VTA dopamine neurons are hyperexcitable in 3xTg-AD mice due to casein kinase 2-dependent SK channel dysfunction.

Alzheimer's disease (AD) patients exhibit neuropsychiatric symptoms that extend beyond classical cognitive deficits, suggesting involvement of subcortical areas. Here, we investigated the role of midbrain dopamine (DA) neurons in AD using the amyloid + tau-driven 3xTg-AD mouse model. We found deficits in reward-based operant learning in AD mice, suggesting possible VTA DA neuron dysregulation. Physiological assessment revealed hyperexcitability and disrupted firing in DA neurons caused by reduced activity of small-conductance calcium-activated potassium (SK) channels. RNA sequencing from contents of single patch-clamped DA neurons (Patch-seq) identified up-regulation of the SK channel modulator casein kinase 2 (CK2). Pharmacological inhibition of CK2 restored SK channel activity and normal firing patterns in 3xTg-AD mice. These findings shed light on a complex interplay between neuropsychiatric symptoms and subcortical circuits in AD, paving the way for novel treatment strategies.

Lymphatics in bone arise from pre-existing lymphatics.

Bones do not normally have lymphatics. However, individuals with generalized lymphatic anomaly (GLA) or Gorham-Stout disease (GSD) develop ectopic lymphatics in bone. Despite growing interest in the development of tissue-specific lymphatics, the cellular origin of bone lymphatic endothelial cells (bLECs) is not known and the development of bone lymphatics has not been fully characterized. Here, we describe the development of bone lymphatics in mouse models of GLA and GSD. Through lineage-tracing experiments, we show that bLECs arise from pre-existing Prox1-positive LECs. We show that bone lymphatics develop in a stepwise manner where regional lymphatics grow, breach the periosteum and then invade bone. We also show that the development of bone lymphatics is impaired in mice that lack osteoclasts. Last, we show that rapamycin can suppress the growth of bone lymphatics in our models of GLA and GSD. In summary, we show that bLECs can arise from pre-existing LECs and that rapamycin can prevent the growth of bone lymphatics.

Thiolated bone and tendon tissue particles covalently bound in hydrogels for in vivo calvarial bone regeneration.

Bone regeneration of large cranial defects, potentially including traumatic brain injury (TBI) treatment, presents a major problem with non-crosslinking, clinically available products due to material migration outside the defect. Commercial products such as bone cements are permanent and thus not conducive to bone regeneration, and typical commercial bioactive materials for bone regeneration do not crosslink. Our previous work demonstrated that non-crosslinking materials may be prone to material migration following surgical placement, and the current study attempted to address these problems by introducing a new hydrogel system where tissue particles are themselves the crosslinker. Specifically, a pentenoate-modified hyaluronic acid (PHA) polymer was covalently linked to thiolated tissue particles of demineralized bone matrix (TDBM) or devitalized tendon (TDVT), thereby forming an interconnected hydrogel matrix for calvarial bone regeneration. All hydrogel precursor solutions exhibited sufficient yield stress for surgical placement and an adequate compressive modulus post-crosslinking. Critical-size calvarial defects were filled with a 4% PHA hydrogel containing 10 or 20% TDBM or TDVT, with the clinical product DBXⓇ being employed as the standard of care control for the in vivo study. At 12 weeks, micro-computed tomography analysis demonstrated similar bone regeneration among the experimental groups, TDBM and TDVT, and the standard of care control DBXⓇ. The group with 10% TDBM was therefore identified as an attractive material for potential calvarial defect repair, as it additionally exhibited a sufficient initial recovery after shearing (i.e., > 80% recovery). Future studies will focus on applying a hydrogel in a rat model for treatment of TBI. STATEMENT OF SIGNIFICANCE: Non-crosslinking materials may be prone to material migration from a calvarial bone defect following surgical placement, which is problematic for materials intended for bone regeneration. Unfortunately, typical crosslinking materials such as bone cements are permanent and thus not conducive to bone regeneration, and typical bioactive materials for bone regeneration such as tissue matrix are not crosslinked in commercial products. The current study addressed these problems by introducing a new biomaterial where tissue particles are themselves the crosslinker in a hydrogel system. The current study successfully demonstrated a new material based on pentenoate-modified hyaluronic acid with thiolated demineralized bone matrix that is capable of rapid crosslinking, with desirable paste-like rheology of the precursor material for surgical placement, and with bone regeneration comparable to a commercially available standard-of-care product. Such a material may hold promise for a single-surgery treatment of severe traumatic brain injury (TBI) following hemicraniectomy.

Glyoxal cross-linking of solubilized extracellular matrix to produce highly porous, elastic, and chondro-permissive scaffolds for orthopedic tissue engineering.

Extracellular matrix (ECM)-derived implants hold great promise for tissue repair, but new strategies are required to produce efficiently decellularized scaffolds with the necessary porosity and mechanical properties to facilitate regeneration. In this study, we demonstrate that it is possible to produce highly porous, elastic, articular cartilage (AC) ECM-derived scaffolds that are efficiently decellularized, nonimmunogenic, and chondro-permissive. Pepsin solubilized porcine AC was cross-linked with glyoxal, lyophilized and then subjected to dehydrothermal treatment. The resulting scaffolds were predominantly collagenous in nature, with the majority of sulphated glycosaminoglycan (sGAG) and DNA removed during scaffold fabrication. Four scaffold variants were produced to examine the effect of both ECM (10 or 20 mg/mL) and glyoxal (5 or 10 mM) concentration on the mechanical and biological properties of the resulting construct. When seeded with human infrapatellar fat pad-derived stromal cells, the scaffolds with the lowest concentration of both ECM and glyoxal were found to promote the development of a more hyaline-like cartilage tissue, as evident by increased sGAG and type II collagen deposition. Furthermore, when cultured in the presence of human macrophages, it was found that these ECM-derived scaffolds did not induce the production of key proinflammatory cytokines, which is critical to success of an implantable biomaterial. Together these findings demonstrate that the novel combination of solubilized AC ECM and glyoxal crosslinking can be used to produce highly porous scaffolds that are sufficiently decellularized, highly elastic, chondro-permissive and do not illicit a detrimental immune response when cultured in the presence of human macrophages.