Hydroxychloroquine and a low activity bisphosphonate conjugate prevent and reverse ovariectomy-induced bone loss in mice through dual antiresorptive and anabolic effects.

Osteoporosis is incurable because there are no dual antiresorptive and anabolic therapeutic agents that can be administered long-term. The most widely used antiresorptive agents, bisphosphonates (BPs), also inhibit bone formation and thus have limited effect in preventing osteoporotic fracture. Hydroxychloroquine (HCQ), which is used to treat rheumatoid arthritis, prevents the lysosomal degradation of TNF receptor-associated factor 3 (TRAF3), an NF-κB adaptor protein that limits bone resorption and maintains bone formation. We attempted to covalently link HCQ to a hydroxyalklyl BP (HABP) with anticipated low antiresorptive activity, to target delivery of HCQ to bone to test if this targeting increases its efficacy to prevent TRAF3 degradation in the bone microenvironment and thus reduce bone resorption and increase bone formation, while reducing its systemic side effects. Unexpectedly, HABP-HCQ was found to exist as a salt in aqueous solution, composed of a protonated HCQ cation and a deprotonated HABP anion. Nevertheless, it inhibited osteoclastogenesis, stimulated osteoblast differentiation, and increased TRAF3 protein levels in vitro. HABP-HCQ significantly inhibited both osteoclast formation and bone marrow fibrosis in mice given multiple daily PTH injections. In contrast, HCQ inhibited fibrosis, but not osteoclast formation, while the HABP alone inhibited osteoclast formation, but not fibrosis, in the mice. HABP-HCQ, but not HCQ, prevented trabecular bone loss following ovariectomy in mice and, importantly, increased bone volume in ovariectomized mice with established bone loss because HABP-HCQ increased bone formation and decreased bone resorption parameters simultaneously. In contrast, HCQ increased bone formation, but did not decrease bone resorption parameters, while HABP also restored the bone lost in ovariectomized mice, but it inhibited parameters of both bone resorption and formation. Our findings suggest that the combination of HABP and HCQ could have dual antiresorptive and anabolic effects to prevent and treat osteoporosis.

TGFß1+CCR5+ neutrophil subset increases in bone marrow and causes age-related osteoporosis in male mice.

TGFß1 induces age-related bone loss by promoting degradation of TNF receptor-associated factor 3 (TRAF3), levels of which decrease in murine and human bone during aging. We report that a subset of neutrophils (TGFß1+CCR5+) is the major source of TGFß1 in murine bone. Their numbers are increased in bone marrow (BM) of aged wild-type mice and adult mice with TRAF3 conditionally deleted in mesenchymal progenitor cells (MPCs), associated with increased expression in BM of the chemokine, CCL5, suggesting that TRAF3 in MPCs limits TGFß1+CCR5+ neutrophil numbers in BM of young mice. During aging, TGFß1-induced TRAF3 degradation in MPCs promotes NF-κB-mediated expression of CCL5 by MPCs, associated with higher TGFß1+CCR5+ neutrophil numbers in BM where they induce bone loss. TGFß1+CCR5+ neutrophils decreased bone mass in male mice. The FDA-approved CCR5 antagonist, maraviroc, reduced TGFß1+CCR5+ neutrophil numbers in BM and increased bone mass in aged mice. 15-mon-old mice with TGFßRII specifically deleted in MPCs had lower numbers of TGFß1+CCR5+ neutrophils in BM and higher bone volume than wild-type littermates. We propose that pharmacologic reduction of TGFß1+CCR5+ neutrophil numbers in BM could treat or prevent age-related osteoporosis.

Biofabrication and preclinical evaluation of a large-sized human self-assembled skin substitute.

Severe skin burns are widely treated using split-thickness skin autografts. However, the accessibility of the donor site may be limited depending on the size of the injured surface. As an alternative to skin autografts, our laboratory is clinically investigating a model of human self-assembled skin substitute (SASS) with a standard size of 35 cm2. For the management of extensive skin wounds, multiple grafts are required to cover the entire wound bed. Even if SASSs could provide an adequate and efficient treatment, in some cases, the long-term follow-up of the skin graft site reveals the appearance of marks at the junction between SASSs. This study aims to produce a large-sized self-assembled skin substitute (L-SASS; 289 cm2) and evaluate its preclinical potential for skin wound coverage. The L-SASSs and SASSs shared similar contraction behavior on an agar surface, thickness, and epidermal differentiation in vitro. After grafting, similar histological results were obtained for skin substitutes produced with both methods. Hence, the self-assembly approach of tissue engineering is a scaffold-free method that allows the production of living skin substitutes in a large format.

TGFß-induced degradation of TRAF3 in mesenchymal progenitor cells causes age-related osteoporosis.

Inflammaging induces osteoporosis by promoting bone destruction and inhibiting bone formation. TRAF3 limits bone destruction by inhibiting RANKL-induced NF-κB signaling in osteoclast precursors. However, the role of TRAF3 in mesenchymal progenitor cells (MPCs) is unknown. Mice with TRAF3 deleted in MPCs develop early onset osteoporosis due to reduced bone formation and enhanced bone destruction. In young mice TRAF3 prevents ß-catenin degradation in MPCs and maintains osteoblast formation. However, TRAF3 protein levels decrease in murine and human bone samples during aging when TGFß1 is released from resorbing bone. TGFß1 induces degradation of TRAF3 in murine MPCs and inhibits osteoblast formation through GSK-3ß-mediated degradation of ß-catenin. Thus, TRAF3 positively regulates MPC differentiation into osteoblasts. TRAF3 deletion in MPCs activated NF-κB RelA and RelB to promote RANKL expression and enhance bone destruction. We conclude that pharmacologic stabilization of TRAF3 during aging could treat/prevent age-related osteoporosis by inhibiting bone destruction and promoting bone formation.

α-2-Macroglobulin induces the shedding of microvesicles from cutaneous wound myofibroblasts.

Microvesicles (MVs) are recognized as an important class of cell-to-cell messengers. Although the properties of MVs are increasingly documented, the mechanisms regulating MV biogenesis remain debated. Myofibroblasts are a key cellular component of wound healing and have been shown to produce MVs upon stimulation with serum. However, the mediator(s) responsible for the observed effect of serum on MV release have yet to be identified. To isolate the molecule(s) of interest, serum proteins were sequentially separated using chromatography, selective precipitation, and electrophoresis. MV production was assessed throughout the purification and after stimulation of myofibroblasts with two potent purified molecules. α-2-Macroglobulin (A2M) was thereby found to dose-dependently stimulate MV release. We confirmed the presence of the A2M receptor, low-density lipoprotein receptor-related protein-1 (LRP1), on myofibroblasts. Inhibition of LRP1 resulted in a significant decrease in MV production. Together, our results suggest that A2M positively regulates MV shedding through the activation of LRP1 on myofibroblasts.

Improved Methods to Produce Tissue-Engineered Skin Substitutes Suitable for the Permanent Closure of Full-Thickness Skin Injuries.

There is a clinical need for skin substitutes to replace full-thickness skin loss. Our group has developed a bilayered skin substitute produced from the patient's own fibroblasts and keratinocytes referred to as Self-Assembled Skin Substitute (SASS). After cell isolation and expansion, the current time required to produce SASS is 45 days. We aimed to optimize the manufacturing process to standardize the production of SASS and to reduce production time. The new approach consisted in seeding keratinocytes on a fibroblast-derived tissue sheet before its detachment from the culture plate. Four days following keratinocyte seeding, the resulting tissue was stacked on two fibroblast-derived tissue sheets and cultured at the air-liquid interface for 10 days. The resulting total production time was 31 days. An alternative method adapted to more contractile fibroblasts was also developed. It consisted in adding a peripheral frame before seeding fibroblasts in the culture plate. SASSs produced by both new methods shared similar histology, contractile behavior in vitro and in vivo evolution after grafting onto mice when compared with SASSs produced by the 45-day standard method. In conclusion, the new approach for the production of high-quality human skin substitutes should allow an earlier autologous grafting for the treatment of severely burned patients.

Role of seaweed laminaran from Saccharina longicruris on matrix deposition during dermal tissue-engineered production.

Our laboratory has developed a technique to reconstruct in vitro tissue from human cells using the self-assembly tissue-engineering method, which utilizes the ability of fibroblasts to deposit the matrix they secrete. The time necessary for tissue construction, several weeks, is a drawback for many clinical uses. We hypothesized that the addition of laminaran can increase the deposition of matrix, speeding up the production of the tissue. Laminaran was isolated from the brown seaweed Saccharina longicruris harvested in Canada and its structure was evaluated. Laminaran is a small molecular weight polysaccharide composed of linear glucose chains. Monolayer-cultured human skin fibroblasts were cultured in the presence of laminaran with ascorbate for 7 or 35 days to produce a dermis. Treatment did not induce any variation in the growth rate or alpha smooth muscle actin content but it did increase the deposition of collagen I in a dose-dependent manner. After 35 days, the reconstructed dermal thickness was increased when laminaran was added, and collagen I deposition and MMP activity were also significantly increased. Thus, laminaran can be used to increase the rate of production of reconstructed self-assembled dermis and can also potentially be used in cosmetic or therapeutic creams to stimulate matrix production.