Photocatalyst-free visible-light-promoted C(sp2)-P coupling: efficient synthesis of aryl phosphonates.

A novel and efficient method for the synthesis of aryl phosphonates from aryl halides and trialkylphosphites via EDA complex-based photochemistry has been developed. It is demonstrated that aryl radicals, generated from the photoexcitation of the EDA complex formed by aryl halide and potassium thioacetate, could be intercepted with trialkylphosphite to produce the corresponding aryl phosphonates in moderate to good yields. It should be noted that the reaction is performed at room temperature in the absence of any transition metal catalyst, oxidant and photocatalyst, exhibiting high efficiency, high selectivity, and operational simplicity.

The development of a mouse model to investigate the formation of heterotopic ossification.

BACKGROUND: Muscle injury and concomitant bone injury are important drivers to induce heterotopic ossification (HO). However, the related roles of muscle and concomitant bone injury in HO formation are still unclear. This study aims to develop a mouse model through the combination of hindlimb amputation (Am) and cardiotoxin (CTX) injection to investigate the mechanism of HO formation. METHOD: The mice were randomly divided into Am group (Am of right hindlimb, n = 12), CTX group (CTX injection in the calf muscle of left hindlimb, n = 12) and Am + CTX group (the combination of Am of right hindlimb and CTX injection of left hindlimb, n = 18). MicroCT was used to evaluate the incidence of HO. Histology was used to investigate the progression of HO. RESULTS: The MicroCT showed that only Am or CTX injection failed to induce HO while the combination of Am and CTX injection successfully induced HO. The incidence of HO was significant in Am + CTX group on day 7 (0% vs 0% vs 83.3%, p = 0.001) and day 14 (0% vs 0% vs 83.3%, p = 0.048). HO was located on the left hindlimb where CTX was injected. Moreover, the bone volume and bone density on day 14 were higher than those on day 7 in Am + CTX group. Histology revealed the evidence of calcification and expression of osteogenic markers in calcification sites in Am + CTX group. CONCLUSION: In summary, the combination of Am and CTX injection could successfully induce dystrophic calcification/HO, which occurs in the location of muscle injury.

In vitro study to identify ligand-independent function of estrogen receptor-α in suppressing DNA damage-induced chondrocyte senescence.

In osteoarthritis (OA), chondrocytes undergo many pathological alternations that are linked with cellular senescence. However, the exact pathways that lead to the generation of a senescence-like phenotype in OA chondrocytes are not clear. Previously, we found that loss of estrogen receptor-α (ERα) was associated with an increased senescence level in human chondrocytes. Since DNA damage is a common cause of cellular senescence, we aimed to study the relationship among ERα levels, DNA damage, and senescence in chondrocytes. We first examined the levels of ERα, representative markers of DNA damage and senescence in normal and OA cartilage harvested from male and female human donors, as well as from male mice. The influence of DNA damage on ERα levels was studied by treating human chondrocytes with doxorubicin (DOX), which is an often-used DNA-damaging agent. Next, we tested the potential of overexpressing ERα in reducing DNA damage and senescence levels. Lastly, we explored the interaction between ERα and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Results indicated that the OA chondrocytes contained DNA damage and displayed senescence features, which were accompanied by significantly reduced ERα levels. Overexpression of ERα reduced the levels of DNA damage and senescence in DOX-treated normal chondrocytes and OA chondrocytes. Moreover, DOX-induced the activation of NF-κB pathway, which was partially reversed by overexpressing ERα. Taken together, our results demonstrated the critical role of ERα in maintaining the health of chondrocytes by inhibiting DNA damage and senescence. This study also suggests that maintaining the ERα level may represent a new avenue to prevent and treat OA.

Differences in the intrinsic chondrogenic potential of human mesenchymal stromal cells and iPSC-derived multipotent cells.

BACKGROUND: Human multipotent progenitor cells (hiMPCs) created from induced pluripotent stem cells (iPSCs) represent a new cell source for cartilage regeneration. In most studies, bone morphogenetic proteins (BMPs) are needed to enhance transforming growth factor-ß (TGFß)-induced hiMPC chondrogenesis. In contrast, TGFß alone is sufficient to result in robust chondrogenesis of human primary mesenchymal stromal cells (hMSCs). Currently, the mechanism underlying this difference between hiMPCs and hMSCs has not been fully understood. METHODS: In this study, we first tested different growth factors alone or in combination in stimulating hiMPC chondrogenesis, with a special focus on chondrocytic hypertrophy. The reparative capacity of hiMPCs-derived cartilage was assessed in an osteochondral defect model created in rats. hMSCs isolated from bone marrow were included in all studies as the control. Lastly, a mechanistic study was conducted to understand why hiMPCs and hMSCs behave differently in responding to TGFß. RESULTS: Chondrogenic medium supplemented with TGFß3 and BMP6 led to robust in vitro cartilage formation from hiMPCs with minimal hypertrophy. Cartilage tissue generated from this new method was resistant to osteogenic transition upon subcutaneous implantation and resulted in a hyaline cartilage-like regeneration in osteochondral defects in rats. Interestingly, TGFß3 induced phosphorylation of both Smad2/3 and Smad1/5 in hMSCs, but only activated Smad2/3 in hiMPCs. Supplementing BMP6 activated Smad1/5 and significantly enhanced TGFß's compacity in inducing hiMPC chondrogenesis. The chondro-promoting function of BMP6 was abolished by the treatment of a BMP pathway inhibitor. CONCLUSIONS: This study describes a robust method to generate chondrocytes from hiMPCs with low hypertrophy for hyaline cartilage repair, as well as elucidates the difference between hMSCs and hiMPCs in response to TGFß. Our results also indicated the importance of activating both Smad2/3 and Smad1/5 in the initiation of chondrogenesis.

Risk factors of opioid use associated with an enhanced-recovery programme after total knee arthroplasty.

BACKGROUND: Characterizing the impacts of postoperative opioid use on total knee arthroplasty (TKA) patients may help optimize the pain management after TKA. The aim of the study is to examine the prevalence and risk factors for opioid use with an enhanced-recovery programme after primary TKA. METHODS: We identified 361 patients undergoing TKA, and separated those on the basis of whether to receive opioid use after surgery. Themultivariate logistic regression model was used to identify independent risk factors for opioid use after primary TKA. Length of stay (LOS) and postoperative complications were also recorded and compared. RESULTS: The prevalence of opioid use after primary TKA was 23.0%. The significant risk factor was the longer operative time (OR [odds ratio] = 1.017, 95% CI [confidence interval] = 1.001 to 1.032, p = 0.034) and the protective factor was the utilization of tranexamic acid(OR= 0.355, 95% CI = 0.161 to 0.780, p = 0.010). In addition, the LOS was longer in opioid group (p < 0.05). CONCLUSION: Considering the adverse health effects of opioid use, strategies need to be developed to prevent persistent opioid use after TKA. Reducing operative time and the application of tranexamic acid could lower the risk of opioid use with an enhanced-recovery programme after primary TKA.

Macrophages Modulate the Function of MSC- and iPSC-Derived Fibroblasts in the Presence of Polyethylene Particles.

Fibroblasts in the synovial membrane secrete molecules essential to forming the extracellular matrix (ECM) and supporting joint homeostasis. While evidence suggests that fibroblasts contribute to the response to joint injury, the outcomes appear to be patient-specific and dependent on interactions between resident immune cells, particularly macrophages (Mφs). On the other hand, the response of Mφs to injury depends on their functional phenotype. The goal of these studies was to further explore these issues in an in vitro 3D microtissue model that simulates a pathophysiological disease-specific microenvironment. Two sources of fibroblasts were used to assess patient-specific influences: mesenchymal stem cell (MSC)- and induced pluripotent stem cell (iPSC)-derived fibroblasts. These were co-cultured with either M1 or M2 Mφs, and the cultures were challenged with polyethylene particles coated with lipopolysaccharide (cPE) to model wear debris generated from total joint arthroplasties. Our results indicated that the fibroblast response to cPE was dependent on the source of the fibroblasts and the presence of M1 or M2 Mφs: the fibroblast response as measured by gene expression changes was amplified by the presence of M2 Mφs. These results demonstrate that the immune system modulates the function of fibroblasts; furthermore, different sources of differentiated fibroblasts may lead to divergent results. Overall, our research suggests that M2 Mφs may be a critical target for the clinical treatment of cPE induced fibrosis.

Graphene oxide-functionalized nanocomposites promote osteogenesis of human mesenchymal stem cells via enhancement of BMP-SMAD1/5 signaling pathway.

Biomaterials that can harness the intrinsic osteogenic potential of stem cells offer a promising strategy to accelerate bone regeneration and repair. Previously, we had used methacrylated gelatin (GelMA)-based scaffolds to achieve bone formation from human mesenchymal stem cells (hMSCs). In this study, we aimed to further enhance hMSC osteogenesis by incorporating graphene oxide (GO)-based nanosheets into GelMA. In vitro results showed high viability and metabolic activities in hMSCs encapsulated in the newly developed nanocomposites. Incorporation of GO markedly increased mineralization within hMSC-laden constructs, which was further increased by replacing GO with silica-coated graphene oxide (SiGO). Mechanistic analysis revealed that the nanosheet enhanced the production, retention, and biological activity of endogenous bone morphogenetic proteins (BMPs), resulting in robust osteogenesis in the absence of exogenous osteoinductive growth factors. Specifically, the osteoinductive effect of the nanosheets was abolished by inhibiting the BMP signaling pathway with LDN-193189 treatment. The bone formation potential of the technology was further tested in vivo using a mouse subcutaneous implantation model, where hMSCs-laden GO/GelMA and SiGO/GelMA samples resulted in bone volumes 108 and 385 times larger, respectively, than the GelMA control group. Taken together, these results demonstrate the biological activity and mechanism of action of GO-based nanosheets in augmenting the osteogenic capability of hMSCs, and highlights the potential of leveraging nanomaterials such as GO and SiGO for bone tissue engineering applications.

Caveolin-1 mediates soft scaffold-enhanced adipogenesis of human mesenchymal stem cells.

BACKGROUND: Human bone marrow-derived mesenchymal stem cells (hBMSCs) can differentiate into adipocytes upon stimulation and are considered an appropriate cell source for adipose tissue engineering. In addition to biochemical cues, the stiffness of a substrate that cells attach to has also been shown to affect hBMSC differentiation potential. Of note, most current studies are conducted on monolayer cultures which do not directly inform adipose tissue engineering, where 3-dimensional (3D) scaffolds are often used to create proper tissue architecture. In this study, we aim to examine the adipogenic differentiation of hBMSCs within soft or stiff scaffolds and investigate the molecular mechanism mediating the response of hBMSCs to substrate stiffness in 3D culture, specifically the involvement of the integral membrane protein, caveolin-1 (CAV1), known to regulate signaling in MSCs via compartmentalizing and concentrating signaling molecules. METHODS: By adjusting the photo-illumination time, photocrosslinkable gelatin scaffolds with the same polymer concentration but different stiffnesses were created. hBMSCs were seeded within soft and stiff scaffolds, and their response to adipogenic induction under different substrate mechanical conditions was characterized. The functional involvement of CAV1 was assessed by suppressing its expression level using CAV1-specific siRNA. RESULTS: The soft and stiff scaffolds used in this study had a compressive modulus of ~0.5 kPa and ~23.5 kPa, respectively. hBMSCs showed high viability in both scaffold types, but only spread out in the soft scaffolds. hBMSCs cultured in soft scaffolds displayed significantly higher adipogenesis, as revealed by histology, qRT-PCR, and immunostaining. Interestingly, a lower CAV1 level was observed in hBMSCs in the soft scaffolds, concomitantly accompanied by increased levels of Yes-associated protein (YAP) and decreased YAP phosphorylation, when compared to cells seeded in the stiff scaffolds. Interestingly, reducing CAV1 expression with siRNA was shown to further enhance hBMSC adipogenesis, which may function through activation of the YAP signaling pathway. CONCLUSIONS: Soft biomaterials support superior adipogenesis of encapsulated hBMSCs in 3D culture, which is partially mediated by the CAV1-YAP axis. Suppressing CAV1 expression levels represents a robust method in the promotion of hBMSC adipogenesis.

Tissue Engineering for Musculoskeletal Regeneration and Disease Modeling.

Musculoskeletal injuries and associated conditions are the leading cause of physical disability worldwide. The concept of tissue engineering has opened up novel approaches to repair musculoskeletal defects in a fast and/or efficient manner. Biomaterials, cells, and signaling molecules constitute the tissue engineering triad. In the past 40 years, significant progress has been made in developing and optimizing all three components, but only a very limited number of technologies have been successfully translated into clinical applications. A major limiting factor of this barrier to translation is the insufficiency of two-dimensional cell cultures and traditional animal models in informing the safety and efficacy of in-human applications. In recent years, microphysiological systems, often referred to as organ or tissue chips, generated according to tissue engineering principles, have been proposed as the next-generation drug testing models. This chapter aims to first review the current tissue engineering-based approaches that are being applied to fabricate and develop the individual critical elements involved in musculoskeletal organ/tissue chips. We next highlight the general strategy of generating musculoskeletal tissue chips and their potential in future regenerative medicine research. Exemplary microphysiological systems mimicking musculoskeletal tissues are described. With sufficient physiological accuracy and relevance, the human cell-derived, three-dimensional, multi-tissue systems have been used to model a number of orthopedic disorders and to test new treatments. We anticipate that the novel emerging tissue chip technology will continually reshape and improve our understanding of human musculoskeletal pathophysiology, ultimately accelerating the development of advanced pharmaceutics and regenerative therapies.

Dead muscle tissue promotes dystrophic calcification by lowering circulating TGF-ß1 level.

AIMS: Dystrophic calcification (DC) is the abnormal appearance of calcified deposits in degenerating tissue, often associated with injury. Extensive DC can lead to heterotopic ossification (HO), a pathological condition of ectopic bone formation. The highest rate of HO was found in combat-related blast injuries, a polytrauma condition with severe muscle injury. It has been noted that the incidence of HO significantly increased in the residual limbs of combat-injured patients if the final amputation was performed within the zone of injury compared to that which was proximal to the zone of injury. While aggressive limb salvage strategies may maximize the function of the residual limb, they may increase the possibility of retaining non-viable muscle tissue inside the body. In this study, we hypothesized that residual dead muscle tissue at the zone of injury could promote HO formation. METHODS: We tested the hypothesis by investigating the cellular and molecular consequences of implanting devitalized muscle tissue into mouse muscle pouch in the presence of muscle injury induced by cardiotoxin. RESULTS: Our findings showed that the presence of devitalized muscle tissue could cause a systemic decrease in circulating transforming growth factor-beta 1 (TGF-ß1), which promoted DC formation following muscle injury. We further demonstrated that suppression of TGF-ß signalling promoted DC in vivo, and potentiated osteogenic differentiation of muscle-derived stromal cells in vitro. CONCLUSION: Taken together, these findings suggest that TGF-ß1 may play a protective role in dead muscle tissue-induced DC, which is relevant to understanding the pathogenesis of post-traumatic HO. Cite this article: Bone Joint Res 2020;9(11):742-750.

Injectable BMP-2 gene-activated scaffold for the repair of cranial bone defect in mice.

Tissue engineering using adult human mesenchymal stem cells (MSCs) seeded within biomaterial scaffolds has shown the potential to enhance bone healing. Recently, we have developed an injectable, biodegradable methacrylated gelatin-based hydrogel, which was especially effective in producing scaffolds in situ and allowed the delivery of high viable stem cells and gene vehicles. The well-demonstrated benefits of recombinant adeno-associated viral (rAAV) vector, including long-term gene transfer efficiency and relative safety, combination of gene and cell therapies has been developed in both basic and translational research to support future bone tissue regeneration clinical trials. In this study, we have critically assessed the applicability of single-step visible light (VL) photocrosslinking fabrication of gelatin scaffold to deliver rAAV encoding human bone morphogenetic protein-2 (BMP-2) gene to address the need for sustained BMP-2 presence localized within scaffolds for the repair of cranial bone defect in mouse model. In this method, rAAV-BMP-2 and human bone marrow-derived MSCs (hBMSCs) were simultaneously included into gelatin scaffolds during scaffold formation by VL illumination. We demonstrated that the subsequent release of rAAV-BMP-2 constructs from the scaffold matrix, which resulted in efficient in situ expression of BMP-2 gene by hBMSCs seeded within the scaffolds, and thus induced their osteogenic differentiation without the supplement of exogenous BMP-2. The reparative capacity of this novel stem cell-seeded and gene-activated scaffolds was further confirmed in the cranial defect in the severe combined immunodeficiency mice, revealed by imaging, histology, and immunohistochemistry at 6 weeks after cranial defect treatment.

Prostate-specific antigen modulates the osteogenic differentiation of MSCs via the cadherin 11-Akt axis.

BACKGROUND: A high prevalence of osteoblastic bone metastases is characteristic of prostate cancer. Prostate-specific antigen (PSA) is a serine protease uniquely produced by prostate cancer cells and is an important serological marker for prostate cancer. However, whether PSA modulates the osteogenic process remains largely unknown. In this study, we explored the effect of PSA on modulating the osteoblastic differentiation of mesenchymal stem cells (MSCs). In this study, we used flow cytometry, CCK-8 assay, Alizarin red S (ARS) staining and quantification, alkaline phosphatase (ALP) activity and staining, Western blotting, and quantitative real-time PCR (qRT-PCR) to explore the effect of PSA on osteogenic differentiation of MSCs. RESULTS: We first demonstrated that although PSA did not affect the proliferation, morphology, or phenotype of MSCs, it significantly promoted the osteogenic differentiation of MSCs in a concentration-dependent manner. Furthermore, we demonstrated that PSA promoted the osteogenic differentiation of MSCs by elevating the expression of Cadherin 11 in MSCs and, thus, activating the Akt signaling pathway. CONCLUSIONS: In conclusion, we demonstrated that PSA could promote the osteogenesis of MSCs through Akt signaling pathway activation by elevating the expression of cadherin-11 in MSCs. These findings imply a possible role of PSA in osteoblastic bone metastases in prostate cancer.

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.

Repeated exposure to propofol impairs spatial learning, inhibits LTP and reduces CaMKIIα in young rats.

Propofol is one of the most widely used intravenous anesthetics. We investigated the effects of propofol injected during development on synaptic plasticity and long-term spatial learning and memory in young rats. Propofol (75 mg/kg) was administered to 7-day-old rats either as a single dose or in 7 doses at 24-h intervals. At postnatal day 28, spatial learning in the Morris water maze and long-term potentiation (LTP) in the CA1 region of the hippocampus were significantly reduced in the rats that had received 7 doses of propofol. This treatment also significantly decreased the expression of CaMKIIα and pCaMKIIα in the hippocampus, and reduced the pCaMKIIα/CaMKIIα ratio, as measured by immunochemistry and Western blotting. We conclude that repeated exposure to propofol impairs learning and memory in the developing rat brain, and this finding may be associated with down-regulation of CaMKIIα and pCaMKIIα.