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.

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.

ATM is a key driver of NF-κB-dependent DNA-damage-induced senescence, stem cell dysfunction and aging.

NF-κB is a transcription factor activated in response to inflammatory, genotoxic and oxidative stress and important for driving senescence and aging. Ataxia-telangiectasia mutated (ATM) kinase, a core component of DNA damage response signaling, activates NF-κB in response to genotoxic and oxidative stress via post-translational modifications. Here we demonstrate that ATM is activated in senescent cells in culture and murine tissues from Ercc1-deficient mouse models of accelerated aging, as well as naturally aged mice. Genetic and pharmacologic inhibition of ATM reduced activation of NF-κB and markers of senescence and the senescence-associated secretory phenotype (SASP) in senescent Ercc1-/- MEFs. Ercc1-/Δ mice heterozygous for Atm have reduced NF-κB activity and cellular senescence, improved function of muscle-derived stem/progenetor cells (MDSPCs) and extended healthspan with reduced age-related pathology especially age-related bone and intervertebral disc pathologies. In addition, treatment of Ercc1-/∆ mice with the ATM inhibitor KU-55933 suppressed markers of senescence and SASP. Taken together, these results demonstrate that the ATM kinase is a major mediator of DNA damage-induced, NF-κB-mediated cellular senescence, stem cell dysfunction and aging and thus represents a therapeutic target to slow the progression of aging.

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.

Biomechanical and Histopathological Analysis of a Retrieved Dermal Allograft After Superior Capsule Reconstruction: A Case Report.

The purpose of this study was to evaluate biomechanical and histopathological results of a retrieved acellular human dermal allograft (AHDA) after superior capsule reconstruction (SCR). A 67-year-old man with pseudoparalysis was treated with SCR for an irreparable posterosuperior rotator cuff tear. The patient failed clinically 4.5 months postoperatively and elected to undergo reverse total shoulder arthroplasty (RTSA). At the time of RTSA, the AHDA was harvested. Biomechanical and histopathologic analyses were performed and compared to native grafts. Failure loads for the explanted graft and native grafts 1 and 2 were 158, 790, and 749 N, respectively. The stiffness values were 20.2, 73, and 100.5 N/mm. The displacement at failure for each graft was 10.1, 27.9, and 17.0 mm. Hematoxylin and eosin and Masson's trichrome staining revealed the presence of cells in all portions of the AHDA. The medial portion presented extensive cellular infiltration, the middle portion moderate, and the lateral portion the least infiltration. Although the only identifiable cells in the lateral portions were found in pockets on the interior of the graft, cells were mainly localized on the exterior. Postoperative cell incorporation could be found in acellular dermal allograft after SCR. However, biomechanical properties in the early postoperative phase were inferior compared with unimplanted allografts.

Characterization of the chondrogenic and osteogenic potential of male and female human muscle-derived stem cells: Implication for stem cell therapy.

People of all backgrounds are susceptible to bone and cartilage damage, and these injuries can be debilitating. Current treatments for bone and cartilage injuries are less than optimal, and we are interested in developing new approaches to treat these diseases, specifically using human muscle-derived stem cells (hMDSCs). Our lab previously demonstrated that sex differences exist between male and female murine MDSCs; thus, this paper sought to investigate whether sex differences also exist in hMDSCs. In the present study, we characterized the chondrogenic and osteogenic sex differences of hMDSCs in vitro and in vivo. We performed in vitro osteogenic and chondrogenic differentiation using hMDSC pellet cultures. As demonstrated by microCT, histology, and immunohistochemistry, male hMDSCs were more chondrogenic and osteogenic than their female counterparts in vitro. No differences were observed based on the sex of hMDSCs in osteogenic and chondrogenic gene expression and cell surface markers. For our in vivo study, we transduced hMDSCs with lenti-BMP2/GFP and transplanted these cells into critical-sized calvarial defects in mice. MicroCT results revealed that male hMDSCs regenerated more bone at 2 weeks and demonstrated higher bone density at 4 and 6 weeks than female hMDSCs. Histology demonstrated that both male and female hMDSCs regenerated functional bone. Clinical relevance: These studies reinforce that stem cells isolated from male and female patients differ in function, and we should disclose the sex of cells used in future studies. Considering sex differences of hMDSCs may help to improve cell-based therapies for autologous cell treatment of bone and cartilage damage. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1339-1349, 2019.

Influences of donor and host age on human muscle-derived stem cell-mediated bone regeneration.

BACKGROUND: Human muscle-derived stem cells (hMDSCs) have been shown to regenerate bone efficiently when they were transduced with Lenti-viral bone morphogenetic protein 2 (LBMP2). However, whether the age of hMDSCs and the animal host affect the bone regeneration capacity of hMDSCs and mechanism are unknown which prompted the current study. METHODS: We isolated three gender-matched young and old populations of skeletal muscle stem cells, and tested the influence of cells' age on in vitro osteogenic differentiation using pellet culture before and after Lenti-BMP2/green fluorescent protein (GFP) transduction. We further investigated effects of the age of hMDSCs and animal host on hMDSC-mediated bone regeneration in a critical-size calvarial bone defect model in vivo. Micro-computer tomography (CT), histology, and immunohistochemistry were used to evaluate osteogenic differentiation and mineralization in vitro and bone regeneration in vivo. Western blot, quantitative polymerase chain reaction (PCR), and oxidative stress assay were performed to detect the effects of age of hMDSCs on cell survival and osteogenic-related genes. Serum insulin-like growth factor 1 (IGF1) and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured with an enzyme-linked immunosorbent assay (ELISA). RESULTS: We found LBMP2/GFP transduction significantly enhanced osteogenic differentiation of hMDSCs in vitro, regardless of donor age. We also found old were as efficient as young LBMP2/GFP-transduced hMDSCs for regenerating functional bone in young and old mice. These findings correlated with lower phosphorylated p38MAPK expression and similar expression levels of cell survival genes and osteogenic-related genes in old hMDSCs relative to young hMDSCs. Old cells exhibited equivalent resistance to oxidative stress. However, both young and old donor cells regenerated less bone in old than young hosts. Impaired bone regeneration in older hosts was associated with high bone remodeling due to higher serum levels of RANKL and lower level of IGF-1. CONCLUSION: hMDSC-mediated bone regeneration was not impaired by donor age when hMDSCs were transduced with LBMP2/GFP, but the age of the host adversely affected hMDSC-mediated bone regeneration. Regardless of donor and host age, hMDSCs formed functional bone, suggesting a promising cell resource for bone regeneration.

Evaluation of Endothelial and Vascular-Derived Progenitor Cell Populations in the Proximal and Distal UCL of the Elbow: A Comparative Study.

Background: Vascular-derived progenitor and endothelial cell populations (CD31, CD34, CD146) are capable of multipotent differentiation at the site of injured ligamentous tissue to aid in the intrinsic healing response. Proximal ulnar collateral ligament (UCL) tears have been reported to have better healing capability when compared with distal UCL tears. Purpose: To compare the vascular composition of the proximal and distal insertions of the anterior bundle of the UCL of the elbow via known markers of endothelial and vascular-derived progenitor cells (CD31, CD34, CD146). Study Design: Descriptive laboratory study. Methods: UCLs were harvested from 10 nonpaired fresh-frozen human cadaveric elbows and transected into proximal and distal portions. Endothelial and vascular-derived progenitor cell densities were assessed with 4 staining groups: CD31 (immunohistochemistry) and CD31/α-smooth muscle actin (α-SMA), CD34/α-SMA, and CD146/α-SMA (immunofluorescence). CD31 immunohistochemistry identified endothelial progenitor cells in the UCL. Later staining of the same slides with α-SMA demonstrated the relationship of progenitor cells to the surrounding vasculature. Fluorescent staining was quantified by calculating the proportion of positively stained nuclei versus the total number of nuclei in the proximal and distal UCL. Results: CD31+ cells were present in the proximal and distal sections of all 10 UCLs. Fluorescent staining revealed no significant differences in the ratio of CD31 to total nuclei between the distal (median, 36% [range, 23%-53%]) and proximal UCL (39% [22%-56%]) (P = .432, Wilcoxon signed-rank test). Similarly, no differences were seen between CD34 distal (39% [24%-64%]) and proximal regions (46% [28%-63%]) (P = .846, Wilcoxon signed-rank test) or CD146 distal (40% [12%-65%]) and proximal regions (40% [22%-51%]) (P ≥ .999, Wilcoxon signed-rank test). Conclusion: Analysis of UCL tissues demonstrated equal distributions of vascular endothelial and vascular-derived progenitor cell markers throughout the proximal and distal UCL. Unlike that of the medial collateral ligament of the knee, the microvascular composition of the proximal and distal UCL insertions was not different, suggesting a well-vascularized ligament throughout its course. Clinical Relevance: These findings investigate one of the possible contributors to UCL healing after injury, which may provide insight into operative and nonoperative management of UCL injuries in the future. This study also indicates that reasons other than differences in progenitor cell density alone may explain the clinical healing differences seen between proximal and distal UCL tears. A better understanding of the microvascular environment and associated blood supply is warranted to understand the healing capability of the UCL.

Inhibition of NF-κB improves the stress resistance and myogenic differentiation of MDSPCs isolated from naturally aged mice.

A decline in the regenerative capacity of adult stem cells with aging is well documented. As a result of this decline, the efficacy of autologous stem cell therapies is likely to decline with increasing donor age. In these cases, strategies to restore the function of aged stem cells would have clinical utility. Globally, the transcription factor NF-κB is up-regulated in aged tissues. Given the negative role that NF-κB plays in myogenesis, we investigated whether the age-related decline in the function of muscle-derived stem/progenitor cells (MDSPCs) could be improved by inhibition of NF-κB. Herein, we demonstrate that pharmacologic or genetic inhibition of NF-κB activation increases myogenic differentiation and improves resistance to oxidative stress. Our results suggest that MDSPC "aging" may be reversible, and that pharmacologic targeting of pathways such as NF-κB may enhance the efficacy of cell-based therapies.