14 days compared to the control group. CALM also demonstrated statistically significant improvements in brighter and more youthful appearing skin. CALM is safe and well tolerated.

Abstract Background Chronic injuries to the scapholunate ligament (SLIL) alter carpal kinematics and may progress to early degenerative osteoarthritis. To date, there is no consensus for the best method for SLIL reconstruction. This study aims to assess the use of growth factors (bone morphogenetic protein [BMP]2 and growth and differentiation factor 5 [GDF5]) for compartmentalized regeneration of bone and ligament in this multiphasic scaffold in a rabbit knee model. Case Description A total of 100 µg of BMP2 and 30 µg of GDF5 were encapsulated into a heparinized gelatin–hyaluronic acid hydrogel and loaded into the appropriate compartment of the multiphasic scaffold. The multiphasic scaffold was implanted to replace the native rabbit medial collateral ligament (n = 16). The rabbits were randomly assigned to two different treatment groups. The first group was immobilized postoperatively with the knee pinned in flexion with K-wires for 4 weeks (n = 8) prior to sacrifice. The second group was immobilized for 4 weeks, had the K-wires removed followed by a further 4 weeks of mobilization prior to sample harvesting. Literature Review Heterotopic ossification as early as 4 weeks was noted on gross dissection and confirmed by microcomputed tomography and histological staining. This analysis revealed formation of a bony bridge located within and over the ligament compartment in the intra-articular region. Biomechanical testing showed increased ultimate force of the ligament compartment at 4 weeks postimplantation consistent with the presence of bone formation and higher numbers of scaffold failures at the bone–tendon junction. This study has demonstrated that the addition of BMP2 and GDF5 in the bone–ligament–bone (BLB) scaffold resulted in heterotopic bone formation and failure of the ligament compartment. Clinical Relevance The implantation of a three-dimensional-printed BLB scaffold alone demonstrated superior biomechanical and histological results, and further investigation is needed as a possible clinical reconstruction for the SLIL.