A bioactive supramolecular and covalent polymer scaffold for cartilage repair in a sheep model.

Regeneration of hyaline cartilage in human-sized joints remains a clinical challenge, and it is a critical unmet need that would contribute to longer healthspans. Injectable scaffolds for cartilage repair that integrate both bioactivity and sufficiently robust physical properties to withstand joint stresses offer a promising strategy. We report here on a hybrid biomaterial that combines a bioactive peptide amphiphile supramolecular polymer that specifically binds the chondrogenic cytokine transforming growth factor ß-1 (TGFß-1) and crosslinked hyaluronic acid microgels that drive formation of filament bundles, a hierarchical motif common in natural musculoskeletal tissues. The scaffold is an injectable slurry that generates a porous rubbery material when exposed to calcium ions once placed in cartilage defects. The hybrid material was found to support in vitro chondrogenic differentiation of encapsulated stem cells in response to sustained delivery of TGFß-1. Using a sheep model, we implanted the scaffold in shallow osteochondral defects and found it can remain localized in mechanically active joints. Evaluation of resected joints showed significantly improved repair of hyaline cartilage in osteochondral defects injected with the scaffold relative to defects injected with the growth factor alone, including implantation in the load-bearing femoral condyle. These results demonstrate the potential of the hybrid biomimetic scaffold as a niche to favor cartilage repair in mechanically active joints using a clinically relevant large-animal model.

Fisetin Attenuates Cellular Senescence Accumulation During Culture Expansion of Human Adipose-Derived Stem Cells.

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 ß-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.

Losartan in Combination With Bone Marrow Stimulation Showed Synergistic Effects on Load to Failure and Tendon Matrix Organization in a Rabbit Model.

PURPOSE: To investigate the effects of combining bone marrow stimulation (BMS) with oral losartan to block transforming growth factor ß1 (TGF-ß1) on biomechanical repair strength in a rabbit chronic injury model. METHODS: Forty rabbits were randomly allocated into 4 groups (10 in each group). The supraspinatus tendon was detached and left alone for 6 weeks to establish a rabbit chronic injury model and was then repaired in a surgical procedure using a transosseous, linked, crossing repair construct. The animals were divided into the following groups: control group (group C), surgical repair only; BMS group (group B), surgical repair with BMS of the tuberosity; losartan group (group L), surgical repair plus oral losartan (TGF-ß1 blocker) for 8 weeks; and BMS-plus-losartan group (group BL), surgical repair plus BMS plus oral losartan for 8 weeks. At 8 weeks after repair, biomechanical and histologic evaluations were performed. RESULTS: The biomechanical testing results showed significantly higher ultimate load to failure in group BL than in group B (P = .029) but not compared with group C or group L. A 2 × 2 analysis-of-variance model found that the effect of losartan on ultimate load significantly depended on whether BMS was performed (interaction term F1,28 = 5.78, P = .018). No difference was found between the other groups. No difference in stiffness was found between any groups. On histologic assessment, groups B, L, and BL showed improved tendon morphology and an organized type I collagen matrix with less type III collagen compared with group C. Group BL showed the most highly organized tendon matrix with more type I collagen and less type III collagen, which indicates less fibrosis. Similar results were found at the bone-tendon interface. CONCLUSIONS: Rotator cuff repair combined with oral losartan and BMS of the greater tuberosity showed improved pullout strength and a highly organized tendon matrix in this rabbit chronic injury model. CLINICAL RELEVANCE: Tendon healing or scarring is accompanied by the formation of fibrosis, which has been shown to result in compromised biomechanical properties, and is therefore a potential limiting factor in healing after rotator cuff repair. TGF-ß1 expression has been shown to play an important role in the formation of fibrosis. Recent studies focusing on muscle healing and cartilage repair have found that the downregulation of TGF-ß1 by losartan intake can reduce fibrosis and improve tissue regeneration in animal models.

Enhancement of myogenic potential of muscle progenitor cells and muscle healing during pregnancy.

The decline of muscle regenerative potential with age has been attributed to a diminished responsiveness of muscle progenitor cells (MPCs). Heterochronic parabiosis has been used as a model to study the effects of aging on stem cells and their niches. These studies have demonstrated that, by exposing old mice to a young systemic environment, aged progenitor cells can be rejuvenated. One interesting idea is that pregnancy represents a unique biological model of a naturally shared circulatory system between developing and mature organisms. To test this hypothesis, we evaluated the muscle regeneration potential of pregnant mice using a cardiotoxin (CTX) injury mouse model. Our results indicate that the pregnant mice demonstrate accelerated muscle healing compared to nonpregnant control mice following muscle injury based on improved muscle histology, superior muscle regeneration, and a reduction in inflammation and necrosis. Additionally, we found that MPCs isolated from pregnant mice display a significant improvement of myogenic differentiation capacity in vitro and muscle regeneration in vivo when compared to the MPCs from nonpregnant mice. Furthermore, MPCs from nonpregnant mice display enhanced myogenic capacity when cultured in the presence of serum obtained from pregnant mice. Our proteomics data from these studies provides potential therapeutic targets to enhance the myogenic potential of progenitor cells and muscle repair.

The effects of fisetin on bone and cartilage: A systematic review.

As the worldwide population progresses in age, there is an increasing need for effective treatments for age-associated musculoskeletal conditions such as osteoporosis and osteoarthritis (OA). Fisetin, a natural flavonoid, has garnered attention as a promising pharmaceutical option for treating or delaying the progression of osteoporosis and OA. However, there is no systematic review of the effects of fisetin on bone and cartilage. The aim of this review is to report the latest evidence on the effects of fisetin on bone and cartilage, with a focus on clinical significance. The PubMed, Embase, and Cochrane Library databases were searched up to December 9th 2021 to evaluate the effects of fisetin on bone and cartilage in in vitro studies and in vivo preclinical animal studies. The risk of bias, quality, study design, sample characteristics, dose and duration of fisetin treatment, and outcomes of the 13 eligible studies were analyzed in this systematic review. Qualitative evaluation was conducted for each study due to differences in animal species, cell type, created disease model, dose and duration of fisetin treatment, and time between intervention and assessment among the eligible studies. The beneficial effects of fisetin on osteoporosis have been demonstrated in in vitro and in vivo preclinical studies across animal species. Similarly, the beneficial effects of fisetin on OA have been demonstrated in in vivo preclinical animal studies, but the reports on OA are still limited. Fisetin, a natural supplement can be use in orthobiologics treatment, as adjuvant to orthopaedic surgery, to improve clinical outcome.

Effect of Oral Losartan on Orthobiologics: Implications for Platelet-Rich Plasma and Bone Marrow Concentrate-A Rabbit Study.

Recent efforts have focused on customizing orthobiologics, such as platelet-rich plasma (PRP) and bone marrow concentrate (BMC), to improve tissue repair. We hypothesized that oral losartan (a TGF-ß1 blocker with anti-fibrotic properties) could decrease TGF-ß1 levels in leukocyte-poor PRP (LP-PRP) and fibrocytes in BMC. Ten rabbits were randomized into two groups (N = 5/group): osteochondral defect + microfracture (control, group 1) and osteochondral defect + microfracture + losartan (losartan, group 2). For group 2, a dose of 10mg/kg/day of losartan was administrated orally for 12 weeks post-operatively. After 12 weeks, whole blood (WB) and bone marrow aspirate (BMA) samples were collected to process LP-PRP and BMC. TGF-ß1 concentrations were measured in WB and LP-PRP with multiplex immunoassay. BMC cell populations were analyzed by flow cytometry with CD31, CD44, CD45, CD34, CD146 and CD90 antibodies. There was no significant difference in TGF-ß1 levels between the losartan and control group in WB or LP-PRP. In BMC, the percentage of CD31+ cells (endothelial cells) in the losartan group was significantly higher than the control group (p = 0.008), while the percentage of CD45+ cells (hematopoietic cells-fibrocytes) in the losartan group was significantly lower than the control group (p = 0.03).

Therapeutic potential of senolytic agent quercetin in osteoarthritis: A systematic review and meta-analysis of preclinical studies.

BACKGROUND: Quercetin, a natural flavonoid, has shown promise as a senolytic agent for various degenerative diseases. Recently, its protective effect against osteoarthritis (OA), a representative age-related disease of the musculoskeletal system, has attracted much attention. The aim of this study is to summarize and analyze the current literature on the effects of quercetin on OA cartilage in in vivo preclinical studies. METHODS: The Medline (via/using PubMed), Embase, and Web of Science databases were searched up to March 10th, 2023. Risk of bias and the qualitative assessment including mechanisms of all eligible studies and a meta-analysis of cartilage histological scores among the applicable studies was performed. RESULTS: A total of 12 in vivo animal studies were included in this systematic review. A random-effects meta-analysis was performed on six studies using the Osteoarthritis Research Society International (OARSI) scoring system, revealing that quercetin significantly improved OA cartilage OARSI scores (SMD, -6.30 [95% CI, -9.59 to -3.01]; P = 0.0002; heterogeneity: I2 = 86%). The remaining six studies all supported quercetin's protective effects against OA during disease and aging. CONCLUSIONS: Quercetin has shown beneficial effects on cartilage during OA across animal species. Future double-blind randomized controlled clinical trials are needed to verify the efficacy of quercetin in the treatment of OA in humans.

Mechanical strain drives exosome production, function, and miRNA cargo in C2C12 muscle progenitor cells.

Mesenchymal stem cells (MSCs) have been proven to promote tissue repair. However, concerns related to their clinical application and regulatory hurdles remain. Recent data has demonstrated the proregenerative secretome of MSCs can result in similar effects in the absence of the cells themselves. Within the secretome, exosomes have emerged as a promising regenerative component. Exosomes, which are nanosized lipid vesicles secreted by cells, encapsulate micro-RNA (miRNA), RNA, and proteins that drive MSCs regenerative potential with cell specific content. As such, there is an opportunity to optimize the regenerative potential of MSCs, and thus their secreted exosome fraction, to improve clinical efficacy. Exercise is one factor that has been shown to improve muscle progenitor cell function and regenerative potential. However, the effect of exercise on MSC exosome content and function is still unclear. To address this, we used an in vitro culture system to evaluate the effects of mechanical strain, an exercise mimetic, on C2C12 (muscle progenitor cell) exosome production and proregenerative function. Our results indicate that the total exosome production is increased by mechanical strain and can be regulated with different tensile loading regimens. Furthermore, we found that exosomes from mechanically stimulated cells increase proliferation and myogenic differentiation of naïve C2C12 cells. Lastly, we show that exosomal miRNA cargo is differentially expressed following strain. Gene ontology mapping suggests positive regulation of bone morphogenetic protein signaling, regulation of actin-filament-based processes, and muscle cell apoptosis may be at least partially responsible for the proregenerative effects of exosomes from mechanically stimulated C2C12 muscle progenitor cells.

Sustained-release losartan from peptide nanofibers promotes chondrogenesis.

Introduction: The central pathologic feature of osteoarthritis (OA) is the progressive loss of articular cartilage, which has a limited regenerative capacity. The TGF-ß1 inhibitor, losartan, can improve cartilage repair by promoting hyaline rather that fibrous cartilage tissue regeneration. However, there are concerns about side effects associated with oral administration and short retention within the joint following intra-articular injections. To facilitate local and sustained intra-articular losartan delivery we have designed an injectable peptide amphiphile (PA) nanofiber that binds losartan. The aims of this study are to characterize the release kinetics of losartan from two different PA nanofiber compositions followed by testing pro-regenerative bioactivity on chondrocytes. Methods: We tested the impact of electrostatic interactions on nanostructure morphology and release kinetics of the negatively charged losartan molecule from either a positively or negatively charged PA nanofiber. Subsequently, cytotoxicity and bioactivity were evaluated in vitro in both normal and an IL-1ß-induced OA chondrocyte model using ATDC5. Results: Both nanofiber systems promoted cell proliferation but that the positively-charged nanofibers also significantly increased glycosaminoglycans production. Furthermore, gene expression analysis suggested that losartan-encapsulated nanofibers had significant anti-inflammatory, anti-degenerative, and cartilage regenerative effects by significantly blocking TGF-ß1 in this in vitro system. Discussion: The results of this study demonstrated that positively charged losartan sustained-release nanofibers may be a novel and useful treatment for cartilage regeneration and OA by blocking TGF-ß1.

Effects of oral losartan administration on homeostasis of articular cartilage and bone in a rabbit model.

Background and aims: Previous work has shown that oral losartan can enhance microfracture-mediated cartilage repair in a rabbit osteochondral defect injury model. In this study, we aimed to determine whether oral losartan would have a detrimental effect on articular cartilage and bone homeostasis in the uninjured sides. Methods: New Zealand rabbits were divided into 4 groups including normal uninjured (Normal), contralateral uninjured side of osteochondral defect (Defect), osteochondral defect plus microfracture (Microfracture) and osteochondral defect plus microfracture and losartan oral administration (10 mg/kg/day) (Losartan). Rabbits underwent different surgeries and treatment and were sacrificed at 12 weeks. Both side of the normal group and uninjured side of treatment groups tibias were harvested for Micro-CT and histological analysis for cartilage and bone including H&E staining, Herovici's staining (bone and cartilage) Alcian blue and Safranin O staining (cartilage) as well as immunohistochemistry of losartan related signaling pathways molecules for both cartilage and bone. Results: Our results showed losartan oral treatment at 10 mg/kg/day slightly increase Alcian blue positive matrix as well as decrease collagen type 3 in articular cartilage while having no significant effect on articular cartilage structure, cellularity, and other matrix. Losartan treatment also did not affect angiotensin receptor type 1 (AGTR1), angiotensin receptor type 2 (AGTR2) and phosphorylated transforming factor ß1 activated kinase 1 (pTAK1) expression level and pattern in the articular cartilage. Furthermore, losartan treatment did not affect microarchitecture of normal cancellous bone and cortical bone of tibias compared to normal and other groups. Losartan treatment slightly increased osteocalcin positive osteoblasts on the surface of cancellous bone and did not affect bone matrix collagen type 1 content and did not change AGTR1, AGTR2 and pTAK1 signal molecule expression. Conclusion: Oral losartan used as a microfracture augmentation therapeutic does not have significant effect on uninjured articular cartilage and bone based on our preclinical rabbit model. These results provided further evidence that the current regimen of using losartan as a microfracture augmentation therapeutic is safe with respect to bone and cartilage homeostasis and support clinical trials for its application in human cartilage repair.

VEGF-attenuated platelet-rich plasma improves therapeutic effect on cartilage repair.

Autologous platelet-rich plasma (PRP) has gained popularity as a less invasive treatment for various musculoskeletal tissue injuries and conditions due to its favorable safety profile, minimal manipulation and cost-effectiveness. Although PRP treatment has been clinically used for the treatment of osteoarthritis (OA) and damaged cartilage, evidence on therapeutic efficacy has been inconsistent, which calls for a methodology to achieve consistent and improved treatment outcomes. Given that PRP contains numerous proteins, we hypothesized that attenuation of a growth factor known to be detrimental to the healing tissue would enhance efficacy of PRP treatment. Considering that VEGF-mediated angiogenesis inhibits the repair of articular cartilage, we developed VEGF-attenuated PRP by sequestering VEGF in PRP using VEGF-binding microspheres. We demonstrated that VEGF attenuation in PRP did not inhibit the effect of PRP on chondrogenic differentiation of stem cells in vitro. In addition, healing of rat OA cartilage was significantly improved after treatment with VEGF-attenuated PRP when compared to the PRP treatment group or PBS control group. We expect that attenuation of unwanted biological activity using growth factor-binding microspheres could provide a new PRP customization method broadly applicable to various tissue repair processes.

The use of heparin/polycation coacervate sustain release system to compare the bone regenerative potentials of 5 BMPs using a critical sized calvarial bone defect model.

Nonunion following bone fracture and segmental bone defects are challenging clinical conditions. To combat this clinical dilemma, development of new bone tissue engineering therapies using biocompatible materials to deliver bone growth factors is desirable. This aim of this study is to use a heparin/polycation coacervate sustained-release platform to compare 5 bone morphogenetic proteins (BMPs) for promoting bone defect healing in a critical sized calvarial defect model. The in vitro 3D osteogenic pellet cultures assays demonstrated that BMPs 2, 4, 6, 7 and 9 all enhanced mineralization in vitro compared to the control group. BMP2 resulted in higher mineralized volume than BMP4 and BMP6. All BMPs and the control group activated the pSMAD5 signaling pathway and expressed osterix (OSX). The binding of BMP2 with coacervate significantly increased the coacervate average particle size. BMP2, 4, 6, & 7 bound to coacervate significantly increased the Zeta potential of the coacervate while BMP9 binding showed insignificant increase. Furthermore, using a monolayer culture osteogenic assay, it was found that hMDSCs cultured in the coacervate BMP2 osteogenic medium expressed higher levels of RUNX2, OSX, ALP and COX-2 compared to the control and BMPs 4, 6, 7 & 9. Additionally, the coacervate complex can be loaded with up to 2 µg of BMP proteins for sustained release. In vivo, when BMPs were delivered using the coacervate sustained release system, BMP2 was identified to be the most potent BMP promoting bone regeneration and regenerated 10 times of new bone than BMPs 4, 6 & 9. BMP7 also stimulated robust bone regeneration when compared to BMPs 4, 6 & 9. The quality of the newly regenerated bone by all BMPs delivered by coacervate is equivalent to the host bone consisting of bone matrix and bone marrow with normal bone architecture. Although the defect was not completely healed at 6 weeks, coacervate sustain release BMPs, particularly BMP2 and BMP7, could represent a new strategy for treatment of bone defects and non-unions.

Rejuvenated Stem/Progenitor Cells for Cartilage Repair Using the Pluripotent Stem Cell Technology.

It is widely accepted that chondral defects in articular cartilage of adult joints are never repaired spontaneously, which is considered to be one of the major causes of age-related degenerative joint disorders, such as osteoarthritis. Since mobilization of subchondral bone (marrow) cells and addition of chondrocytes or mesenchymal stromal cells into full-thickness defects show some degrees of repair, the lack of self-repair activity in adult articular cartilage can be attributed to lack of reparative cells in adult joints. In contrast, during a fetal or embryonic stage, joint articular cartilage has a scar-less repair activity, suggesting that embryonic joints may contain cells responsible for such activity, which can be chondrocytes, chondroprogenitors, or other cell types such as skeletal stem cells. In this respect, the tendency of pluripotent stem cells (PSCs) to give rise to cells of embryonic characteristics will provide opportunity, especially for humans, to obtain cells carrying similar cartilage self-repair activity. Making use of PSC-derived cells for cartilage repair is still in a basic or preclinical research phase. This review will provide brief overviews on how human PSCs have been used for cartilage repair studies.

Improved Bone Quality and Bone Healing of Dystrophic Mice by Parabiosis.

Duchenne muscular dystrophy (DMD) is a degenerative muscle disorder characterized by a lack of dystrophin expression in the sarcolemma of muscle fibers. DMD patients acquire bone abnormalities including osteopenia, fragility fractures, and scoliosis indicating a deficiency in skeletal homeostasis. The dKO (dystrophin/Utrophin double knockout) is a more severe mouse model of DMD than the mdx mouse (dystrophin deficient), and display numerous clinically-relevant manifestations, including a spectrum of degenerative changes outside skeletal muscle including bone, articular cartilage, and intervertebral discs. To examine the influence of systemic factors on the bone abnormalities and healing in DMD, parabiotic pairing between dKO mice and mdx mice was established. Notably, heterochronic parabiosis with young mdx mice significantly increased bone mass and improved bone micro-structure in old dKO-hetero mice, which showed progressive bone deterioration. Furthermore, heterochronic parabiosis with WT C56/10J mice significantly improved tibia bone defect healing in dKO-homo mice. These results suggest that systemic blood-borne factor(s) and/or progenitors from WT and young mdx mice can influence the bone deficiencies in dKO mice. Understanding these circulating factors or progenitor cells that are responsible to alleviate the bone abnormalities in dKO mice after heterochronic parabiosis might be useful for the management of poor bone health in DMD.

Intra-articular Injection of Bevacizumab Enhances Bone Marrow Stimulation-Mediated Cartilage Repair in a Rabbit Osteochondral Defect Model.

BACKGROUND: Bone marrow stimulation (BMS) via microfracture historically has been a first-line treatment for articular cartilage lesions. However, BMS has become less favorable because of resulting fibrocartilage formation. Previous studies have shown that angiogenesis blockade promotes cartilage repair. Bevacizumab is a Food and Drug Administration-approved medication used clinically to prevent angiogenesis. HYPOTHESIS: The intra-articular injection of bevacizumab would prevent angiogenesis after BMS and lead to improved cartilage repair with more hyaline-like cartilage. STUDY DESIGN: Controlled laboratory study. METHODS: The dose of bevacizumab was first optimized in a rabbit osteochondral defect model with BMS. Then, 48 rabbits (n = 8/group/time point) were divided into 3 groups: osteochondral defect (defect), osteochondral defect + BMS (BMS group), and osteochondral defect + BMS + bevacizumab intra-articular injection (bevacizumab group). Rabbits were sacrificed at either 6 or 12 weeks after surgery. Three-dimensional (3D) micro-computed tomography (microCT), macroscope score, modified O'Driscoll histology scores, collagen type 2, Herovici staining, and hematoxylin and eosin staining were performed. Angiogenesis markers were also evaluated. RESULTS: The intra-articular dose of 12.5 mg/0.5 mL bevacizumab was found to be effective without deleteriously affecting the subchondral bone. Intra-articular injection of bevacizumab resulted in significantly improved cartilage repair for the bevacizumab group compared with the BMS or the defect group based on 3D microCT, the macroscope score (both P < .05), the modified O'Driscoll histology score (P = .0034 and P = .019 vs defect and BMS groups, respectively), collagen type 2, Herovici staining, and hematoxylin and eosin staining at 6 weeks. Cartilage in the bevacizumab group had significantly more hyaline cartilage than did that in other groups. At 12 weeks, the cartilage layer regenerated in all groups; however, the bevacizumab group showed more hyaline-like morphology, as demonstrated by microCT, histology scores (P < .001 and .0225 vs defect and BMS groups, respectively), histology, and immunohistochemistry. The bevacizumab injection did not significantly change mRNA expressions of smooth muscle actin, vascular endothelial growth factor, or hypoxia-inducible factor-1 alpha. CONCLUSION: Intra-articular injection of bevacizumab significantly enhanced the quality and quantity of hyaline-like cartilage after BMS in a rabbit model. Future large-animal and human studies are necessary to evaluate the clinical effect of this therapy, which may lead to improved BMS outcomes and thus the durability of the regenerated cartilage. CLINICAL RELEVANCE: The use of bevacizumab may be an important clinical adjunct to improve BMS-mediated cartilage repair.

The Beneficial Effect of an Intra-articular Injection of Losartan on Microfracture-Mediated Cartilage Repair Is Dose Dependent.

BACKGROUND: A previous publication demonstrated that the oral intake of losartan promoted microfracture-mediated hyaline-like cartilage repair in osteochondral defects of a rabbit knee model. However, an intra-articular (IA) injection of losartan may have direct beneficial effects on cartilage repair and has not been studied. PURPOSE: To determine the dosage and beneficial effects of an IA injection of losartan on microfracture-mediated cartilage repair and normal cartilage homeostasis. STUDY DESIGN: Controlled laboratory study. METHODS: Rabbits were divided into 5 groups (n = 6 each): a microfracture group (MFX group) and 4 different losartan treatment groups that received varying doses of IA losartan (0.1, 1, 10, and 100 mg per knee). An osteochondral defect (5 mm) was created in the trochlear groove cartilage of 1 limb in each rabbit, and 5 microfracture perforations were made in the osteochondral defect. Both the injured and the contralateral knee joints were injected with IA losartan immediately after microfracture and at 2 and 4 weeks after surgery. Rabbits were sacrificed at 6 weeks after surgery for analysis including gross observation, micro-computed tomography, histology, and reverse transcription quantitative polymerase chain reaction. RESULTS: Micro-computed tomography and gross observation demonstrated comparable subchondral bone healing and hyaline-like cartilage morphology in the 0.1-, 1-, and 10-mg losartan groups relative to the MFX group. Conversely, the 100-mg losartan group showed neither bony defect healing nor cartilage repair. Histology revealed higher O'Driscoll scores and hyaline-like cartilage regeneration in the 1-mg losartan group compared with the MFX group. In contrast, the 100-mg losartan group showed the lowest histology score and no cartilage repair. An IA injection of losartan at the doses of 0.1, 1, and 10 mg did not cause adverse effects on uninjured cartilage, while the 100-mg dose induced cartilage damage. Quantitative polymerase chain reaction results showed downregulation of the transforming growth factor ß (TGF-ß) signaling pathway after IA losartan injection. CONCLUSION: An IA injection of losartan at the dose of 1 mg was most effective for the enhancement of microfracture-mediated cartilage repair without adversely affecting uninjured cartilage. Conversely, a high dose (100 mg) IA injection of losartan inhibited cartilage repair in the osteochondral defect and was chondrotoxic to normal articular cartilage. CLINICAL RELEVANCE: An IA injection of losartan at an optimal dosage represents a novel microfracture enhancement therapy and warrants a clinical trial for future clinical applications.

Influence of fixation on CA4+ contrast enhanced microCT of articular cartilage and subsequent feasibility for histological evaluation.

CA4+ is a novel cationic iodinated contrast agent utilized for contrast-enhanced microCT (CECT). In this study, we compared CA4+ CECT for cartilage quantification of unfixed and neutral buffered formalin (NBF)-fixed rabbit distal femur cartilage after 8-, 24- and 30-hours of contrast agent diffusion. The stability of CA4+ binding to cartilage after PBS soak and decalcification was also investigated by CECT. We further assessed the feasibility of cartilage histology and immunohistochemistry after CA4+ CECT. Contrast-enhanced CA4+ labeled unfixed and NBF-fixed cartilage tissues facilitate articular cartilage quantification and accurate morphological assessment. The NBF fixed tissues demonstrate higher cartilage intensity and imaging characteristics distinct from subchondral bone than unfixed tissues while maintaining stable binding even after decalcification with 10% EDTA. The unfixed tissues labeled with CA4+, after CECT imaging and decalcification, are amenable to H&E, Alcian blue, and Safranin O staining, as well as Col2 immunohistochemistry. In contrast, only H&E and Alcian blue staining can be accomplished with CA4+ labeled NBF fixed cartilage, and CA4+ labeling interferes with downstream immunohistochemistry and Safranin O staining, likely due to its positive charge. In conclusion, CA4+ CECT of NBF fixed tissues provides high quality microCT cartilage images and allows for convenient quantification along with feasible downstream H&E and Alcian blue staining after decalcification. CA4+ CECT of unfixed tissues enables researchers to obtain both quantitative microCT as well as cartilage histology and immunohistochemistry data from one set of animals in a cost-, time-, and labor-efficient manner.

Aberrant RhoA activation in macrophages increases senescence-associated secretory phenotypes and ectopic calcification in muscular dystrophic mice.

Duchenne Muscular Dystrophy (DMD) patients often suffer from both muscle wasting and osteoporosis. Our previous studies have revealed reduced regeneration potential in skeletal muscle and bone, concomitant with ectopic calcification of soft tissues in double knockout (dKO, dystrophin-/-; utrophin-/-) mice, a severe murine model for DMD. We found significant involvement of RhoA/ROCK (Rho-Associated Protein Kinase) signaling in mediating ectopic calcification of muscles in dKO mice. However, the cellular identity of these RhoA+ cells, and the role that RhoA plays in the chronic inflammation-associated pathologies has not been elucidated. Here, we report that CD68+ macrophages are highly prevalent at the sites of ectopic calcification of dKO mice, and that these macrophages highly express RhoA. Macrophages from dKO mice feature a shift towards a more pro-inflammatory M1 polarization and an increased expression of various senescence-associated secretory phenotype (SASP) factors that was reduced with the RhoA/ROCK inhibitor Y-27632. Further, systemic inhibition of RhoA activity in dKO mice led to reduced number of RhoA+/CD68+ cells, as well as a reduction in fibrosis and ectopic calcification. Together, these data revealed that RhoA signaling may be a key regulator of imbalanced mineralization in the dystrophic musculoskeletal system and consequently a therapeutic target for the treatment of DMD or other related muscle dystrophies.

Biologically Regulated Marrow Stimulation by Blocking TGF-ß1 With Losartan Oral Administration Results in Hyaline-like Cartilage Repair: A Rabbit Osteochondral Defect Model.

BACKGROUND: Microfracture or bone marrow stimulation (BMS) is often the first choice for clinical treatment of cartilage injuries; however, fibrocartilage, not pure hyaline cartilage, has been reported because of the development of fibrosis in the repair tissue. Transforming growth factor ß1 (TGF-ß1), which can promote fibrosis, can be inhibited by losartan and potentially be used to reduce fibrocartilage. HYPOTHESIS: Blocking TGF-ß1 would improve cartilage healing in a rabbit knee BMS model via decreasing the amount of fibrocartilage and increasing hyaline-like cartilage formation. STUDY DESIGN: Controlled laboratory study. METHODS: An osteochondral defect was made in the patellar groove of 48 New Zealand White rabbits. The rabbits were divided into 3 groups: a defect group (defect only), a BMS group (osteochondral defect + BMS), and a BMS + losartan group (osteochondral defect + BMS + losartan). For the rabbits in the BMS + losartan group, losartan was administrated orally from the day after surgery through the day of euthanasia. Rabbits were sacrificed 6 or 12 weeks postoperatively. Macroscopic appearance, microcomputed tomography, histological assessment, and TGF-ß1 signaling pathway were evaluated at 6 and 12 weeks postoperatively. RESULTS: The macroscopic assessment of the repair revealed that the BMS + losartan group was superior to the other groups tested. Microcomputed tomography showed superior healing of the bony defect in the BMS + losartan group in comparison with the other groups. Histologically, fibrosis in the repair tissue of the BMS + losartan group was significantly reduced when compared with the other groups. Results obtained with the modified O'Driscoll International Cartilage Repair Society grading system yielded significantly superior scores in the BMS + losartan group as compared with both the defect group and the BMS group (F value: 15.8, P < .001, P = .012, respectively). TGF-ß1 signaling and TGF-ß-activated kinase 1 of the BMS + losartan group were significantly suppressed in the synovial tissues. CONCLUSION: By blocking TGF-ß1 with losartan, the repair cartilage tissue after BMS was superior to the other groups and consisted primarily of hyaline cartilage. These results should be easily translated to the clinic because losartan is a Food and Drug Administration-approved drug and it can be combined with the BMS technique for optimal repair of chondral defects. CLINICAL RELEVANCE: Biologically regulated marrow stimulation by blocking TGF-ß1 (oral intake of losartan) provides superior repair via decreasing fibrocartilage formation and resulting in hyaline-like cartilage as compared with outcomes from BMS only.

Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the accumulation of mutant prelamin A (progerin) in the nuclear lamina, resulting in increased nuclear stiffness and abnormal nuclear architecture. Nuclear mechanics are tightly coupled to cytoskeletal mechanics via lamin A/C. However, the role of cytoskeletal/nuclear mechanical properties in mediating cellular senescence and the relationship between cytoskeletal stiffness, nuclear abnormalities, and senescent phenotypes remain largely unknown. Here, using muscle-derived mesenchymal stromal/stem cells (MSCs) from the Zmpste24-/- (Z24-/- ) mouse (a model for HGPS) and human HGPS fibroblasts, we investigated the mechanical mechanism of progerin-induced cellular senescence, involving the role and interaction of mechanical sensors RhoA and Sun1/2 in regulating F-actin cytoskeleton stiffness, nuclear blebbing, micronuclei formation, and the innate immune response. We observed that increased cytoskeletal stiffness and RhoA activation in progeria cells were directly coupled with increased nuclear blebbing, Sun2 expression, and micronuclei-induced cGAS-Sting activation, part of the innate immune response. Expression of constitutively active RhoA promoted, while the inhibition of RhoA/ROCK reduced cytoskeletal stiffness, Sun2 expression, the innate immune response, and cellular senescence. Silencing of Sun2 expression by siRNA also repressed RhoA activation, cytoskeletal stiffness and cellular senescence. Treatment of Zmpste24-/- mice with a RhoA inhibitor repressed cellular senescence and improved muscle regeneration. These results reveal novel mechanical roles and correlation of cytoskeletal/nuclear stiffness, RhoA, Sun2, and the innate immune response in promoting aging and cellular senescence in HGPS progeria.