TGF-ß1 plays a protective role in glucocorticoid-induced dystrophic calcification.

Dystrophic calcification (DC) is the deposition of calcium in degenerated tissue which occurs as a reaction to tissue damage. Sometimes if tissue repair fails, it can progress into heterotopic ossification (HO), a pathological condition of abnormal bone formation. HO happens frequently in severe trauma patients such as in blast injury, central nervous system injury and burn injury, in which excessive endogenous glucocorticoid production has always been found. Glucocorticoids have a big impact on bone and muscle. However, few studies have investigated the impact of glucocorticoids on DC/HO formation in muscle. This study aimed to determine the role of glucocorticoids in DC/HO pathogenesis following muscular injury and the possible underlying mechanism. In this study, we administered a high dose of a synthetic glucocorticoid, dexamethasone (DEX), to animals with muscle injury induced by cardiotoxin (CTX) injection to mimic a glucocorticoid excess state following severe muscle trauma. The findings reported here showed that DEX treatment together with CTX-induced muscle injury led to a significant amount of DC in muscle. This effect was likely related to protein level alterations in the fibrinolytic system and resultant decreased circulating transforming growth factor-beta 1 (TGF-ß1), given that supplementation of recombinant TGF-ß1 markedly rescued this phenomenon. In summary, our results suggest that glucocorticoid excess impairs muscle regeneration and promotes DC/HO, and that TGF-ß1 could be a key factor in modulating this process.

The effect of adipose-derived stem cells on enthesis healing after repair of acute and chronic massive rotator cuff tears in rats.

BACKGROUND: Chronic massive rotator cuff tears heal poorly and often retear. This study investigated the effect of adipose-derived stem cells (ADSCs) and transforming growth factor-ß3 (TGF-ß3) delivered in 1 of 2 hydrogels (fibrin or gelatin methacrylate [GelMA]) on enthesis healing after repair of acute or chronic massive rotator cuff tears in rats. METHODS: Adult male Lewis rats underwent bilateral transection of the supraspinatus and infraspinatus tendons with intramuscular injection of botulinum toxin A (n = 48 rats). After 8 weeks, animals received 1 of 8 interventions (n = 12 shoulders/group): (1) no repair, (2) repair only, or repair augmented with (3) fibrin, (4) GelMA, (5) fibrin + ADSCs, (6) GelMA + ADSCs, (7) fibrin + ADSCs + TGF-ß3, or (8) GelMA + ADSCs + TGF-ß3. An equal number of animals underwent acute tendon transection and immediate application of 1 of 8 interventions. Enthesis healing was evaluated 4 weeks after the repair by microcomputed tomography, histology, and mechanical testing. RESULTS: Increased bone loss and reduced structural properties were seen in chronic compared with acute tears. Bone mineral density of the proximal humerus was higher in repairs of chronic tears augmented with fibrin + ADSCs and GelMA + ADSCs than in unrepaired chronic tears. Similar improvement was not seen in acute tears. No intervention enhanced histologic appearance or structural properties in acute or chronic tears. CONCLUSIONS: Surgical repair augmented with ADSCs may provide more benefit in chronic tears compared with acute tears, although there was no added benefit to supplementing ADSCs with TGF-ß3.

Engineering hyaline cartilage from mesenchymal stem cells with low hypertrophy potential via modulation of culture conditions and Wnt/ß-catenin pathway.

Mesenchymal stem cells (MSCs) represent a promising cell source to regenerate articular cartilage, but current chondroinduction protocols, commonly using transforming growth factor-ß (TGFß), lead to concomitant chondrocytic hypertrophy with ossification risk. Here, we showed that a 14-day culture of MSC-laden hyaluronic acid hydrogel in the presence of TGFß, followed by 7 days culture in TGFß-free medium, with the supplement of Wnt/ß-catenin inhibitor XAV939 from day 10-21, resulted in significantly reduced hypertrophy phenotype. The stability of the hyaline phenotype of the MSC-derived cartilage, generated with a standard protocol (Control) or the optimized (Optimized) method developed in this study, was further examined through intramuscular implantation in nude mice. After 4 weeks, constructs from the Control group showed obvious mineralization; in contrast, the Optimized group displayed no signs of mineralization, and maintained cartilaginous histology. Further analysis showed that TGFß treatment time affected p38 expression, while exposure to XAV939 significantly inhibited P-Smad 1/5 level, which together resulted in decreased level of Runx2. These findings suggest a novel treatment regimen to generate hyaline cartilage from human MSCs-loaded scaffolds, which have a minimal risk of eliciting endochondral ossification.

Engineering in-vitro stem cell-based vascularized bone models for drug screening and predictive toxicology.

The production of veritable in-vitro models of bone tissue is essential to understand the biology of bone and its surrounding environment, to analyze the pathogenesis of bone diseases (e.g., osteoporosis, osteoarthritis, osteomyelitis, etc.), to develop effective therapeutic drug screening, and to test potential therapeutic strategies. Dysregulated interactions between vasculature and bone cells are often related to the aforementioned pathologies, underscoring the need for a bone model that contains engineered vasculature. Due to ethical restraints and limited prediction power of animal models, human stem cell-based tissue engineering has gained increasing relevance as a candidate approach to overcome the limitations of animals and to serve as preclinical models for drug testing. Since bone is a highly vascularized tissue, the concomitant development of vasculature and mineralized matrix requires a synergistic interaction between osteogenic and endothelial precursors. A number of experimental approaches have been used to achieve this goal, such as the combination of angiogenic factors and three-dimensional scaffolds, prevascularization strategies, and coculture systems. In this review, we present an overview of the current models and approaches to generate in-vitro stem cell-based vascularized bone, with emphasis on the main challenges of vasculature engineering. These challenges are related to the choice of biomaterials, scaffold fabrication techniques, and cells, as well as the type of culturing conditions required, and specifically the application of dynamic culture systems using bioreactors.

Promotion of human mesenchymal stem cell osteogenesis by PI3-kinase/Akt signaling, and the influence of caveolin-1/cholesterol homeostasis.

INTRODUCTION: Stem cells are considered an important resource for tissue repair and regeneration. Their utilization in regenerative medicine will be aided by mechanistic insight into their responsiveness to external stimuli. It is likely that, similar to all other cells, an initial determinant of stem cell responsiveness to external stimuli is the organization of signaling molecules in cell membrane rafts. The clustering of signaling molecules in these cholesterol-rich membrane microdomains can affect the activity, specificity, cross-talk and amplification of cell signaling. Membrane rafts fall into two broad categories, non-caveolar and caveolar, based on the absence or presence, respectively, of caveolin scaffolding proteins. We have recently demonstrated that caveolin-1 (Cav-1) expression increases during, and knockdown of Cav-1 expression enhances, osteogenic differentiation of human bone marrow derived mesenchymal stem cells (MSCs). The increase in Cav-1 expression observed during osteogenesis is likely a negative feedback mechanism. We hypothesize that focal adhesion signaling pathways such as PI3K/Akt signaling may be negatively regulated by Cav-1 during human MSC osteogenesis. METHODS: Human bone marrow MSCs were isolated from femoral heads obtained after total hip arthroplasty. MSCs were incubated in standard growth medium alone or induced to osteogenically differentiate by the addition of supplements (ß-glycerophosphate, ascorbic acid, dexamethasone, and 1,25-dihydroxyvitamin D3). The activation of and requirement for PI3K/Akt signaling in MSC osteogenesis were assessed by immunoblotting for phosphorylated Akt, and treatment with the PI3K inhibitor LY294002 and Akt siRNA, respectively. The influences of Cav-1 and cholesterol membrane rafts on PI3K/Akt signaling were investigated by treatment with Cav-1 siRNA, methyl-ß-cyclodextrin, or cholesterol oxidase, followed by cellular sub-fractionation and/or immunoblotting for phosphorylated Akt. RESULTS: LY294002 and Akt siRNA inhibited MSC osteogenesis. Methyl-ß-cyclodextrin, which disrupts all membrane rafts, inhibited osteogenesis. Conversely, Cav-1 siRNA and cholesterol oxidase, which displaces Cav-1 from caveolae, enhanced Akt signaling induced by osteogenic supplements. In control cells, phosphorylated Akt began to accumulate in caveolae after 10 days of osteogenic differentiation. CONCLUSIONS: PI3K/Akt signaling is a key pathway required for human MSC osteogenesis, and it is likely that localization of active Akt in non-caveolar and caveolar membrane rafts positively and negatively contributes to osteogenesis, respectively.

Differential regulation of osteogenic marker gene expression by Wnt-3a in embryonic mesenchymal multipotential progenitor cells.

The Wnt family of secreted glycoproteins plays an integral role in embryonic development and differentiation. To explore the role of Wnt's in one aspect of differentiation, namely osteogenesis, we employed a retroviral gene transfer approach to express Wnt-3a in the multipotent murine embryonic mesenchymal cell line C3H10T1/2. We found that expression of Wnt-3a in these cells had a significant, positive effect on cell growth in serum-containing medium, in that the cells grew to very high densities compared to the control cells. Additionally, apoptosis was markedly inhibited by Wnt-3a. However, when the cells were grown in serum-deficient medium, the Wnt-3a-expressing cells arrested efficiently in G1 phase, indicating that serum growth factors were needed in addition to Wnt-3a for enhanced proliferation. Wnt-3a-expressing cells exhibited high levels of alkaline phosphatase gene expression and enzymatic activity, but did not show any matrix mineralization. Unexpectedly, basal expression of bone sialoprotein, osteocalcin, and osteopontin were markedly inhibited by Wnt-3a, as were other known target genes of Wnt-3a, such as Brachyury, FGF-10, and Cdx1. When Wnt-3a-expressing cells were treated with osteogenic supplements in the presence of BMP-2, alkaline phosphatase gene expression and activity were further elevated. Additionally, BMP-2 was able to reverse the inhibitory effect of Wnt-3a on osteocalcin and osteopontin gene expression. These results indicate that while Wnt-3a represses basal expression of some osteogenic genes, this repression can be partially reversed by BMP-2. Finally, the enhanced gene expression of alkaline phosphatase induced by Wnt-3a could be effectively suppressed by the combined action of dexamethasone and 1,25-dihydroxyvitamin D(3). These data show for the first time that Wnt-3a has an unusual effect on multipotential embryonic cells, in that it enhances cellular proliferation and expression of alkaline phosphatase, while it represses most other marker genes of osteogenic differentiation.

Titanium particles suppress expression of osteoblastic phenotype in human mesenchymal stem cells.

Long-term stability of arthroplasty prosthesis depends on the integration between osseous tissue and the implant biomaterial. Integrity of the osseous tissue requires the contribution of mesenchymal stem cells and their continuous differentiation into an osteoblastic phenotype. This study aims to investigate the hypothesis that exposure to wear debris particles derived from orthopaedic biomaterials affects the osteoblastic differentiation of human mesenchymal stem cells (hMSC). Upon in vitro culture in the presence of osteogenic supplements (OS), we observe that cultures of hMSCs isolated from femoral head bone marrow are capable of osteogenic differentiation, expressing alkaline phosphatase, osteocalcin, and bone sialoprotein (BSP), in addition to producing collagen type I and BSP accompanied by extracellular matrix mineralization. Exposure of OS-treated hMSCs to submicron commercially pure titanium (cpTi) particles suppresses BSP gene expression, reduces collagen type I and BSP production, decreases cellular proliferation and viability, and inhibits matrix mineralization. In comparison, exposure to zirconium oxide (ZrO2) particles of similar size did not alter osteoblastic gene expression and resulted in only a moderate decrease in cellular proliferation and mineralization. Confocal imaging of cpTi-treated hMSC cultures revealed patchy groups of cells displaying disorganized cytoskeletal architecture and low levels of extracellular BSP. These in vitro findings suggest that chronic exposure of marrow cells to titanium wear debris in vivo may contribute to decreased bone formation at the bone/implant interface by reducing the population of viable hMSCs and compromising their differentiation into functional osteoblasts. Understanding the nature of hMSC bioreactivity to orthopaedic wear debris should provide additional insights into mechanisms underlying aseptic loosening.

Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells.

Explant cultures of adult human trabecular bone fragments give rise to osteoblastic cells, that are known to express osteoblast-related genes and mineralize extracellular matrix. These osteoblastic cells have also been shown to undergo adipogenesis in vitro and chondrogenesis in vivo. Here we report the in vitro developmental potential of adult human osteoblastic cells (hOB) derived from explant cultures of collagenase-pretreated trabecular bone fragments. In addition to osteogenic and adipogenic differentiation, these cells are capable of chondrogenic differentiation in vitro in a manner similar to adult human bone marrow-derived mesenchymal progenitor cells. High-density pellet cultures of hOB maintained in chemically defined serum-free medium, supplemented with transforming growth factor-beta1, were composed of morphologically distinct, chondrocyte-like cells expressing mRNA transcripts of collagen types II, IX and X, and aggrecan. The cells within the high-density pellet cultures were surrounded by a sulfated proteoglycan-rich extracellular matrix that immunostained for collagen type II and proteoglycan link protein. Osteogenic differentiation of hOB was verified by an increased number of alkaline phosphatase-positive cells, that expressed osteoblast-related transcripts such as alkaline phosphatase, collagen type I, osteopontin and osteocalcin, and formed mineralized matrix in monolayer cultures treated with ascorbate, beta-glycerophosphate, and bone morphogenetic protein-2. Adipogenic differentiation of hOB was determined by the appearance of intracellular lipid droplets, and expression of adipocyte-specific genes, such as lipoprotein lipase and peroxisome proliferator-activated receptor gamma2, in monolayer cultures treated with dexamethasone, indomethacin, insulin and 3-isobutyl-1-methylxanthine. Taken together, these results show that cells derived from collagenase-treated adult human trabecular bone fragments have the potential to differentiate into multiple mesenchymal lineages in vitro, indicating their developmental plasticity and suggesting their mesenchymal progenitor nature.