Culture Condition of Bone Marrow Stromal Cells Affects Quantity and Quality of the Extracellular Vesicles.

Extracellular vesicles (EVs) released by bone marrow stromal cells (BMSCs) have been shown to act as a transporter of bioactive molecules such as RNAs and proteins in the therapeutic actions of BMSCs in various diseases. Although EV therapy holds great promise to be a safer cell-free therapy overcoming issues related to cell therapy, manufacturing processes that offer scalable and reproducible EV production have not been established. Robust and scalable BMSC manufacturing methods have been shown to enhance EV production; however, the effects on EV quality remain less studied. Here, using human BMSCs isolated from nine healthy donors, we examined the effects of high-performance culture media that can rapidly expand BMSCs on EV production and quality in comparison with the conventional culture medium. We found significantly increased EV production from BMSCs cultured in the high-performance media without altering their multipotency and immunophenotypes. RNA sequencing revealed that RNA contents in EVs from high-performance media were significantly reduced with altered profiles of microRNA enriched in those related to cellular growth and proliferation in the pathway analysis. Given that pre-clinical studies at the laboratory scale often use the conventional medium, these findings could account for the discrepancy in outcomes between pre-clinical and clinical studies. Therefore, this study highlights the importance of selecting proper culture conditions for scalable and reproducible EV manufacturing.

4PBA reduces growth deficiency in osteogenesis imperfecta by enhancing transition of hypertrophic chondrocytes to osteoblasts.

Short stature is a major skeletal phenotype in osteogenesis imperfecta (OI), a genetic disorder mainly caused by mutations in genes encoding type I collagen. However, the underlying mechanism is poorly understood, and no effective treatment is available. In OI mice that carry a G610C mutation in COL1A2, we previously found that mature hypertrophic chondrocytes (HCs) are exposed to cell stress due to accumulation of misfolded mutant type I procollagen in the endoplasmic reticulum (ER). By fate mapping analysis of HCs in G610C OI mice, we found that HCs stagnate in the growth plate, inhibiting translocation of HC descendants to the trabecular area and their differentiation to osteoblasts. Treatment with 4-phenylbutyric acid (4PBA), a chemical chaperone, restored HC ER structure and rescued this inhibition, resulting in enhanced longitudinal bone growth in G610C OI mice. Interestingly, the effects of 4PBA on ER dilation were limited in osteoblasts, and the bone fragility was not ameliorated. These results highlight the importance of targeting HCs to treat growth deficiency in OI. Our findings demonstrate that HC dysfunction induced by ER disruption plays a critical role in the pathogenesis of OI growth deficiency, which lays the foundation for developing new therapies for OI.

Analysis of Association between Morphometric Parameters of Growth Plate and Bone Growth of Tibia in Mice and Humans.

OBJECTIVE: The purposes of this study are to evaluate which growth plate parameters are associated with bone growth in mice and to compare the mouse results with those in humans. DESIGN: The sagittal sections of the proximal growth plate of the mouse tibia from neonate to young adult stages were subjected to histomorphometric and functional analyses. The radiographic images of tibias of human patients until puberty were analyzed to obtain the tibia length and the proximal growth plate height. It was found that a linear correlation best modeled the relationship between the growth plate variables with the tibia growth rate and length. RESULTS: In mice, total height, resting zone height, combined height of the proliferation and prehypertrophic zones, proliferation activity, and the total width of tibia growth plate showed high linear correlation with tibia bone length and bone growth rate, but the hypertrophic zone height and the growth plate area did not. In both mice and humans, the total growth plate width of tibia was found to have the strongest correlation with tibia length and growth rate. CONCLUSIONS: The results validated that growth plate total height, the height of the resting zone and cell proliferation activity are appropriate parameters to evaluate the balance between growth plate activity and bone growth in mice, consistent with previous reports. The study also provided a new growth plate parameter candidate, growth plate width for growth plate activity evaluation in both mouse and human tibia bone.

Exosomes Isolated From Platelet-Rich Plasma and Mesenchymal Stem Cells Promote Recovery of Function After Muscle Injury.

BACKGROUND: Clinical use of platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) has gained momentum as treatment for muscle injuries. Exosomes, or small cell-derived vesicles, could be helpful if they could deliver the same or better physiological effect without cell transplantation into the muscle. HYPOTHESIS: Local delivery of exosomes derived from PRP (PRP-exos) or MSCs (MSC-exos) to injured muscles hastens recovery of contractile function. STUDY DESIGN: Controlled laboratory study. METHODS: In a rat model, platelets were isolated from blood, and MSCs were isolated from bone marrow and expanded in culture; exosomes from both were isolated through ultracentrifugation. The tibialis anterior muscles were injured in vivo using maximal lengthening contractions. Muscles were injected with PRP-exos or MSC-exos (immediately after injury and 5 and 10 days after injury); controls received an equal volume of saline. Histological and biochemical analysis was performed on tissues for all groups. RESULTS: Injury resulted in a significant loss of maximal isometric torque (66% ± 3%) that gradually recovered over 2 weeks. Both PRP-exos and MSC-exos accelerated recovery, with similar faster recovery of contractile function over the saline-treated group at 5, 10, and 15 days after injury (P < .001). A significant increase in centrally nucleated fibers was seen with both types of exosome groups by day 15 (P < .01). Genes involved in skeletal muscle regeneration were modulated by different exosomes. Muscles treated with PRP-exos had increased expression of Myogenin gene (P < .05), whereas muscles treated with MSC-exos had reduced expression of TGF-ß (P < .05) at 10 days after muscle injury. CONCLUSION: Exosomes derived from PRP or MSCs can facilitate recovery after a muscle strain injury in a small-animal model likely because of factors that can modulate inflammation, fibrosis, and myogenesis. CLINICAL RELEVANCE: Given their small size, low immunogenicity, and ease with which they can be obtained, exosomes could represent a novel therapy for many orthopaedic ailments.

Sclerostin Antibody-Induced Changes in Bone Mass Are Site Specific in Developing Crania.

Sclerostin antibody (Scl-Ab) is an anabolic bone agent that has been shown to increase bone mass in clinical trials of adult diseases of low bone mass, such as osteoporosis and osteogenesis imperfecta (OI). Its use to decrease bone fragility in pediatric OI has shown efficacy in several growing mouse models, suggesting translational potential to pediatric disorders of low bone mass. However, the effects of pharmacologic inhibition of sclerostin during periods of rapid growth and development have not yet been described with respect to the cranium, where lifelong deficiency of functioning sclerostin leads to patterns of excessive bone growth, cranial compression, and facial palsy. In the present study, we undertook dimensional and volumetric measurements in the skulls of growing Brtl/+ OI mice treated with Scl-Ab to examine whether therapy-induced phenotypic changes were similar to those observed clinically in patients with sclerosteosis or Van Buchem disorder. Mice treated between 3 and 14 weeks of age with high doses of Scl-Ab show significant calvarial thickening capable of rescuing OI-induced deficiencies in skull thickness. Other changes in cranial morphology, such as lengths and distances between anatomic landmarks, intracranial volume, and suture interdigitation, showed minimal effects of Scl-Ab when compared with growth-induced differences over the treatment duration. Treatment-induced narrowing of foramina was limited to sites of vascular but not neural passage, suggesting patterns of local regulation. Together, these findings reveal a site specificity of Scl-Ab action in the calvaria with no measurable cranial nerve impingement or brainstem compression. This differentiation from the observed outcomes of lifelong sclerostin deficiency complements reports of Scl-Ab treatment efficacy at other skeletal sites with the prospect of minimal cranial secondary complications. © 2019 American Society for Bone and Mineral Research. © 2019 American Society for Bone and Mineral Research.

Endoplasmic reticulum stress is induced in growth plate hypertrophic chondrocytes in G610C mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a hereditary bone disorder most commonly caused by autosomal dominant mutations in genes encoding type I collagen. In addition to bone fragility, patients suffer from impaired longitudinal bone growth. It has been demonstrated that in OI, an accumulation of mutated type I collagen in the endoplasmic reticulum (ER) induces ER stress in osteoblasts, causing osteoblast dysfunction leading to bone fragility. We hypothesize that ER stress is also induced in the growth plate where bone growth is initiated, and examined a mouse model of dominant OI that carries a G610C mutation in the procollagen α2 chain. The results demonstrated that G610C OI mice had significantly shorter long bones with growth plate abnormalities including elongated total height and hypertrophic zone. Moreover, we found that mature hypertrophic chondrocytes expressed type I collagen and ER dilation was more pronounced compared to wild type littermates. The results from in vitro chondrocyte cultures demonstrated that the maturation of G610C OI hypertrophic chondrocytes was significantly suppressed and ER stress related genes were upregulated. Given that the alteration of hypertrophic chondrocyte activity often causes dwarfism, our findings suggest that hypertrophic chondrocyte dysfunction induced by ER stress may be an underlying cause of growth deficiency in G610C OI mice.