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In: Orthopaedic Journal of Sports Medicine, SAGE Publications, Vol. 10, No. 7_suppl5 ( 2022-07-01), p. 2325967121S0066-

Abstract: Bone morphogenetic proteins (BMPs) belong to the transforming growth factor superfamily that were first discovered by Marshall Urist. There are 14 BMPs identified to date, each with distinct and versatile functional roles. Two pioneering studies compared the effects of 14 different BMPs on bone regeneration using an ectopic bone formation model and found that BMPs 2, 6, 7, and 9 efficiently induce bone formation using an adeno-viral BMP-transduced C2C12 cell line. This study also found that BMP3 can exert an inhibitory effect on bone formation induced by BMP2, 6, and 7, but not BMP9 [1,2]. Our research team previously showed that human muscle derived stem cells (hMDSCs) can promote bone regeneration in critical size calvarial bone defect when transduced with lenti-viral BMP2 [3-5] . However, the gene transduction of stem cells may limit its clinical translation due to safety concerns. Coacervate is a polymer designed to achieve local and sustained release of growth factors including for tissue engineering applications for bone and cartilage repair [6-8]. The aim of this study is to use the coacervate sustained release platform to identify the most potent BMPs for enhancement of bone regeneration in a critical sized calvarial bone defect. Methods: 1. Synthesis of poly (ethylene argininylaspartate diglyceride) (PEAD) was performed as previously described[9]. To form coacervate, 12.5ml FDA approved heparin (2mg/ml) was added to 2µg of BMPs and allowed to bind at least 1 minute and then PEAD was added to the heparin-BMP complex allowing for BMPs to be sustain released. 2. In vivo bone formation using calvarial bone defect model: male ISCRSCID mice were divided into 6 groups (N=6): (1) PBS+coacervate group; (2) 2 µg BMP2+coacervate (3) 2 µg BMP4+coacervate; (4) 2 µg BMP6+coacervate; (5) 2 µg BMP7+coacervate;(6) 2 µg BMP9+coacervate. Fibrin sealant was used as scaffold. Critical size 5mm calvarial bone defects were created on the right parietal bone of mice as previously described [5] . After creation of the defect, each group received respective coacervate BMPs. The coacervate with BMPs or PBS was first mixed with 20ml thrombin, added to the defect, and thereafter, 20ml fibrinogen was added to the defect and allowed 1-2 minutes to form fibrin gel. 3. MicroCT and histology: Bone regeneration was quantified using the Viva-CT 80 at days 1, 14, 28 and 42 respectively. Mice were sacrificed at 6 weeks after surgery and skulls were harvested and fixed in formalin for histology. After decalcification, tissues were paraffin embedded and sectioned. H & E, Herovici’s staining, and TRAP staining were performed to reveal general morphology, collagen type 1 and osteoclasts. Immunohistochemistry was performed for osterix to stain osteogenic progenitor cells. Statistical analysis was performed using one-way analysis of variance using Graphpad Prism 9 followed by Tukey-post hoc pairwise comparisons. P 〈 0.05 was considered statistical difference. Results: MicroCT results showed almost no bone formation in the PBS coacervate group (Fig.1A). All coacervate delivered BMPs groups regenerated new bone in the defect area. The BMP2 and BMP7 groups showed more robust new bone formation than BMPs 4, 6, & 9. However, none of the BMP groups completely healed the critical sized bone defect (Fig.1A). Quantification indicated that all BMP groups regenerated significantly more bone than the PBS group. The BMP2 group regenerated the highest amount of new bone at all time points tested which was approximately 10 folds more bone than that of BMP 4, 6, & 9 groups. BMP2 also regenerated more new bone than the BMP7 group. BMP7 regenerated more bone when compared to the BMP4, 6, & 9 groups and resulted in approximately 3 folds more new bone than the BMP 4, 6, & 7 groups at any time points (Fig.1B). Herovici’s staining showed red collagen I bone matrix in all groups at the edge (1/4 from front of the defect) and middle (1/2 from front of the defect) of the defect. The BMP2 group showed complete healing at the edge and more bone at the middle of the defect. Scar tissues showed blue fiber like structure (Fig.2A). H & E staining demonstrated the new bone is functional trabecular bone with bone matrix while the bone marrow contained cells of the myeloid lineage, red blood cells lineage and megakaryocytes (Fig.2B). Immunohistochemistry revealed OSX + osteogenic progenitor cell on the new bone surface in all BMPs groups. Quantification showed no statistical differences. PBS group showed only residual host bone due to minimal new bone available to analyze (Fig.3A and C). Furthermore, TRAP staining for osteoclasts indicated the new bone formation after BMP treatment indicating a normal bone remodeling. The BMP2 group showed significantly lower TRAP + cells on the bone surface compared to the PBS group (Fig.3B and D). Conclusions: The current study reveals that the use of coacervate to deliver BMPs induced bone formation to different extents in a critical sized calvarial defect. BMP2 and BMP7 were the most potent BMPs in promoting bone formation. The new bone regenerated by the BMP2 group is equivalent to new bone regenerated using lenti-BMP2 transduced human muscle derived stem cells. More importantly, the newly regenerated bones are the same as natural host trabecular bone without any residual material. [Figure: see text] [Figure: see text][Figure: see text]

Type of Medium: Online Resource

ISSN: 2325-9671 , 2325-9671

URL: Article

DOI: 10.1177/2325967121S00668

Language: English

Publisher: SAGE Publications

Publication Date: 2022

detail.hit.zdb_id: 2706251-X

SSG: 31

In: Orthopaedic Journal of Sports Medicine, SAGE Publications, Vol. 10, No. 7_suppl5 ( 2022-07-01), p. 2325967121S0076-

Abstract: It has been reported that pain occurs after the onset of OA and is often associated with inflammatory synovial expression of tumor necrotic factor (TNFα), suggesting that TNFα is one of the main factors causing inflammation, pain and OA development in the joints (1). Inhibition of TNFα could be a potential approach to reduce inflammation in patients with OA. However, most anti-TNFα treatments in clinical trials with antibodies or inhibitors to reduce inflammation in OA have yielded conflicting results (2). It is essential to explore novel and more efficient approaches to modulate the expression of TNFα and inflammation in patients with painful OA. TNFα-induced protein 8-like 2 (TIPE2) was found to regulate the immune system’s homeostasis, and thus regulate inflammation. Zmpste24 deficient mouse (Z24-/-) is a reliable model of human Hutchinson-Gilford progeria. They are incapable of producing lamin A, an essential component of the nuclear envelope, and exhibit profound nuclear architecture abnormalities and multiple histopathological defects that phenocopy an accelerated aging process (3). Z24-/- mice are spontaneously and progressively developed OA around three months old. To investigate the role of TIPE2 on OA in this accelerated aging model, we have performed intra-articular injections of adeno-associated virus(AAV) carrying the TIPE2 gene in the knee of Z24-/- mice. Our results indicate that the overexpression of TIPE2 ameliorates osteoarthritis phenotype through the reduction of inflammation and senescent cells, suggesting that TIPE2 gene delivery may provide a novel anti-inflammatory therapy to alleviate the aging related OA in the clinic. Methods: Construction of the AAV-TIPE2 gene expression vector. Construction was performed as previously described (4). AAV production and titration. Viral production and purification of AAV2 were carried out as previously described (5). The concentration was determined using an AAV titration kit (Takara). The titer for AAV2-TIPE2 was 4.7 × 10⁁12 GCP/ml. Animals. Both male and female Z24-/- (B6.129SZmpste24tm1Sgy/Mmucd) mice at 12 weeks old were used for this study. The animal protocol was approved by the Institutional Animal Welfare Committee at the Colorado State University. TIPE2 treatment. AAV2-TIPE2 (20ul) was intra-articular injected into the right knee joint of Z24-/- mice and the same volume of PBS was injected into their left knee joint, which was used as control. At four weeks post-injection, mice were euthanized, and the knee joints were excised for decalcification, paraffin embedding, sectioning and histology analysis. Histology. H & E and Safranin O staining were performed following the manufacture protocol. The AC damage shown in the Safranin O staining images will be further evaluated using the Mankin scoring system(6). Immune staining. TNF-α and p16 immune staining was performed as the previous description(7). Immunofluorescence images were taken using a Nikon upright microscope. β-Gal staining. β-Gal staining was performed using the senescence staining kit (Cell Signaling). Statistical analysis. All values are expressed as the means ± SD. Statistical analyses were performed using Microsoft Excel software. Data were analyzed by the independent samples t-test compared between 2 groups at each time point using 2 tails. A value of P 〈 0.05 was considered statistically significant. Results: TIPE2 alleviates OA progression by regulating cell inflammatory responses. Since the inflammatory factor TNFα plays a key role in OA progression we first confirmed that TNFα is highly expressed in OA chondrocytes in the knee joint of Z24-/- mice at three months old (Fig.1). As we expected, TIPE2 treatment significantly decreased TNFα expression in chondrocytes of the OA knee (Fig. 1). TIPE2 treatment prevents cartilage degradation. We saw the difference in Safranin O–positive staining intensity in the AC (articular cartilage) of TIPE2 treated mice and PBS control mice. The glycosaminoglycan content (red) is degraded in the Z24-/- cartilage (PBS control), but cartilage degradation was reduced in Z24-/- mice treated with TIPE2 (Fig.2). TIPE2 treatment decreased β-Gal + senescent cells in the OA knee. OA as a whole joint disease, we observed the Z24 -/- mice chondrocytes exhibiting a variety of senescent-associated phenotypes. β-gal staining was observed in a subset of chondrocytes and subchondral bone close to the lesion sites of the OA. TIPE2 alleviates OA progression by targeting senescent cells. We observed that a significant decrease of TNFα positive cells (Fig. 1) and β-Gal positive cells (yellow arrow indicated in Fig.3, # p 〈 0.05) by intra-articular injection of AAV TIPE2. This decrease is further confirmed by the determination of p16 expression in the Z24 -/- mice by TIPE2 treatment (Fig.3, *p 〈 0.03). These findings suggest that TIPE2 can suppress OA progression via targeting inflammation factor TNFα in vivo. Conclusions: OA has become an emerging health challenge that occurs in older people. TNF-α is one of the main factors causing inflammation, pain and OA development in the joints. Here we first report that OA spontaneously occurred in aging joints of Z24-/- mice, and using a TNF-α antagonist, TIPE2, could attenuate OA development by decreasing inflammation through a reduction in the number of senescent cells. It has been reported that cellular senescence and the senescence-associated secretory phenotype (SASP) that drive and promote chronic inflammation in multiple age-related chronic diseases(8). Our results provide evidence that the downregulation of the inflammation factor TNF-α is capable to reduce cell senescence in this OA animal model. In conclusion, our research demonstrated a connection between TNF-α and senescence in aging caused OA. Our results indicate that TIPE2, as a TNF-α regulator, modulates inflammation can be used as a treatment for OA. [Figure: see text][Figure: see text] [Figure: see text]

Type of Medium: Online Resource

ISSN: 2325-9671 , 2325-9671

URL: Article

DOI: 10.1177/2325967121S00767

Language: English

Publisher: SAGE Publications

Publication Date: 2022

detail.hit.zdb_id: 2706251-X

SSG: 31

In: Orthopaedic Journal of Sports Medicine, SAGE Publications, Vol. 10, No. 7_suppl5 ( 2022-07-01), p. 2325967121S0080-

Abstract: Anterior cruciate ligament (ACL) reconstruction is the 6th most common orthopedic procedure performed in the United States (1,2). There is substantial evidence to suggest that muscle weakness significantly contributes to adverse outcomes after ACL injury/reconstruction (3). Despite efforts to improve rehabilitation methods, there are currently no effective strategies for restoring pre-injury muscle strength in ACL-injured limbs. Our team has identified that estrogen-related receptor gamma (ERRγ) is a crucial regulator of paracrine angiogenesis in the skeletal muscle (4). Selective over-expression of ERRγ in the skeletal muscle [ERRGO mice] activates a robust paracrine angiogenic gene program involving myofibrillar induction and secretion of a battery of angiogenic factors resulting in muscle vascularization (4). To determine if muscle ERRγ-driven angiogenesis can mitigate muscle atrophy after ACL injury, we performed ACL injury on the ERRGO mice, as well as age-matched wild-type (WT) littermate control mice. In this model, we found that ERRGO mice with muscle ERRγ overexpression significantly mitigated muscle atrophy compared to WT control mice 4 weeks after ACL injury. This finding strongly suggests that muscle-specific ERRγ activation may reduce muscle atrophy after ACL injury as a consequence of increased muscle angiogenesis. This preventive effect is potentially linked to developing a therapeutic approach to reverse these muscle changes after ACL surgery. Methods:Animals: 12 weeks old male and female ERRGO and WT mice obtained from Dr. Narkar’s laboratory were used for this study. The ACL injury was conducted as previously described (5). We performed ACL injury on the right leg, and the left leg was used as non-injured control. The mice were euthanized four weeks after injury. The muscle tissues were harvested, the gastrocnemius muscle (GM) mass was weighted, flash-frozen in liquid nitrogen-cooled 2-methylbutane, and cryo-sectioned. H & E staining was performed on 10 µm cryosections from GM according to the manufacturer’s instructions. Immunohistochemical staining: The muscle sections were fixed with 4% paraformaldehyde. A Mouse on Mouse kit (Vector) was used for anti-muscle RING-finger protein-1 (MuRF1, marker for muscle atrophy) staining according to the manufacturer’s protocol. Statistical analysis: All results are presented as mean ± standard deviation (SD). Means from ACL injured and non-injured of WT and ERRGO mice were compared using Student’s t-test. Differences were considered statistically significant when the P-value was 〈 0.05. Results: We performed the following experiments to determine if ERRγ overexpression in the muscle can prevent muscle weakness after ACL injury. The ACLs on the right leg of ERRGO and WT mice were excised. 4 weeks after injury, the mice were sacrificed, and muscle tissues were collected for histology analysis. First, we observed that muscles in the hindlimbs of WT mice were atropied, as expected, after ACL injury compared to the muscles in the non-injured hindlimb (Fig.1A). Strikingly, after ACL injury, the hindlimb muscles in ERRGO mice were resiliant to atrophy (Fig.1A). Quantitatively, we found that the gastrocnemius muscles weights were significantly reduced in WT mice after ACL injury compared to the GM weights from the non-injured leg. However, this ACL injury-induced reduction in gastrocnemius weight was not observed in ERRGO mice after ACL injury (Fig.1 B). The myofiber cross-sectional area (CSA) was measured based on the H & E staining on the GM muscle of ERRGO and WT mice to evaluate the muscle atrophy further. We found that the CSA of muscle fibers in WT mice was significantly smaller after ACL injury than in the non-injured control muscle (Fig. 2A, 2C, P 〈 0.05). The average size of muscle fibers was not significantly decreased in the muscle of ERRGO mice after ACL injury compared to non-injured muscle. (Fig. 2A, 2C, P 〉 0.05). Since MuRF1 is a biomarker of myofiber atrophy (6), we evaluated the MuRF1 expression in muscle sections by immunostaining. The result showed an increase in MuRF1 expression in the WT muscle compared to ERRGO muscle after ACL injury (Fig. 2B). Together, those results demonstrated that muscle-specific ERRγ activation mitigates muscle atrophy after ACL injury. Conclusions: Skeletal muscle is adversely affected by the ACL injury, and post-reconstruction recovery is limited by muscle weakness. It has been reported that ERRγ expression in the skeletal muscle directly correlates with vascular density, and ERRγ is highly expressed in well-vascularized muscle beds (4). Based on our preliminary data, we observed that the ERRGO mice with muscle-specific ERRγ activation have the capacity to mitigate the muscle atrophy after ACL injury. As we know, exercise induces muscle angiogenesis, and regular physical activity has been considered a therapeutic modality for preventing aging-related muscle wasting. Although exercise is the primary method for alleviating muscle weakness, many patients cannot achieve the exercise intensity that is necessary to prevent or reverse muscle atrophy. Drugs targeting ERRγ will likely be safe as ERRγ belongs to the nuclear receptor superfamily, which are excellent ‘druggable’ targets with unique ligand-binding pockets that facilitate selective and specific drug design. Future studies will investigate the beneficial effects of ERRγ overexpression in ERRGO mice on muscle atrophy after ACL injury at different time points and determine if muscle-specific activation of ERRγ can mitigate age-related muscle progenitor cells dysfunction and offset the infiltration and activation of FAPs and senescent cells after ACL injury

Type of Medium: Online Resource

ISSN: 2325-9671 , 2325-9671

URL: Article

DOI: 10.1177/2325967121S00808

Language: English

Publisher: SAGE Publications

Publication Date: 2022

detail.hit.zdb_id: 2706251-X

SSG: 31

In: Orthopaedic Journal of Sports Medicine, SAGE Publications, Vol. 10, No. 7_suppl5 ( 2022-07-01), p. 2325967121S0060-

Abstract: Microfracture (MFx) technique is the most commonly used first-line treatment for cartilage injuries; however, it has been shown to have inferior long-term clinical outcomes as the repaired tissue is predominantly fibrocartilage. Bone Marrow Aspirate Concentrate (BMAC) treatment has been shown to enhance the healing ability of cartilage repair, superior to MFx treatment alone, although chondral defect filling was achieved with fibrocartilage or “hyaline-like” cartilage. Therefore, new therapeutic strategies to further improve cartilage healing following defects are in need. Fisetin (FIS) is a compound with antioxidant, anti-inflammatory, and senolytic activity capable of eliminating senescent cells systemically. Previous studies have reported that FIS attenuates the progression of osteoarthritis and osteoporosis in aged mice, however, whether FIS treatment improves the quality of repaired cartilage in MFx-treated acute osteochondral defects augmented with BMAC has not yet been investigated. We hypothesized that FIS or autologous BMAC, or a combination of the two, would enhance MFx procedure both histologically and mechanically in the repair of osteochondral defects in a rabbit model. Methods: All surgical procedures were performed by an experienced orthopaedic surgeon and followed Institutional IACUC approved protocols. Sixty-four skeletally mature New Zealand White rabbits at seven months old were used in this study. Animal procedure: Before surgery, bone marrow aspirate was collected through the iliac crests in each rabbit under anesthesia and processed via a two-step centrifugation method to prepare BMAC. After exposing the bilateral knee joints through the medial parapatellar approach, osteochondral defects (diameter: 5 mm, depth: 2 mm) were created bilaterally in the patellar groove of each rabbit, followed by the MFx procedure (5 holes with 2 mm depth) to allow bleeding at each MFx hole as previously described (Fig.1). BMACs were injected into the left knee joint as an autograft immediately after closing the joint capsule in all rabbits, with the right knee as a control (no BMAC transplantation). Rabbits were then randomly divided into 4 groups (N=8/group): MFx alone, MFx+FIS, MFx+BMAC and MFx+FIS+BMAC. FIS-treated rabbits were given FIS orally via drinking water at a dose of 20 mg/kg/day daily from immediately after surgery until euthanasia. Rabbits were sacrificed at 6 and 12 weeks post-op. The macroscopic appearance was evaluated using the International Cartilage Repair Society (ICRS) macroscopic assessment grading. ΜicroCT and histology: Microcomputed tomography (μCT) was performed to evaluate subchondral bone healing. Cartilage healing was assessed histologically on de-calcified tissue at 6 and 12 weeks post-op (H & E, Safranin O, and Alcian blue) and with immunohistochemistry for collagen II and p16. Regenerated cartilage was scored using the Modified O’Driscoll ICRS grading system (max 27 points). Biomechanical tests: The strength of the regenerated cartilage was analyzed by measuring the instantaneous elastic modulus of the regenerated cartilage in each group of samples collected at 12 weeks. (N=6/group). Results: Macroscopic assessment and μCT: At both 6 and 12 weeks postoperatively, MFx+BMAC and MFx+FIS+BMAC groups scored significantly higher than MFx alone group in the ICRS macroscopic evaluation. ( p 〈 0.01, Fig. 2 A, D and Fig. 3 A, D). At both 6- and 12-week time points after surgery, μCT showed favorable healing of the bone defect in MFx+FIS, MFx+BMAC, and MFx+FIS+BMAC groups compared to MFx alone group. (Fig. 2 B and Fig. 3 B). Histology: At both 6- and 12- week time points, the Modified O’Driscoll score was significantly higher in the MFx+BMAC and MFx+FIS+BMAC groups than in the MFx alone group ( p 〈 0.01), and at 12 weeks, the MFx+FIS group had a significantly higher score than the MFx alone group. ( p 〈 0.05, Fig. 2E and Fig. 3E). In addition, immunohistochemistry showed stronger staining of type II collagen (brown) in the MFx+FIS, MFx+BMAC, and MFx+FIS+BMAC groups than in the MFx alone group at both time points, with a stronger reduction in staining of the cellular senescence marker p16 (brown) in FIS-treated group compared to MFx alone or MFx+BMAC group. (Fig. 2C and Fig. 3C). Biomechanical analysis: The instantaneous elastic modulus (cartilage’s strength) was significantly increased in the MFx+FIS+BMAC group compared to MFx alone group. ( p 〈 0.05, Fig. 4). Gene expression analyses : qPCR showed the expression level of SOD1 in synovium was significantly higher in the MFx+FIS group at 6 weeks ( p 〈 0.01) and in the MFx+FIS+BMAC group at 12 weeks ( p 〈 0.05) compared to the MFx alone group. Conclusions: Our results showed that BMAC treatment enhanced the healing of MFx-treated osteochondral defects, macroscopically, biomechanically, and histologically, compared to MFx alone. Furthermore, FIS treatment improved MFx-treated cartilage repair, and its combined use with BMAC led to significantly higher quality cartilage regeneration with stronger mechanical properties. Oxidative stress, which is one of the inducers of cell senescence, has been noted as the primary factor contributing to age-related changes in cartilage homeostasis, function, and response to injury. Given the increase in SOD1 expression commensurate with p16 reduction in the FIS-treated group (Figs 2-3), our results suggest that FIS may improve cartilage healing via reducing cellular senescence. These results support the clinical use of FIS combined with BMAC to enhance the effect of MFx in the repair of osteochondral defects and highlight cellular senescence as a novel therapeutic target for cartilage repair following injury. [Figure: see text][Figure: see text] [Figure: see text][Figure: see text]

Type of Medium: Online Resource

ISSN: 2325-9671 , 2325-9671

URL: Article

DOI: 10.1177/2325967121S00604

Language: English

Publisher: SAGE Publications

Publication Date: 2022

detail.hit.zdb_id: 2706251-X

SSG: 31

In: Stem Cell Research & Therapy, Springer Science and Business Media LLC, Vol. 10, No. 1 ( 2019-12)

Abstract: Osteoarthritis and cartilage injury treatment is an unmet clinical need. Therefore, development of new approaches to treat these diseases is critically needed. Previous work in our laboratory has shown that murine muscle-derived stem cells (MDSCs) can efficiently repair articular cartilage in an osteochondral and osteoarthritis model. However, the cartilage repair capacity of human muscle-derived stem cells has not been studied which prompt this study. Method In this study, we tested the in vitro chondrogenesis ability of six populations of human muscle-derived stem cells (hMDSCs), before and after lenti-BMP2/GFP transduction using pellet culture and evaluated chondrogenic differentiation of via histology and Raman spectroscopy. We further compared the in vivo articular cartilage repair of hMDSCs stimulated with BMP2 delivered through coacervate sustain release technology and lenti-viral gene therapy-mediated gene delivery in a monoiodoacetate (MIA)-induced osteoarthritis (OA) model. We used microCT and histology to evaluate the cartilage repair. Results We observed that all hMDSCs were able to undergo chondrogenic differentiation in vitro. As expected, lenti-BMP2/GFP transduction further enhanced the chondrogenic differentiation capacities of hMDSCs, as confirmed by Alcian blue and Col2A1staining as well as Raman spectroscopy analysis. We observed through micro-CT scanning, Col2A1 staining, and histological analyses that delivery of BMP2 with coacervate could achieve a similar articular cartilage repair to that mediated by hMDSC-LBMP2/GFP. We also found that the addition of soluble fms-like tyrosine kinase-1 (sFLT-1) protein further improved the regenerative potential of hMDSCs/BMP2 delivered through the coacervate sustain release technology. Donor cells did not primarily contribute to the repaired articular cartilage since most of the repair cells are host derived as indicated by GFP staining. Conclusions We conclude that the delivery of hMDSCs and BMP2 with the coacervate technology can achieve a similar cartilage repair relative to lenti-BMP2/GFP-mediated gene therapy. The use of coacervate technology to deliver BMP2/sFLT1 with hMDSCs for cartilage repair holds promise for possible clinical translation into an effective treatment modality for osteoarthritis and traumatic cartilage injury.

Type of Medium: Online Resource

ISSN: 1757-6512

URL: Article

DOI: 10.1186/s13287-019-1434-3

Language: English

Publisher: Springer Science and Business Media LLC

Publication Date: 2019

detail.hit.zdb_id: 2548671-8

In: Antioxidants, MDPI AG, Vol. 12, No. 8 ( 2023-08-21), p. 1646-

Abstract: Fisetin has been shown to be beneficial for brain injury and age-related brain disease via different mechanisms. The purpose of this study was to determine the presence of senescent cells and the effects of fisetin on cellular senescence in the brain and other vital organs in old sheep, a more translational model. Female sheep 6–7 years old (N = 6) were treated with 100 mg/kg fisetin or vehicle alone on two consecutive days a week for 8 weeks. All vital organs were harvested at the time of sacrifice. Histology, immunofluorescence staining, and RT-Q-PCR were performed on different regions of brain tissues and other organs. Our results indicated that fisetin treatment at the current regimen did not affect the general morphology of the brain. The presence of senescent cells in both the cerebral brain cortex and cerebellum and non-Cornu Ammonis (CA) area of the hippocampus was detected by senescent-associated β-galactosidase (SA-β-Gal) staining and GL13 (lipofuscin) staining. The senescent cells detected were mainly neurons in both gray and white matter of either the cerebral brain cortex, cerebellum, or non-CA area of the hippocampus. Very few senescent cells were detected in the neurons of the CA1-4 area of the hippocampus, as revealed by GL13 staining and GLB1 colocalization with NEUN. Fisetin treatment significantly decreased the number of SA-β-Gal+ cells in brain cortex white matter and GL13+ cells in the non-CA area of the hippocampus, and showed a decreasing trend of SA-β-Gal+ cells in the gray matter of both the cerebral brain cortex and cerebellum. Furthermore, fisetin treatment significantly decreased P16+ and GLB1+ cells in neuronal nuclear protein (NEUN)+ neurons, glial fibrillary acidic protein (GFAP)+ astrocytes, and ionized calcium binding adaptor molecule 1 (IBA1)+ microglia cells in both gray and white matter of cerebral brain cortex. Fisetin treatment significantly decreased GLB1+ cells in microglia cells, astrocytes, and NEUN+ neurons in the non-CA area of the hippocampus. Fisetin treatment significantly decreased plasma S100B. At the mRNA level, fisetin significantly downregulated GLB1 in the liver, showed a decreasing trend in GLB1 in the lung, heart, and spleen tissues, and significantly decreased P21 expression in the liver and lung. Fisetin treatment significantly decreased TREM2 in the lung tissues and showed a trend of downregulation in the liver, spleen, and heart. A significant decrease in NRLP3 in the liver was observed after fisetin treatment. Finally, fisetin treatment significantly downregulated SOD1 in the liver and spleen while upregulating CAT in the spleen. In conclusion, we found that senescent cells were widely present in the cerebral brain cortex and cerebellum and non-CA area of the hippocampus of old sheep. Fisetin treatment significantly decreased senescent neurons, astrocytes, and microglia in both gray and white matter of the cerebral brain cortex and non-CA area of the hippocampus. In addition, fisetin treatment decreased senescent gene expressions and inflammasomes in other organs, such as the lung and the liver. Fisetin treatment represents a promising therapeutic strategy for age-related diseases.

Type of Medium: Online Resource

ISSN: 2076-3921

URL: Article

DOI: 10.3390/antiox12081646

Language: English

Publisher: MDPI AG

Publication Date: 2023

detail.hit.zdb_id: 2704216-9

SSG: 15,3

In: Sensors, MDPI AG, Vol. 22, No. 16 ( 2022-08-19), p. 6233-

Abstract: There is an unmet need for improved, clinically relevant methods to longitudinally quantify bone healing during fracture care. Here we develop a smart bone plate to wirelessly monitor healing utilizing electrical impedance spectroscopy (EIS) to provide real-time data on tissue composition within the fracture callus. To validate our technology, we created a 1-mm rabbit tibial defect and fixed the bone with a standard veterinary plate modified with a custom-designed housing that included two impedance sensors capable of wireless transmission. Impedance magnitude and phase measurements were transmitted every 48 h for up to 10 weeks. Bone healing was assessed by X-ray, µCT, and histology. Our results indicated the sensors successfully incorporated into the fracture callus and did not impede repair. Electrical impedance, resistance, and reactance increased steadily from weeks 3 to 7—corresponding to the transition from hematoma to cartilage to bone within the fracture gap—then plateaued as the bone began to consolidate. These three electrical readings significantly correlated with traditional measurements of bone healing and successfully distinguished between union and not-healed fractures, with the strongest relationship found with impedance magnitude. These results suggest that our EIS smart bone plate can provide continuous and highly sensitive quantitative tissue measurements throughout the course of fracture healing to better guide personalized clinical care.

Type of Medium: Online Resource

ISSN: 1424-8220

URL: Article

DOI: 10.3390/s22166233

Language: English

Publisher: MDPI AG

Publication Date: 2022

detail.hit.zdb_id: 2052857-7

In: Biomaterials, Elsevier BV, Vol. 288 ( 2022-09), p. 121708-

Type of Medium: Online Resource

ISSN: 0142-9612

URL: Article

DOI: 10.1016/j.biomaterials.2022.121708

Language: English

Publisher: Elsevier BV

Publication Date: 2022

detail.hit.zdb_id: 2004010-6

SSG: 12

In: Scientific Reports, Springer Science and Business Media LLC, Vol. 10, No. 1 ( 2020-04-27)

Abstract: Duchenne muscular dystrophy (DMD) is a progressive muscle disease, characterized by mutations in the X-linked dystrophin, that has several therapeutic options but no curative treatment. Transplantation of muscle progenitor cells for treatment of DMD has been widely investigated; however, its application is hindered by limited cell survival due to the harmful dystrophic microenvironment. An alternative approach to utilize progenitor cells and circulatory factors and to improve the dystrophic muscle pathology and microenvironment is through parabiotic pairing, where mice are surgically sutured to create a joint circulatory system. Parabiotic mice were generated by surgically joining wild type (WT) mice expressing green fluorescent protein (GFP) with mdx mice. These mice developed a common circulation (approximately 50% green cells in the blood of mdx mice) 2-weeks after parabiotic pairing. We observed significantly improved dystrophic muscle pathology, including decreased inflammation, necrotic fibers and fibrosis in heterogenetic parabionts. Importantly, the GFP + cells isolated from the mdx mice (paired with GFP mice) underwent myogenic differentiation in vitro and expressed markers of mesenchymal stem cells and macrophages, which may potentially be involved in the improvement of dystrophic muscle pathology. These observations suggest that changing the dystrophic microenvironment can be a new approach to treat DMD.

Type of Medium: Online Resource

ISSN: 2045-2322

URL: Article

DOI: 10.1038/s41598-020-64042-z

Language: English

Publisher: Springer Science and Business Media LLC

Publication Date: 2020

detail.hit.zdb_id: 2615211-3

In: Stem Cells, Oxford University Press (OUP), Vol. 41, No. 7 ( 2023-07-14), p. 698-710

Abstract: Mesenchymal stem cells (MSCs) have long been viewed as a promising therapeutic for musculoskeletal repair. However, regulatory concerns including tumorgenicity, inconsistencies in preparation techniques, donor-to-donor variability, and the accumulation of senescence during culture expansion have hindered the clinical application of MSCs. Senescence is a driving mechanism for MSC dysfunction with advancing age. Often characterized by increased reactive oxygen species, senescence-associated heterochromatin foci, inflammatory cytokine secretion, and reduced proliferative capacity, senescence directly inhibits MSCs efficacy as a therapeutic for musculoskeletal regeneration. Furthermore, autologous delivery of senescent MSCs can further induce disease and aging progression through the secretion of the senescence-associated secretory phenotype (SASP) and mitigate the regenerative potential of MSCs. To alleviate these issues, the use of senolytic agents to selectively clear senescent cell populations has become popular. However, their benefits to attenuating senescence accumulation in human MSCs during the culture expansion process have not yet been elucidated. To address this, we analyzed markers of senescence during the expansion of human primary adipose-derived stem cells (ADSCs), a population of fat-resident MSCs commonly used in regenerative medicine applications. Next, we used the senolytic agent fisetin to determine if we can reduce these markers of senescence within our culture-expanded ADSC populations. Our results indicate that ADSCs acquire common markers of cellular senescence including increased reactive oxygen species, senescence-associated β-galactosidase, and senescence-associated heterochromatin foci. Furthermore, we found that the senolytic agent fisetin works in a dose-dependent manner and selectively attenuates these markers of senescence while maintaining the differentiation potential of the expanded ADSCs.

Type of Medium: Online Resource

ISSN: 1066-5099 , 1549-4918

URL: Article

DOI: 10.1093/stmcls/sxad036

Language: English

Publisher: Oxford University Press (OUP)

Publication Date: 2023

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SSG: 12